February 11, 2009
January 14, 2009
January 12, 2009
Monday, January 12th - Germany
Had a challenging day (legs have peripheral neuropothy and I was just tired). I slept all afternoon but we had a good night. Drove around some and had Indian food that was excellent (German food for dinner last night and breakfast/lunch today.
Berlin is very nice and we're going to take a bus tour tomorrow and then drive to Duderstadt (this is the town where the "Klinik" is located) late afternoon on Tuesday. 3 hours according to GPS on my BlackBerry Storm which has been great except for lousy phone reception in the hotel room and running out of battery today.
I couldn't find my charger before we left but we bought a new one tonight. Boy did I go through BlackBerry withdraw today. That won't happen again!
Appointment is on Wednesday at 8:30AM on Wednesday in Duderstadt to harvest my Dendritic Cells. The first injection of the vaccination will be on 1/21 at 1:30PM at Dr. Chang's office in New York and will be administered by one of the doctors from Germany who will bring it over along with those for other patients.
Thanks to ALL who have helped make this possible! It's not likely that I would be having this potentially life saving treatment without the overwhelming support of my many friends and family.
Dan
Monday, January 12th - Germany
Had a challenging day (legs have peripheral neuropothy and I was just tired). I slept all afternoon but we had a good night. Drove around some and had Indian food that was excellent (German food for dinner last night and breakfast/lunch today.
Berlin is very nice and we're going to take a bus tour tomorrow and then drive to Duderstadt (this is the town where the "Klinik" is located) late afternoon on Tuesday. 3 hours according to GPS on my BlackBerry Storm which has been great except for lousy phone reception in the hotel room and running out of battery today.
I couldn't find my charger before we left but we bought a new one tonight. Boy did I go through BlackBerry withdraw today. That won't happen again!
Appointment is on Wednesday at 8:30AM on Wednesday in Duderstadt to harvest my Dendritic Cells. The first injection of the vaccination will be on 1/21 at 1:30PM at Dr. Chang's office in New York and will be administered by one of the doctors from Germany who will bring it over along with those for other patients.
Thanks to ALL who have helped make this possible! It's not likely that I would be having this potentially life saving treatment without the overwhelming support of my many friends and family.
Dan
January 10, 2009
What Is Dendritic Cell Therapy?
Dendritic Cell (DC) Therapy or so-called Dendritic Cell vaccine is a newly emerging and potent form of immune therapy used to treat cancer, AIDS and other serious conditions. In case of cancer, Dendritic Cell therapy is an immune therapy which harnesses the body's own immune system to fight cancer. The Dendritic Cell itself is an immune cell whose role is the recognition, processing and presentation of foreign antigens to the T-cells in the effector arm of the immune system. Although Dendritic Cells are potent cells, they are not usually present in adequate quantity to allow for a potent immune response. Dendritic Cell Therapy thus involves the harvesting of blood cells (i.e. monocytes or macrophages) from a patient and processing them in the laboratory to produce Dendritic Cells which are then given back to a patient in order to allow massive Dendritic participation in optimally activating the immune system. To learn more about vaccine and Dendritic Cell therapy for cancer, please read the following articles:
The National Cancer Institute has a very concise primer on Treating and Preventing Cancer with Vaccines on their site.
“Dendritic Cells (I): Biological functions” and “Dendritic Cells (II) Role and therapeutic implications in cancer” by S. Satthaporn and O. Eremin of the U. of Nottingham and Lincoln County Hospital, UK as published in J. of the Royal College Surgeons, Edinb.
46:9-20 and 159-167,2001.
“Clinical Applications of Dendritic Cell Cancer Vaccines” by Dr. Joseph Barr of the U. of Pittsburgh Cancer Institute, Pittsburgh, PA USA in The Oncologist 4(2): 140-144, 1999.
Also, a slide show / lecture presentation by Dr. Michael Morse on “Current Status of Dendritic-Cell Vaccines” is available on the Medscape site from WebMD as part of an educational session on “Therapeutic Cancer Vaccines: Targeting the Future of Cancer Treatment” but requires registration to enter the site.
Abstracts of recent reviews on Pubmed include articles by IG Schmidt-Wolf et al. on “Dendritic Cell, the immunotherapeutic cell for cancer”, TL Whiteside and C Odous from U. of Pittsburgh Cancer Institute on “Dendritic cell biology and cancer therapy”, EM Hersh et al. on “Clinical Applications of dendritic cell vaccination in the treatment of cancer”
Also, please refer to our Research Archives for many related abstracts on the therapy.
About Vaccines
What is a Vaccine?
What are Cancer Vaccines?
Substances Used to Make Vaccines
What Is a Vaccine?
A vaccine is a substance designed to stimulate the immune system to launch an immune response. This response is directed against specific targets, or antigens, that are part of the vaccine. An antigen is any substance that the immune system recognizes as foreign.
The flu vaccine, for example, contains copies of the flu virus that cannot cause the flu. Antigens on the viruses in the vaccine stimulate the immune system to produce cells that can fight the flu virus if it shows up in your nose or throat.
The flu vaccine only works if it is given at least two weeks before exposure to infectious flu virus. The immune system needs those two weeks to produce immune cells that can attack the flu virus.
Because the flu virus changes from year to year, a new flu vaccine is needed every year. Your immune system, however, still protects you against last year's flu type. This type of vaccine is called a preventive vaccine - it stimulates a long-lasting immunity that helps protect you from getting sick for years or even for a lifetime.
What Are Cancer Vaccines?
Cancer vaccines are intended either to treat existing cancer or to prevent the development of cancer. Cancer treatment vaccines are designed to strengthen the body's natural defenses against a cancer that has already developed. These vaccines may stop an existing tumor from growing, stop a tumor from coming back after it has been treated, or eliminate cancer cells not killed by previous treatments.
Cancer preventive vaccines are given to healthy people and are designed to target infectious agents that can cause cancer. The HPV vaccine is an example of a cancer preventive vaccine. It is used to help prevent cervical cancer.
Substances Used to Make Vaccines
Vaccines can be made using specific types of molecules from viruses or cells, including molecules from bacterial cells or human cells. These molecules may contain a single antigen or several different antigens. Carbohydrates (sugars), proteins, and peptides (pieces of proteins) are among the types of molecules that have been used to make vaccines. Molecules of DNA or RNA that contain genetic instructions for one or more antigens can also be used as vaccines (for more information, see Cancer Vaccine Strategies.)
In addition, whole viruses or cells, or parts of viruses or cells that contain different types of molecules, can be used to make vaccines. The flu vaccine, for example, is made using inactive whole flu viruses. If whole human cells are used as vaccines, they are usually treated with enough radiation to keep them from dividing (growing and multiplying) or enough to kill them.
Immune Cells Reveal Fancy Footwork
ScienceDaily (Dec. 7, 2008) — Our immune system plays an essential role in protecting us from diseases, but how does it do this exactly? Dutch biologist Suzanne van Helden discovered that before dendritic cells move to the lymph nodes they lose their sticky feet. This helps them to move much faster. Immature dendritic cells patrol the tissues in search of antigens.
After exposure to such antigens they undergo a rigorous maturation process. During this maturation the dendritic cells migrate to the lymph nodes to activate T cells. Suzanne van Helden studied the adhesion and migration of both immature and mature dendritic cells.
Dendritic cell as a general
A dendritic cell can be compared with a pocket-sized general. As an immature cell he is on patrol in the bloodstream and in tissues in search of foreign bodies. The feet, or podosomes, help the cell to move around at a slow pace. As soon as immature dendritic cells detect a problem they must report back quickly to the T cells to warn them of impending danger. The dendritic cells are then hindered by their adhesive feet.
This is the reason why at this point the cell undergoes modifications and loses its feet. In this way the mature dendritic cell can wing its way to the T cells at full speed. Once alerted, the T cells can intervene and tackle the problem in the body's infected tissues.
Van Helden not only demonstrated that dendritic cells lose their podosomes very quickly during maturation but she also identified the substances that are responsible for their disappearance. The presence of prostaglandin E2 is indispensable for this disassembly. In addition, it appears that dendritic cells lose their podosomes after interaction with certain bacteria. What is striking is that only gram-negative bacteria lead to podosome loss. Gram-positive bacteria do not have this effect. Van Helden concludes that dendritic cells can apparently distinguish between different pathogens.
Dendritic cells in action
The immune system can act in different ways to keep the body healthy. Unfortunately the working of the immune system is not perfect. In cancer for example, the immune system does not respond to the altered cells that make up the tumor. It is possible that this knowledge about the adhesion and migration of dendritic cells could contribute to future developments in a new approach to cancer treatment.
Van Helden carried out her research within a group of scientists that study the function of dendritic cells in different ways. The research comprises not only fundamental research, as in Van Helden's case, but also preclinical and clinical trials. The research was made possible by a grant from NWO. Spinoza Prize winner Carl Figdor supervised Van Helden during her research.
Adapted from materials provided by NWO (Netherlands Organization for Scientific Research).
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FREQUENTLY ASKED QUESTIONS | |
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SCIENTIFIC REVIEW
Dendritic cells (I) : biological functions
S. SATTHAPORN* and O. EREMIN*# Medical Center University Nottingham U.K. Lincolnshire Lincoln , U.K.
*Section of Surgery, E Floor, West Block, Queen's
Dendritic cells (DCs) are potent antigen presenting cells (APCs) that possess the ability to stimulate naïve T cells. They comprise a system of leukocytes widely distributed in all tissues, especially in those that provide an environmental interface. DCs posses a heterogeneous haemopoietic lineage, in that subsets from different tissues have been shown to posses a differential morphology, phenotype and function. The ability to stimulate naïve T cell proliferation appears to be shared between these various DC subsets. It has been suggested that the so-called myeloid and lymphoid-derived subsets of DCs perform specific stimulatory or tolerogenic function, respectively. DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens (Ags), and their subsequent presentation on the cell surface linked to major histocompatibility (MHC) molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues where they present Ag to T cells and induce an immune response. DCs are receiving increasing scientific and clinical interest due to their key role in anti-cancer host responses and potential use as biological adjuvants in tumor vaccines, as well as their involvement in the immunobiology of tolerance and autoimmunity.
J.R.Coll.Surg.Edinb., 46, February 2001, 9-20
Dendritic cells (DCs), originally identified by Steinman and his colleagues (1972) represent the pacemakers of the immune response.1 They are crucial to the presentation of peptides and proteins to T and B lymphocytes and are widely recognized as the key antigen presenting cells (APCs). They are critical for the induction of T cell responses resulting in cell-mediated immunity (CMI). The T cell receptors (TCRs) on T lymphocytes recognize fragments of antigens (Ags) bound to molecules of the major histocompatibility complex (MHC) on the surfaces of APCs. The peptide binding proteins are of two types, MHC class I and II, which interact with and stimulate cytotoxic T lymphocytes (CTLs) and T helper cells (Ths), respectively. On entry into APCs, Ags are processed, spliced into peptides in the cytosol and then reexpressed on the cell surface linked to MHC proteins. When bound to MHC class I molecules, CTLs are generated and activated and cells in tissues expressing the Ags (e.g. virus infected cells, cancer cells) are recognized and destroyed. Antigens reexpressed on the cell surface linked to MHC class II molecules interact with Th cells which when activated have profound immune-regulatory effects.2 Thus, DCs play a key role in host defenses and a crucial role in putative anti-cancer immune responses.
DENDRITIC CELL DEVELOPMENT
Introduction
An important advance in DC biology, within the past few years, has been the ability to propagate in vitro large numbers of DCs, using defined growth factors. One of the most important findings is that DCs are not a single cell type, but a heterogeneous collection of cells that have arisen from distinct, bone marrow-derived, hematopoietic lineages.3-7 To date, at least three different pathways have been described. The emerging concepts are that each pathway develops from unique progenitors that particular cytokine combinations drive developmental events within each pathway and that cells developing within a particular pathway exhibit distinct specialized functions.3-7
The ability to propagate DC subtypes, at various stages of development in vitro, from early progenitors has been critical in assessing the developmental and functional characteristics of DCs. Together with in situ histochemical analyses and genetically modified animal models, in vitro studies have shown that the earliest DC progenitors/precursors are released from the bone marrow and circulate through the blood and lymphoid organs ready to receive differentiation signals.4-6
Several studies have been carried out suggesting that there are different pathways for the formation of mature DCs from CD34+ or other primitive progenitors. Each pathway differs in terms of progenitors and intermediate stages, cytokine requirements, surface marker expression and, probably most importantly, biological function.
Lymphoid-related DC pathway
The close lineage relationship of DCs and monocytes has been challenged in recent years by the findings of several groups who have described the development of DCs and lymphoid cells from the same precursors. The term lymphoid DC is meant to describe a distinct DC subtype that is closely linked to the lymphocyte lineage.
Initially described in the mouse, the term ‘lymphoid’ refers to several features that suggest a precursor in common with T cells. This pathway appears to lack a number of characteristics found in myeloid cells, in particular, lack of defined surface phenotypes-CD11b, CD13, CD14, and CD33. 7 In blood, the lymphoid precursors may be the CD4+ CD11c+ ‘plasma-like cell’. There is now some evidence that an equivalent lymphoid lineage DC exists in humans.7-12 Lymphoid DCs may also arise from progenitors that also have the potential to mature into T and natural killer (NK) cells.7-12 Such progenitors are distributed in the thymus and in the T cell areas of secondary lymphoid tissues.5,7-12 Lymphoid DCs may develop from thymic progenitors when stimulated with Interleukin 3 (IL-3), but not granulocyte macrophage colony-stimulating factor (GM-CSF), and from lymphoid precursors in human tonsil treated with CD40 ligand (CD40L).7,10 More recently, IL-2 and IL-15 have been shown to drive NK cell-associated (IL-2R+) DCs from CD34+ progenitors.11,12
A variety of functions have been attributed to lymphoid DCs. They promote negative selection in the thymus (possibly by inducing fas-mediated apoptosis) and are costimulatory for CD4+ and CD8+T cells.7, 13-15 More recently, lymphoid-like DCs derived from human progenitors have also been shown to preferentially activate the Th2 response.14 Because of their capacity to induce apoptosis and their role in eliminating potentially self-reactive T cells, it has been suggested that lymphoid DCs primarily mediate regulatory rather than stimulatory immune effector functions.7, 14, 16
In the human bone marrow, Galy et al (1995) identified a subset of progenitor cells defined by the phenotype CD34+ CD38+ Thy-1- CD10+. The latter, when cultured under appropriate conditions, were capable of giving rise to T, B, NK and DCs but not to myeloid cell types.8 Progenitors with a similar phenotype but lacking CD10 expression could give rise to myeloid cells and more prolonged thymopoiesis suggesting that acquisition of CD10 corresponded to a maturation step and commitment to T, B, NK and DC lineages. Similarly, a CD34+ Thy-1- but CD38dim foetal thymic precursor gave rise to T, NK and DCs but not other (myeloid) lineages, whilst the more mature CD34+ CD38+ thymic precursor was shown to have less DC potential.17 These results suggest that thymic DCs and thymocytes are derived from a common precursor that migrates to the thymus prior to lineage commitment and terminal differentiation. Lymphoid DCs include those in the thymic medulla and many of the DCs in the T cell areas of all peripheral lymphoid organs. Dendritic cells in the latter T cell areas, however, are heterogeneous and include other types of DCs, for example, sentinel and migratory DCs that have brought Ags from peripheral tissues. Lymphoid DCs in T cell areas have the capacity to regulate self-reactive T cells, for example, by the production of IL-10 or other cytokines, or to delete them, for example, by induction of apoptosis by a member of the tumor necrosis factor (TNF) family like fasL18 or CD30L.19
Myeloid DC pathway: CD34+ haemopoietic progenitors
The myeloid pathway is distinguished by a development stage in which there is expression of certain features associated with phagocytes. Studies with multipotent CD34+ progenitors and peripheral blood mononuclear cells (PBMCs) have described different DC pathways, both associated with the myeloid lineage.
The skin contains a prominent supply of tissue DCs, termed Langerhan cells (LCs), which have typical DC morphology and contain characteristic Birbeck granules (BGs), seen on electron microscopy or by staining, using a specific monoclonal antibody (Mab).20 Evidence from murine bone marrow transplantation studies suggests that LCs are derived from the donor and that they are presumably of myeloid origin.21 In the rat, Bowers and Berkowitz (1986) demonstrated DCs in myeloid colonies in semi-solid cultures of Ia- bone marrow precursors and this was noted also in clonal assays of human bone marrow.22,23 More recent studies, using methylcellulose culture assays of human bone marrow or PBMCs, identified colonies both of pure DCs and also of mixed dendritic/macrophage cell types. Colonies were observed after 14 days of culture when stimulated by leukocyte conditioned medium.24 The resultant cells resembled LCs in their gross morphology and ultrastructure though they lacked the BGs typical of skin LCs. They were CD34+, had high levels of HLA-DR but, unlike the macrophages from the same cultures, were also HLA-DQ+ and lacked both a strong non-specific esterase expression and the monocyte-associated cytoplasmic antigen CD68. Most characteristically, they strongly expressed CD1a+ and their DC phenotype was confirmed by high allostimulatory activity in mixed leukocyte reactions (MLRs)-greater than either macrophages or even fresh blood DCs.
Myeloid DC pathway: PBMCs and CD14+cells
There is considerable evidence from culture studies for a close developmental relationship between DCs and cells of the monocyte/macrophage lineage.6,25 Adherent PBMCs are enriched for monocytes, and this fraction may develop a LC phenotype and function if cultured in the presence of foetal calf serum (FCS).26,27 Further, amongst the PBMCs, only purified monocytes are capable of expressing the LC marker CD1a if cultured in GM-CSF.28 The cytokines required for the in vitro production of DCs from the adherent fraction of PBMCs were first documented by Romani et al (1994) and by Sallusto and Lanzavecchia (1994).29,30 They demonstrated that cultures of PBMCs in GM-CSF and IL-4 produced cells that were CD1a+ CD14- and capable of Ag uptake and processing, the typical profile of immature DCs. Yields of up to 8x106 DCs were obtained from 40 ml of blood.29,30 The possibility that the DCs were derived from contaminating progenitor cells had been excluded by using highly purified CD14Bright monocytes31 and the absence of cellular proliferation in culture.20, 32, 33 Although the resulting cells resembled immature DCs they were atypical because of the presence of lysozyme, myeloperoxidase, non-specific esterase and their lack of CD83.20, 21, 31 In some studies, further differentiation into fully mature DCs could be induced by exposing these cells to a or CD40.21 This final maturation was characterised by down regulation of the ability to take up and process Ag whilst CD54, HLA-DR, CD83 and CD80 expression increased in parallel with the Ag presenting function.21 Rozenwaig et al. (1996) demonstrated a well ordered phenotypic evolution of DC precursors via CD13Low progenitors to CD13High CD1a- and then CD13High CD1a+ intermediates that also expressed variable levels of CD14.23 Using a similar approach, two pathways of DC maturation from cord blood CD34+ progenitors were identified. After 5 days in culture with GMCSF, SCF and TNF-a, cells were sorted into either CD14+CD1a- or CD14-CD1a+ populations (see Table 1).3
Table 1: Comparison of the different developmental pathways of DCs of myeloid origin
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Derived from CD14+CD1a - |
Derived from CD14-CD1a+ |
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Related to interstitial and/or circulating blood DCs |
Related to epidermal DCs (LCs) |
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Nonspecific esterase activity; complement receptors (CD11b, CD15b) |
Intracelular BGs, Lag moléculas, E-cadherin |
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Phagocytic properties |
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CD68+ and express coagulation factor XIIIa |
Lack CD68 and factor XIIIa expression |
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TNF, GM-CSF and IL-13 or IL-4 induce maturation |
TGF-b induces maturation |
FUNCTIONAL DIFFERENCES BETWEEN THE DIFFERENT DC SUBSETS
Although functional differences exist between the myeloid and lymphoid DCs, functional segregation within the myeloid DC lineage system also exists. Several in vitro studies have shown that CD14-derived DCs prime T cells to preferentially activate Th1 responses and IL-12 appears implicated in this process.34, 35 CD14-derived DCs from CD34+ progenitors, but not CD1a-derived DCs, also activate naive B cells to secrete IgM in the presence of CD40L and IL-2.36 In psoriasis and atopic asthma, distinct DC subtypes activate either Th1 or Th2 responses, respectively supporting the existence of pathophysiological associations between DC subtypes and Th cell subsets.37, 38 This raises the possibility of redirecting tissue destructive T cell responses to nondamaging T cell responses in certain diseases. Additional observations indicate that CD14-derived DCs are increased in rheumatoid arthritis as it is predominantly associated with an inflammatory Th1 response.39-41
The thymic DCs expressing CD8a+ appear to be functionally different from CD8a- DCs in that they express Fas-L and can induce T cell apoptosis. Thymic DCs are also far less efficient at inducing T cell IL-2 cytokine production.18 Thus, CD8a+ DCs may have regulatory properties, whereas CD8a- DCs seem to exert T cell stimulatory function. More functional studies have to be performed in order to fully evaluate the pro B lymphocyte DCs, and whether the pro B lymphocyte-derived DCs have unique functions that are not exhibited by the thymic DCs, remains to be elucidated.
It is clear from the forgoing discussion that DCs may develop from a myeloid or lymphoid lineage. The myeloid pathway of differentiation gives rise to DCs that home to peripheral tissues to take up and process exogenous Ags prior to migrating to the secondary lymphoid tissues to present Ags to naïve T cells. Thymic DCs, on the other hand, perform a very different function being involved in the presentation of self-Ag to developing thymocytes and, hence, the subsequent deletion of autoreactive T cells. It would be appropriate for the precursors of thymic DCs to migrate to the thymus in an immature form and undergo development exposed only to self-Ag within the thymus.42 Thus, the existence of alternative developmental pathways would be in keeping with the different functions of DCs in different tissues.
Further studies have revealed that CD8a+ DCs were at least equivalent to CD8a- DCs in stimulating both CD4 and CD8 responses in vivo and in vitro. A surprising recent finding is that, CD8a+ DCs, but not CD8a- DCs, produce significant levels of IL-12 and prime Th1 T cell response.43-46 Thus, the immunoregulatory potential of DCs may depend less on ontology than on recent activatory or downregulatory stimuli. Appropriate induction of T cell tolerance or activation would be ensured by allowing DC behavior to be influenced by environmental signaling at the time of Ag encounter.
MIGRATION AND FUNCTIONAL PROPERTIES OF DENDRITIC CELLS
Myeloid DCs are derived successively from proliferating progenitor cells and non proliferating precursors (especially monocytes). They migrate to and reside as immature DCs at body surfaces and interstitial spaces. Immature DCs have abundant MHC II products within intracellular compartments (MIICs) and respond rapidly to inflammatory cytokines and microbial products to produce mature T cell stimulatory DCs with abundant surface MHC II proteins (see Table 2 and 3); eventually leading to apoptotic death. Some researchers have reported that the Flt-3 ligand can mobilize DCs from proliferating progenitors in humans.47 The immature DCs have many MIICs but require a maturation stimulus to irreversibly differentiate into active T cell stimulatory, mature DCs. Randolph (1998) has described an in vitro system involving monocytes reverse transmigrating across an endothelial monolayer that offers a possible explanation.48 This type of situation would occur when cells move from tissues to afferent lymph. It is possible that veiled DCs in lymph originate from monocytes in tissue that interact with the lymphoid endothelium to acquire the properties of immature DCs. If the monocytes also phagocytose particles before they reverse transmigrate, then the cells become typical mature DCs; the process occurs within 48 hours. The cells posses several markers (p55, DC-LAMP and CD83) that are expressed by mature DCs but are weak or absent in other leukocytes.49-51 Dendritic cells that have matured from monocytes in this in vitro system also express very high levels of surface MHC class II and CD86 and, in complete contrast to monocytes, have lost CD14, CD32 and CD64, all within 48 hours of culture (see Tables 2 and 3).
Table 2: Characteristics of immature dendritic cells
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Characteristics of immature dendritic cells
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Table 3: Characteristics of mature dendritic cells
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Characteristics of mature dendritic cells
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Another source of DCs is the immature LC present within the epidermis. LCs are the prototype immature DC, as revealed by Schuler and Romani (1985 and 1989).52, 53 Immature DCs lack or have low levels of several important accessory molecules that mediate binding and stimulation of T cells -CD40, 54, 58, 80 and 86.52, 53 The MHC II molecules are primarily within the cell in MIICs that coexpress lysosomalassociated membrane proteins and HLA-DM or H-2M.54-56 Randolph et al (1998) have reported that human LCs express high level of MDR-1, a multi-drug resistance receptor. LC migration in vitro from skin organ cultures is blocked by anti-bodies or drugs (verapamil, reserpine) that block MDR-1 or P-glycoprotein.57 There are at least 2 mdr genes, and one controls the extrusion of leukotrienes. Also, recent studies show that specific chemokines and chemokine receptors direct the movements of immature and mature DCs; in particular the inflammatory chemokines macrophage inflammatory protein (MIP)-1a and MIP-3a for mobilizing immature DCs and constitutive lymphoid chemokines (MIP-3b or EBI1-ligand chemokines (ELC), 6C-kine or secondary lymphoid tissue chemokines (SLC), for directing mature DCs to T cell areas in secondary lymphoid compartments.58-60
MATURATION AND FUNCTION OF DENDRITIC CELLS
In most tissues, DCs are present in a so-called ‘immature’ state and are unable to stimulate T cells. Although these DCs lack the requisite accessory signals for T cell activation, such as CD40, CD54, CD80 and CD86, they are extremely well equipped to capture Ags in peripheral sites. Once they have acquired and processed the foreign Ags, they migrate to the T cell areas of lymph nodes (LNs) and the spleen, undergo maturation and stimulate an immune response.
Immature DCs have several features that allow them to capture Ag. Firstly, they can take up particles and microbes by phagocytosis.61, 62 Secondly, they can form large pinocytic vesicles in which extracellular fluid and solutes are sampled; a process called macropinocytosis.55 And thirdly, they express receptors that mediate adsorptive endocytosis, including lectin receptors like the macrophage mannose receptor and DEC-205, as well as Fcg and Fce receptors.30,63
Macropinocytosis and receptor-mediated Ag uptake is very efficient, requiring picomolar and nanomolar concentration of Ag, much less than the micromolar levels typically required by other APCs. However, once DCs have captured Ags, which also provide the signal to mature, their ability to capture more Ag rapidly declines.
The captured Ags enter the endocytic pathway of the cell. In macrophages, most of the protein substrates are directed to the lysosomes, organelles with only a few MHC class II molecules, where the Ags are completely digested into amino acids. By contrast, DCs are able to produce large amounts of MHC class II-peptide complexes. This is due to the specialized, MHC class II rich compartments (MIICs) that are abundant in immature DCs.54, 64, 65 During maturation of DCs, MIICs convert to non-lysosomal vesicles and discharge their MHC-peptide complexes on to the cell surface.65, 66
Once primed, the DCs migrate to secondary lymphoid compartments (e.g. LNs) to present Ag-peptide complexes to naïve CD4+ T cells and CD8+ cytotoxic T cells. Following education by Ag-loaded DCs in LNs, naïve CD4+ T cells differentiate into memory helper T cells, which support the differentiation and expansion of CD8+ CTLs and B cells. Helper T cells exert anti-tumor activity indirectly through the activation of important effector cells such as macrophages and CTLs, which are capable of eradicating tumor cells or virus-infected cells directly. DCs are able also to present Ags via the exogenous class I presentation pathway.67,68 A dedicated peptide transporter translocates these peptides from the cytosol to the endoplasmic reticulum, where they bind to class I molecules. The peptide-bound MHC class I complexes migrate to the cell surface where they are displayed for T cells. This interaction generates CTLs, which have the capacity to eliminate virally infected cells and tumor cells.
It is clear that the maturation of DCs is crucial for the initiation of immunity. This process is characterized by reduced Ag-capture capacity and increased surface expression of MHC and co-stimulatory molecules. However, the maturation of DCs is completed only upon interaction with T cells. It is characterized by loss of phagocytic capacity and expression of many other accessory molecules that interact with receptors on T cells to enhance adhesion and signaling (co-stimulation); for example, LFA-3/CD58, ICAM-1/CD54, B7-1/CD80, B7-2/CD86 and CD83.69,70 (see Table 2 and 3) Expression of one or both of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on the DCs are essential for the effective activation of T lymphocytes, and, for IL-2 production.71 These co-stimulatory molecules bind the CD28 molecules on T lymphocytes. If this fails to occur at the time of Ag recognition by the TCR, an alternative T lymphocyte function may result, namely induction of anergy.54, 71 Another CD80/86 ligand, CTLA-4, is also induced on activated T lymphocytes, and this may contribute a negative regulatory signal.72, 73
Dendritic cells are a major source of many cytokines, namely, interferon-alpha (IFN-a), IL-1, IL-6, IL-7, IL-12 and IL-15 and also produce macrophage inflammatory protein (MIP1g), all of which are important in the elicitation of a primary immune response.74-80 Also, there is evidence that the cytokine secretion pattern of the plastic-adherent monocyte-derived DCs (grown in GM-CSF and IL-4) can be induced along the Th1 (IL-12) or Th 2 (IL-10) cytokine secretory pathway. Interleukin-12 production is critical for the promotion of an effective cellular immune response by activating and differentiating T lymphocyte to the Th 1 pathway. Its secretion appears to be inhibited by various tumor-derived substances, including nitric oxide (NO), prostaglandin E2 (PGE2), IL-10, IFN-a itself, the p40 homodimer of IL-12, and transforming growth factor b (TGF-b), which is regarded predominantly as an immunosuppressive cytokine.74, 78-80
Dendritic cells, grown and matured in vitro, can synthesize IL-10 in a continuous manner. Zhou and Tedder (1995) have found IL-10 transcripts, in CD83+ cells isolated from peripheral blood by RT-PCR analysis, and de Saints-Vis et al. (1998) have observed IL-10 synthesis by CD14+ DCs of myeloid origin. 74, 81 It is well established that monocytes and macrophages synthesize IL-10.82 It is also well known that IL-10 has an important regulatory role on monocyte function and on DC maturation.83-85 Furthermore, IL-10 has been documented to have a significant inhibitory effect on several aspects of APC function, for example, the expression of co-stimulatory molecules and the ability to synthesize IL-12.83, 85, 86 Importantly, IL-10 treated-DCs can be tolerogenic.87-90 Dendritic cells secreting IL-10 exhibit minimal or no stimulatory properties in primary MLRs and are markedly inhibitory to T cell proliferation induced by polyclonal activators91 Thus, IL-10 producing DCs are functionally and phenotypically inhibitory accessory cells and putatively tolerogenic.92
Figure 1: Morphological characteristic of human immature DC from peripheral blood after immuno-magnetic bead isolation (Wright-Giemsa, x 600)
Figure 2: Morphological characteristic of human mature and activated DC from lymph node after immuno-magnetic bead isolation (Wright-Giemsa, x 600)
DC DEATH (APOPTOSIS)
Although previous studies have demonstrated that developmentally and functionally end-stage DCs undergo apoptotic cell death, the possibility that apoptosis contributes to the regulation of the DC pathway at others stages of DC development has not been extensively explored.93
Functionally distinct apoptotic schedules were associated with different phases of DC development when multipotent CD34+ progenitor cells were treated with GM-CSF, TNF ± SCF (c-kit ligand).94 During early phases of growth (days 0-3), unselected progenitors underwent apoptosis. During intermediate stages (days 3-7), high levels of apoptosis resulted in the preferential selection of DC precursors, as revealed by the substantial expansion of DR+ CD33+ CD13+ cells. Late apoptosis (after day 10) was associated with the death of mature DCs. Apoptotic events surrounding the earlier periods were related to the exogenous addition of TNF-a and appeared to be mediated by fas. In contrast, those events associated with terminally differentiated DCs were fas-independent, because there was no correlation between fas expression and cell death. Recent studies by Canque et al (1998) have shown that the inclusion of TNF-a during DC development produces apoptotic events that selectively promote the CD1a-dependent DC pathway from GM-CSF, TNF-a treated CD34+ cord blood progenitors cells, lending support to the above observations.95 The susceptibility to apoptosis was remarkably decreased when DC precursors were treated with GM-CSF or IL-3, further supporting that these cytokines are viability (anti-death) factors for DCs.95
A number of researchers have concentrated on TRANCE or RANK-1, a TNF member identified by several groups recently. It increases the viability of mature myeloid DCs; both mouse DCs generated from the marrow or human DCs developed from monocytes.96, 97 TRANCE does not alter adhesion and co-stimulatory molecules like ICAM and B7, but it does make the mature DCs stay alive longer and express several cytokines genes, including IL-1, IL-6, IL-12 and IL-15. These responses are important features of mature DCs, which are sometimes referred to as activated and super activated. When the mature DCs encounters the correct TNF family member, its viability is improved and cytokine production is enhanced, thus, creating a longer lasting and more effective Ag presenting cell.96, 97
These studies show that, at least within the myeloid lineage, the activation of distinct apoptotic processes regulates DC development and homeostasis. Although suppression of apoptosis may prolong the survival of mature DCs, activation of apoptosis is required for the selective expansion of multipotent DC progenitors. These data also provide insight into the mechanisms of myeloid lineage selection by cytokines such as TNF-a, which may promote both cell death and survival.
DENDRITIC CELL DISTRIBUTION
Dendritic cells in peripheral blood
Although human PBDCs were first isolated in 1982 their phenotype has been poorly defined due to their low numbers and the lack of specific markers by which they could be clearly identified. Hitherto, purification of PBDCs has relied upon either sequential depletion of other PBMC subsets or on their physical properties, such as their capacity for transient adherence to plastic and their low density, thereby, permitting separation over density gradients.98 Using such techniques, it has been demonstrated that the most potent allostimulatory fraction of PBMCs possessed a phenotype characterized by high levels of HLA class II expression and absence of markers for other cell lineages.99 These studies and new techniques, such as immuno-magnetic bead depletion and fluorescence flow cytometry, have enabled the further characterization of PBDCs and the demonstration of at least two subpopulations of cells.100-102
Using three-color flow cytometry, two subpopulations of HLA-DR+ PBDCs, characterized by the phenotypes CD2-CD13- CD33dim CD11c- HLA-DR+ and CD2+ CD13+ CD33bright CD11c+ HLA-DR+ respectively, have been documented.100, 101, 103 The morphology of these two subtypes differ, the CD11c- population possess a lymphoid appearance and the CD11c+ population possess a monocytoid morphology.101, 103 Further, both subsets lack expression of the LC marker CD1a and express only low levels of adhesion and co-stimulation molecules CD80, CD86 and CD40, suggesting that these cells are relatively immature.100, 101, 103 However, when cultured, both populations develop into cells with typical DC morphology that express high levels of adhesion and co-stimulatory molecules and possess potent allostimulatory function.100,101 The CD33dim subset possess a lower allostimulatory activity which increases in culture along with expression of both HLA-DR and CD33.100 However, it has recently been demonstrated that although both subsets are stimulatory in the MLR only the CD2+ subset is capable of presenting Ag to naïve CD4+ T cells suggesting that these subsets of DCs may be functionally distinct.104
In order to facilitate the study of PBDCs, two markers specific for these cells have been identified in recent years. CD83 is a 45 kd member of the immunoglobulin super family that is virtually specific for DCs derived from the peripheral blood.49,105 Although CD83 was not originally found on freshly isolated DCs but only after in vitro culture,49 a later report identified a small subset of DCs that expressed CD83, when freshly isolated, and a larger subset that unregulated CD83 expression upon culture.102 The 55 kd actin bundling protein (p55) is a highly conserved protein important in the rearrangement of the cytoskeleton, cell motility and phagocytosis. Monoclonal antibodies against p55 detect this protein in 96% of PBDCs but not in other PBMC populations and, therefore, may be useful in the quantification and purification of PBDCs.50
While DC subsets have been defined in the peripheral blood their characterization has remained inconsistent reflecting, in part, the different purification protocols and MAbs used to define each subset. It remains to be established if one subset of PBDCs is the precursor for the other or whether each subset has developed along a distinct maturational/functional pathway. It has also been suggested that one subset may be more mature by virtue of being tissue-derived and migrating to lymph nodes or spleen whilst the less mature is directly derived from the bone marrow.101
Dendritic cells in peripheral tissues and lymph nodes
Dendritic cells have been identified within the interstitial space of most human tissues although notable exceptions are the absence of DCs in the cornea and central nervous system.106, 107 Within tissues, DCs exist as trace populations and may be identified by the combination of DC morphology and immunohistochemical labeling to demonstrate the expression of high levels of HLA-DR, CD1a (on LCs in the epidermis) and S100, and the absence of other lineage markers.108, 109 There is evidence that tissue DCs are derived from circulating blood precursors which bind to the endothelial receptors ICAM-1, V-CAM-1 and E-selectin through the expression of CD11a/CD18 and CD49d and cutaneous lymphocyte antigen (CLA), respectively.110, 111 This recruitment of DCs to tissue may be partly mediated by the local production of cytokines such as GM-CSF and by systemic signals such as bacterial lipopolysaccharide (LPS).112, 113 Within tissues, DCs reside in an intermediate stage of maturity as cells specialized for Ag uptake and processing. Tissue DCs take up Ag both in the fluid phase by macropinocytosis and via receptor-mediated endocytosis using the mannose receptor to ingest glycosylated Ag, and via the FcgRII (CD32) cell surface receptors take up antibody-bound Ag.114 Endocytosed particulate matter is channeled via an acidic vacuolar route to the intracellular class II compartment where antigenic peptides are assembled onto MHC class II molecules for presentation to T cells.114 The functional status of the DCs is regulated by a variety of cytokines and upon exposure to TNF-a, IL-1 and bacterial LPS, DCs undergo further maturation and migration.115-117 This involves downregulation of Ag uptake and processing, increased expression of the co-stimulatory molecules CD40, CD54, CD80 and CD86, enhanced Ag presenting function30, 118 and migration from the tissues to the lymph nodes and spleen.119-121 These changes in DC maturity and function, following exposure to cytokines and products of bacterial and cellular degradation, conform to the ‘danger’ hypothesis for activation of the immune response, as proposed by Marzinger (1994).122
DCs migrate to the secondary lymphoid tissues via the afferent lymphatics as veiled cells, so called because of their characteristic sheet-like lamellipodia. Cannulation of dermal afferent lymphatic vessels in human subjects has demonstrated an increase in CD1a+ DCs leaving the skin following exposure to contact sensitizers.123 There is also evidence from animal transplantation models that solid tissue DCs may migrate via the blood to the spleen.121 The mechanisms of homing to the LNs and spleen are not fully understood but recent evidence suggests that expression of certain isoforms of the hyaluronic acid receptor (CD44) may be important.124 In LNs, DCs reside within the T cell paracortical regions as interdigitating DCs (IDCs), whilst in the spleen they are located in the marginal zones at the periphery of the periarterial sheaths.106 The IDCs nonspecifically cluster T cells through their expression of adhesion molecules and present Ag in association with class II molecules. Antigen-specific T cells may then proliferate provided those co-stimulatory signals are communicated via CD40, CD80 and CD86 to their ligands on T cells. Production of cytokines such as IL-12 by the DCs further directs the evolving immune response along a Th1 pathway.75 It is apparent that the responding lymphocytes signal back to the DCs via MHC-TCR and CD40-CD40L interactions to promote further upregulation of DC co-stimulatory function.76 In addition to the IDCs of the T cell regions, DCs that are distinct from follicular DCs have recently been described in the B cell germinal centre.77 This suggests that they may play a role in T-dependent B cell memory immune responses. Kinetic studies in mice demonstrate a rapid DC turnover and life cycle in LNs, with DCs undergoing apoptosis after presentation of Ags to T cells.106, 125
SCIENTIFIC REVIEW
Dendritic cells (II): role and therapeutic implications in cancer
S. SATTHAPORN* and O. EREMIN*#
*Section of Surgery, E Floor, West Block, Queen’s Medical Centre, University of Nottingham and #Department of Surgery, Lincoln County Hospital, Lincoln, UK
The potential to harness the effectiveness and specificity of the immune system underlies the growing interest in cancer immunotherapy. One such approach uses bone marrow-derived dendritic cells (DCs), phenotypically distinct and very potent antigen-presenting cells, to present tumor-associated antigens (TAAgs) and, thereby, generate tumor-specific immunity. Support for this strategy comes from animal studies that have demonstrated that DCs, when loaded ex vivo with tumor Ags or pulsed with peptides and administered to cancer-bearing hosts, can elicit T cell-mediated cancer destruction. These observations have led to clinical trials designed to investigate the immunological and clinical effects of Ag-pulsed DCs administered as a therapeutic vaccine to patients with cancer. In the design and conduct of such trials, important considerations include Ag selection, methods for introducing TAAgs into MHC class I and II processing pathways, methods for isolating and activating DCs, and route of administration. Although current DC-based vaccination methods are cumbersome and complex, promising preliminary results from clinical trials in patients with malignant lymphoma, melanoma, and prostate cancer suggest that immuno-therapeutic strategies, that take advantage of the unique properties of DCs, may ultimately prove both efficacious and widely applicable treatment in patients with cancer.
J.R.Coll.Surg.Edinb., 46, June 2001, 159-167
INTRODUCTION
The interaction between tumor cells and the host immune system are complex, involving a multitude of cell types and mediators.1-3 Several lines of evidence suggest that the immune system has the potential to eliminate neoplastic cells, as evidenced by rare but well documented instances of spontaneous remissions (with no or inadequate treatment) in renal cell carcinoma and melanoma. Also, chronically and severely immunosuppressed individuals (transplantation recipients, congenital immune deficiency states and AIDS patients) exhibit an increased incidence of putative virally induced neoplasm’s; presence of AIDS-associated tumors correlate with the degree of immunosuppression.4-8,9 An intriguing piece of evidence relates to in vivo tumor-related immune responses in patients with paraneoplastic neurological disorders that led to the discovery of onconeural Ags. Paraneoplastic neurological disorders are a rare group of neuronal degenerative diseases that develop as remote effects of malignancies.10,11 The discovery of onconeural antibodies (Abs) led to the proposal that paraneoplastic cerebellar degeneration, associated with breast and ovarian cancer, is an autoimmune disorder mediated by the humoral arm of the immune system.
Induction of effective tumor immunity can be viewed as a three-step process that includes firstly, appropriate presentation of tumor-associated antigens (TAAgs), secondly, selection and activation of TAAg-specific T cells as well as non-Ag-specific effectors and, lastly, homing of TAAg-specific T cells to the tumor site and effective elimination of malignant cells expressing the TAAgs.12-14 Cancers may escape immune surveillance due to changes in and modulation of these various processes. The establishment of an effective anti-tumor response is a complex process. Initially, peptides associated with malignant cells must be located and recognized by T cells circulating in the blood stream and permeating tissues. Most solid cancers express small amounts of TAAgs, which may also be cryptic and not readily available for recognition by rare T cell clones, through a low affinity T cell receptor (TCR) complex. Moreover, tumor cells tend to lack co-stimulatory molecules that drive clonal expansion of T cells, the production of key regulatory cytokines, and development into tumor cell specific cytotoxic T lymphocytes (CTLs).
Dendritic cells (DCs) are the crucial cells providing the necessary components for initiating and developing effective cell-mediated immune (CMI) responses (Satthaporn and Eremin, 2001).15 Dendritic cells, located in most tissues of the body, capture and process Ags, which are then displayed as MHC-peptide complexes on the DC surface.16,17 Essential co-stimulatory molecules are upregulated on DCs as they migrate to secondary lymphoid organs (the spleen and lymph nodes) where they liaise with naïve T cells, inducing the activation and proliferation of Ag specific CTLs.1-3 Thus, effective DC function in cancer involves several interlinked biological processes that occur in sequence: (a) TAAg presentation and recognition in tissues which involves proteolytic intracellular cleavage and peptide surface representation, (b) DC activation and trafficking to regional tumor-draining lymph nodes (LNs), and interaction with CD4+ T cells via the TCR and associated co-stimulatory molecules (CD40, CD80 and CD86), resulting in the generation of Ag-specific CTLs, and (c) migration of CTLs to the tumor site and induction of cancer cell death.
IMMUNE SURVEILLANCE
Eliciting an effective anti-cancer response and removal of malignant cells is a complex biological process. Failure of this process is poorly understood and is believed to be multifactorial, as shown in Table 1.
Table 1: Factors believed to be responsible for failure of immune surveillance
|
Specific cytokines may have important inhibitory effects on DC functions. In particular, IL-10, has been shown to inhibit the differentiation of DCs from circulating precursors.18,19 Also, it has been shown to down regulate the expression of key co-stimulatory molecules and to block the production and secretion of the important DC and T cell regulatory molecules.20,21 The consequences of these various effects are to convert DCs from an immunogenic to a tolerogenic role.22-24 In addition, IL-10 may inhibit DC accumulation within tumors. Production of IL-10 by monocytes/macrophages and/or tumor cells, thus, may be an important mechanism by which malignant cells escape a protective host immune response and proliferate in an uncontrolled manner.25,26
Tumor-associated Ag presentation to naïve T cells, without concomitant co-stimulatory signals, not only fails to active T cells but also may induce tolerance towards the Ags concerned. In the normal setting, this mechanism may protect the host from autoimmune-directed tissue damage; in malignancy, however, it affords a mechanism of evading the immune response despite appropriate presentation of tumor-specific Ag epitopes. In animal models, it has been demonstrated that the introduction of co-stimulatory molecules into tumor cells by gene transfection can restore immunogenicity and elicit specific and effective anti-tumor responses.27,28
The influence of B-7 and CD28 co-stimulation in inducing tolerance or rejection of TAAgs was illustrated in a recent study using Ag presented by mouse EL4 lymphoma cells.29 Because the CTL epitopes presented by EL4 cells had not previously been identified, it was necessary to isolate and purify MHC class I-associated peptides by a combination of affinity chromatography and reversed-phase high-pressure liquid chromatography. Cytotoxic T lymphocytes induced by B7-1 transfected EL4 cells indicated that at least six different epitopes were presented by the parental EL4 cells, but the CTLs induced by the parental EL4 cells recognized a single dominant epitope. Thus, these results suggested that insufficient stimulation contribute to Ag ‘silencing’ and induction of tolerance. It is worth nothing that the induction of CTLs against the dominant epitopes of B7- tumor cells were also dependent on B7 co-stimulation provided by B7+ host cells. The administration of CTLA-4-Ig, a fusion protein that blocks B7-CD28 interaction, eliminated all CTL responses in mice challenged with cells from the B7-P815 tumour.29
Several recent experiments have elegantly demonstrated that there is a critical threshold of TCR molecules to be engaged with peptide-MHC in order for the triggering of a detectable T cell specific response and that the requisite number of occupied TCRs can be significantly decreased if B7-CD28 co-stimulation is provided.30-32 Thus, lack of sufficient co-stimulation through the B7 (on DCs) - CD28 (on T cells) interaction will induce ‘silencing ‘of the TAAgs. The consequences of this are T cell anergy and failure of immune surveillance and removal of proliferating cancer cells.30-32
DENDRITIC CELLS AND CANCER
Escape from immune surveillance is believed to be a fundamental biological feature of malignant disease in man, which contributes to uncontrolled tumor growth, eventually leading to death of the host.33 Defects in immune response in patients with a variety of tumors and in tumor-bearing animals have been well documented. These defects have been ascribed mostly to suppressor cell function.34,35 However, a key element in the induction of the anti-cancer immune response, namely TAAg presentation to T cells in a tumor-bearing host (human and animal), has been poorly documented and inadequately studied. Some authors have shown defective function of macrophages in malignant disease (see review by Al-Sarireh and Eremin (2000)].36-38 Zou et al (1992), however, reported normal and even increased function of Ag presenting cells (APCs), mostly macrophages in tumor-bearing mice.39 In recent studies, it has been shown that a distinct subset of IA+ epidermal APCs appear capable of inducing tolerance to tumor Ags and that activated macrophages may induce structural abnormalities of the TCR-CD3 complex.40,41 Data has also been presented suggesting that bone marrow-derived APCs play an important role in the presentation of TAAgs, a function previously assigned predominately to tumor cells.42,43 Therefore, elucidation of the role of such APCs, in particular DCs (the key APCs), may help to better understand the mechanisms underlying anti-tumor immune responses and, perhaps, improve the effectiveness of anti-cancer immunity in tumor-bearing hosts.
Some studies in humans with solid cancers have investigated DC trafficking in peripheral blood. Radmyar et al (1995) demonstrated in their study that substantial numbers of DCs could be obtained from the peripheral blood of patients with renal cell carcinoma.44 Phase contrast microscopy revealed the typical cytoplasmic processes, whilst phenotypic analysis confirmed expression of DC-associated molecules, including MHC class II, CD1a, CD80 and CD86 and absence of T cell, B cell and monocyte markers.15 Functionally, these DCs, when cultured in vitro, were found to be very potent costimulators of the phytohaemagglutinin-induced proliferation of autologous tumor infiltrating lymphocytes.44 Almand et al (2000), however, showed that DCs in the peripheral blood of patients with head and neck cancer were significantly immunosuppressed.45 There was also an increased intratumoural presence of the immunosuppressive CD34+ progenitor cells. Culturing CD34+ cells with stem cell factor (c-kit ligand) and granulocyte/monocyte-colony stimulating factor (GM-CSF) resulted in the appearance of a substantial number of cells expressing phenotypic markers characteristic of DCs.46 Patients with head and neck squamous cell carcinoma also had increased levels of the immunosuppressive peripheral blood CD34+ cells. However, these latter cells were capable of differentiating into DCs in vitro.47
Gabriolovich et al (1997) evaluated T cell responses to mitogens and to defined Ags in patients with breast cancer. Defects in response to tetanus toxoid and influenza virus were observed in patients with advanced breast cancer. Dendritic cells isolated from patients with breast cancer demonstrated a significantly decreased ability to stimulate control allogeneic T cells but stimulation of the patients T cells with control allogeneic DCs resulted in normal T cell responses. These data suggest that reduced DC function could be a major cause for the observed defect in cellular immunity documented in the patients with breast cancer in this study. In addition, stem cells from these patients could give rise to functional DCs after in vitro growth with GM-CSF and IL-4. Normal levels of control allogeneic and tetanus toxoid dependent T cell proliferation were observed when DCs obtained from progenitor cells were used as stimulators.48
Bell et al (1999) have analysed the presence of immature and mature DCs within adenocarcinoma of the breast using immunohistochemistry.49 Immature DCs were defined by expression of CD1a- and intracellular major histocompatibility complex class II-rich vesicles. Mature DCs were defined by the expression of CD83 and DC-Lamp. These workers demonstrated two levels of heterogeneity of DCs infiltrating breast carcinoma tissue: (a) immature DCs were localised to the tumor bed and (b) mature DCs were confined to peri-tumoural areas.49 Lespagnard et al (1999) evaluated 142 primary breast carcinomas for the presence of DCs, using immunohistochemistry and anti-s100 protein antibody. They showed that 42% of breast carcinomas contained tumor infiltrating DCs expressing S100+ (S100+ TiDCs) and the number of S100+ TiDCs varied according to the grade of the tumors, being highest in grade III cancers. An association was also found between S100+TiDCs and tumor size, lymph node involvement, estrogen/ progesterone receptor status and age.50 Another study identified a population of CD1A+ cells within the lymphoid cell infiltrate in human breast cancer. In the majority of cases the infiltrate was low, compared with the number of macrophages and T cells. There was no correlation between the number of CD1A+ cells and tumor grade, with all tumor grades expressing similar numbers of infiltrating CD1A+ cells. However, the CD1A+ cells were closely associated with tumor cells. It is likely that CD1A+ cells have a role in Ag capture and presentation in human tumours.51
An investigation in patients with melanoma by Enk et al (1997) has shown a differential DC function.52 Patients whose melanoma were responding (rM) to chemotherapy had DCs which were five times more potent inducers of allogeneic T cell proliferation than those patients whose tumors were progressing (pM). Phenotypic analysis showed a marked depression of CD86 expression on DCs in the latter patients. Culture supernatants from pM showed production of a TH2-type cytokine profile (IL-10), whereas a TH1-type cytokine profile (IL-2, IL-12 and interferon-gamma (IFN-gamma) was found predominantly in patients whose melanomas had responded to treatment.52 A recent study by Cayeux et al (1999) have shown that the mixed lymphocyte response in vitro (measure of CMI) was inhibited by anti-CD80, anti-CD86, anti-HLADR, anti-HLA class I antibodies and supernatants of melanoma cultures. The characteristics of DCs generated from patients with melanoma were comparable with those obtained from healthy donors. Dendritic cell function was inhibited by soluble factors present in melanoma cell cultures.53
Ninomiya et al (1999), showed that DCs from patients with hepatocellular carcinoma had significantly lower capacity to stimulate allogeneic T cell proliferation, compared with DCs isolated from patients with liver cirrhosis and normal controls.54 In patients with hepatocellular carcinoma, DCs expressed significantly lower levels of HLA-DR and induction of IL-12 production. On the other hand, DCs from such donors produced significantly higher levels of nitric oxide and tumor necrosis factor-alpha (TNF-alpha) compared with DCs from donors with liver cirrhosis and normal controls. These results confirm a defect of DC maturation in patients with established hepatocellular carcinoma and probably during carcinogenesis and tumor induction.54
Although a dysfunction of DCs is predictable in malignancies and, in fact, is being documented in a number of solid cancers in man, the biological mechanisms underlying these defects of DCs are poorly defined and appear to be heterogeneous in different cancers.
DENDRITIC CELLS AND ANTI-CANCER THERAPY
Dendritic cells are potentially good candidates for immune-based therapies for a variety of reasons. In particular, the following aspects are important, (a) their ability to migrate through tissues and infiltrate into tumors, where they encounter TAAgs which they capture, digest, and re-express for effective induction of a CMI response, (b) their capacity to activate naïve T cells in regional lymph nodes and their differentiation into CTLs, specifically able to interact with cancer cells and lead to tumor cell damage and death, and (c) their role as APCs and capacity to process and present a spectrum of different Ags simultaneously that allows for the induction of a broad repertoire of anti-tumor immune responses to occur.
The ability of DCs to generate anti-tumor immune responses in vivo has been documented in a number of animal tumor models.55,56 Most of these experiments have involved in vitro isolation of DCs, followed by loading of the DCs with tumor Ags and injection of the Ag-bearing DCs into syngeneic animals as a cancer vaccine. Dendritic cells loaded with tumor lysates, tumor Ag-derived peptides, synthetic MHC class I-restricted peptides and whole proteins, have all been demonstrated to generate tumor-specific immune responses and anti-tumor activities.57-60 Furthermore, Ag-loaded DCs can be used therapeutically to induce regression of preexisting tumours.61 Dendritic cells loaded with appropriate TAAgs can induce either protection or rejection of malignant cells in various animal models.62-65
Promising preliminary data has been reported in patients with cancer.66-69 Several systems have been used to deliver TAAgs to DCs, including (a) defined peptides of known sequences, (b) undefined acid-eluted peptides from autologous tumors, (c) whole tumor lysates, (d) retroviral and adenoviral vectors, (e) tumor cell-derived RNA, (f) fusion of DCs with tumor cells, and (g) exosomes derived from DCs pulsed with tumor peptides (subcellular structures containing high levels of MHC molecules and peptides).1-3 These observations have established the rationale for evaluating tumor Ag-bearing DCs as therapeutic vaccines in humans. The unique ability of DCs to induce and sustain primary immune responses makes them optimal candidates for vaccination protocols in cancer (see Table 2).2,70,71
Table 2: Summary of dendritic cell clinical trials
|
Cancer |
Antigen type |
DC type |
Ref) |
|
Volunteer |
Matrix peptide (MP) and keyhole limpet haemocyanin (KLH) |
Immature DC's |
94 |
|
KLH and tetanus toxoid (TT) |
Mature DCs |
95 | |
|
MP, KLH and TT |
Mature DCs (pulsed and unpulsed) |
69 | |
|
Melanoma |
gp100, Mart-1, and tyrosinase |
GM-CSF + IL-4 cultured monocytes |
96 |
|
gp100, Mart-1, and tyrosinase and CD34+ cells |
GM-CSF + IL-4 cultured monocytes |
97 | |
|
Mage-3A1 tumor peptide |
Mature, monocyte-derived DCs |
98 | |
|
Multiple myeloma |
Immunoglobulin idiotype |
Leukopheresis and density gradient centrifugations |
99 |
|
Lymphoma |
Immunoglobulin idiotype |
Density gradient centrifugations |
66 |
|
Prostate |
Prostate-specific membrane antigen-derived peptides, PSM-P1 and PSM-P2 |
GM-CSF + IL-4 cultured monocytes |
100 |
|
Prostate-specific membrane antigen (PSMA) |
GM-CSF + IL-4 cultured monocytes |
101 | |
|
Prostatic acid phosphatase (PAP) |
Leukopheresis and density gradient centrifugations |
102 | |
|
Renal |
Whole tumor lysates |
GM-CSF + IL-4 cultured monocytes + TNF- a and PG E2 |
103 |
|
Whole tumor lysates |
GM-CSF + IL-4 cultured monocytes + TNF- a and PG E2 |
104 | |
|
Hybrid of autologous tumor cell |
Allogeneic DCs |
105 | |
|
Breast and ovary |
HER-2/neu-or MUC1-derived peptides |
GM-CSF + IL-4 and TNF cultured monocytes |
106 |
|
Breast, colorectal, pancreas and lung |
Whole tumor lysates |
GM-CSF + IL-4 cultured monocytes |
60 |
|
Whole tumor lysates |
GM-CSF + IL-4 cultured monocytes +/- adjuvant IL2 |
93 |
However, DC-mediated induction of immunity represents a major therapeutic challenge, and several parameters need to be considered to ensure the optimal outcome of DC-based vaccination protocols including (a) the source of DCs, (b) methods for isolating and activating DCs and (c) route of administration.
Many groups have generated DC-like cells by culturing CD14+ monocyte-enriched PBMCs in vitro. When cultured for 1-2 weeks with media supplemented with GM-CSF and IL-4, monocytes give rise to large numbers of cells that are morphologically and phenotypically similar to the ‘classical’ density-purified DCs (see Figure 1a and 1b). 72,73 These cytokine-generated DCs require additional maturation in vitro with TNF-alpha or INF-alpha in order to fully stimulate in an allogeneic MLR or prime Ag-specific T cell responses in vitro and in vivo.74-76
Figure 1: Light microscopic morphology of DCs. Activated DCs (Figure 1b) were generated from adherent mononuclear cells and incubated with cytokineconditioned medium for 7 days at 37 °C, in a 5% CO2 incubator. DCs show characteristic morphology with irregular shape and cytoplasmic projections or veils
|
|
|
Moreover, without this additional maturation step the DC phenotype can revert to that of a monocyte. Human DCs can also be enriched as circulating precursors from the blood by density-based purification techniques.77 After a period of in vitro culture and maturation, DC precursors become larger and less dense. Gradient solutions lacking potentially immunogenic proteins such as bovine serum albumin,78 have been employed and have included Percoll,79 Nycodenz,80 and metrizamide.81
These different fluids are osmotically active, to varying degrees, and have additional stimulatory properties as well. The use of density-based isolation, however, is limited by the low frequency of DC precursors in blood, representing around 1% of peripheral blood mononuclear cells (PBMCs).77 As a result, leukopheresis has been performed in order to isolate sufficient numbers of DCs for therapeutic vaccination in humans.
In generating DCs for use as cellular vaccines, regardless of source, the infused cells should possess a stable as well as an activated phenotype. In addition to expressing the requisite MHC and co-stimulatory molecules to prime T cells, the cells should express appropriate adhesion molecules and chemokine receptors to attract the DCs to secondary lymphoid organs for priming. Otherwise, ineffective priming may occur, particularly if the DCs are administered systemically rather than locally into the relevant draining lymph nodes. Intra-lymphatic or intra-nodal injection of DCs may be used to deliver DCs directly to secondary lymphoid organs, although these routes of administration have not, as yet, been shown conclusively to produce more effective vaccination.82,83
DC-based immunisation requires that the cells present one or more tumor Ags to the host’s T cells. Truly tumor-specific Ags (TSAgs) offer theoretical advantages as immunotherapy targets. The immune response induced by such Ags presumably would be limited to malignant cells bearing the antigenic epitopes, thereby, limiting the risk of collateral damage to normal tissues.84,85 If such proteins arise in the tumor following thymic development, clones of T cells reactive with such peptides may exist as they probably avoided apoptosis and thymic deletion. Nevertheless, the immunogenicity of these Ags may be limited by the number of epitopes contained within the protein. Moreover, TSAgs may vary between individuals with the same tumor type. Viral tumor Ags can represent desirable immune targets given the inherent immunogenicity of many viral proteins.86
Although the results, to date, of the various DC trials (see Table 2) are exciting and look promising, the current procedures used for isolating and ‘arming’DCs are prolonged and problematic and, as yet, are not applicable to routine clinical practice. As discussed, many groups are currently pursuing techniques for in vitro generation of DCs from CD34+ precursors or CD14+ monocytes. These approaches, while expensive, can generate large numbers of cells for use in clinical trials. Administration of growth factors (e.g., GMCSF, IL-4, TNF-alpha and IFN-alpha have all been used in vitro to generate and activate DCs in patients with cancer; this offers another interesting therapeutic approach. However, whether such an approach will circumvent the inhibitory milieu (both systemic and in situ at the tumor site) in cancer patients requires further careful studies.
In addition to questions regarding the best source of DCs for use in clinical trials, the choice of tumor Ag with which to ‘arm’ the DCs is almost certainly going to have a profound influence on clinical outcome. At present, the choices are limited because only a few TAAgs have been identified and are suitable for loading/priming DCs.87-89 On the other hand, the number of suitable candidate Ags is continually expanding and is expected to accelerate, as a consequence of the intensification of gene mapping and isolation.
Ultimately, combinations of Ags will be used to reduce the risk of generating Ag-loss variants that evade the immune response. The increasing use of microarray technology in assessing tumors may enable individualised Ag combinations to be determined. Antigen delivery also remains to be optimised. Use of protein, whether tumor-derived or produced by recombinant DNA methods, can be cumbersome and potentially limiting, especially at concentrations that may be necessary for MHC class I delivery. Use of specific peptide conjugates or fusion constructs (e.g. with HIV tat) may increase the efficiency of presenting epitopes from these soluble proteins. RNA, DNA, and viral vectors are more easily produced and may offer an alternative approach, although issues of transfection efficiency and DC viability remain unresolved.
The potential benefits of administering cytokines or other DC activators, in combination with DC vaccination, remain relatively unexplored. Clearly, DCs can elaborate their own cytokines for Ag priming, as previously discussed. However, supplementing culture media used for generating DCs in vitro with additional cytokines may enhance the APC functions of these DCs. TNF-• or CD40 ligand are known to activate DCs in vitro and could increase the potency of DC-based therapy.90,91 The addition of IL-12 may aid in Ag priming and generating TH1 responses in vitro or in vivo.92 Synergy between DC vaccination and IL-2 has already been demonstrated in an animal model. 93 No consensus exists on the optimal approach to assessment of immune responses in patients undergoing immunotherapy, let alone DC vaccination. Delayed-type hypersensitivity testing with an Ag challenge injected into the skin has been used to assess gross immune reactivity. Cytokine production by both CD4+ and CD8+ T cells can be detected by cytokine ELISA, ELISPOT, or intracellular cytokine staining. Other techniques for assessing CD4+ T cell responses include measurement of Ag-specific proliferation. CD8+ T cell responses have also been assessed with CTL assays. However, these assays assess the functions of circulating T cells but do not necessarily reflect immune responses in lymphoid organs. Improved immunologic assays that better correlate with clinical outcome will be required before these assays can serve as surrogates for evaluation of tumor status.
By isolating and arming DCs with Ag ex vivo, it may be possible to allow the cells to mature in the absence of an inhibitory milieu. If defective maturation of DCs is a common occurrence in malignancy, then identification of agents that induce their maturation, in vivo, may represent an elegant solution to this problem. In the absence of such agents, administration of DCs grown in vitro from circulating DC precursors, which have been activated and induced to mature in vitro, and armed with appropriate tumor Ags, may prove useful in the treatment of a variety of cancers for which existing therapeutic options have limited use and clinical efficacy.
Dendritic Cell Therapy – With More Graphics
This is similar information to what is presented above but from another source and includes many more illustrations to assist in understand who DC works.
The Immune System
The immune system is the body's defense system. It works on three different levels. The first level is the anatomic response. It consists of anatomical barriers to foreign particles and includes the skin and acid in the stomach. Anatomic barriers prevent foreign substances from entering the body. If foreign particles pass through the first line of defense the second line of defense called the inflammatory response kicks in. The third line of defense is the immune response. It is the main player in specific immune defense.
The cells of the immune system mount the immune response. These cells are also called white blood cells.
There are several types: The neutrophils are responsible for killing bacteria and yeast and are the first white blood cells at the site of an infection. The eosinophils play a part in delayed reactions to foreigners. The key players that will be discussed here are the monocytes and the lymphocytes. Monocytes are scavangers. They scour the body for anything out of place. They can engulf foreign particles and chew off pieces of tumor cells. Lymphocytes are not able to engulf any foreign particles or eat tumor cells. They take the information given to them by monocytes and monocyte-like cells and do their job. There are several types of lymphocytes. The types essential to this topic are the B lymphocytes, and the T lymphocytes. The B lymphocytes get their characteristics after being nurtured in the bone marrow, hence the B. B lymphocytes are primarily responsible for producing antibodies. Antibodies can inactive bacteria, fungi, and viruses and make them and other foreign particles easier to see by the rest of the immune system. T lymphocytes mature in the thymus gland, which is located under the breast bone, hence the T. For the purposes of this topic they can be divided into three major categories: the T helper cells, the T suppressor cells, and the cytotoxic T cells. The T helper and suppressor cells do exactly what their names imply. The cytotoxic T cells are primarily responsible for killing virally infected, and tumor cells.
The Immune System and Cancer
In order for cancer to occur, the immune system must have failed. The normal sequence of events when the immune system comes across tumor cells follows.
An immune cell called the macrophage (also called a monocyte) comes into contact with a cancerous or precancerous cell. This cell has some strange surface features. The strange features signal the macrophage that the cell is not healthy and that the macrophage should take a bite out of it.
The macrophage then begins to digest the bite of the tumor cell. Several little packets of enzymes act like a cellular stomach and break down the piece into smaller and smaller pieces.
There are two possible scenarios that can happen next. The macrophage can either hand off these little pieces of tumor cell to another type of immune cell, or it can transform itself into another, specialized immune cell called a dendritic cell. There is more and more evidence that the latter happens most often.
Dendritic cells are found in all tissues of the body, and many of them began as macrophages. The first dendritic cell discovered is found throughout the skin and is called a Langerhan's cell.
Now that the macrophage has digested pieces of the tumor cell, it transforms into the dendritic cell. The dendritic cell is a much more effective messenger. When it is fully mature, it gives the information about the tumor contained in the small digested packets to the rest of the immune system. A key point here is that the dendritic cell must be mature to effectively present the tumor information. The cell needs to have additional markers on its surface that the other immune cells can recognize. These markers are called co-stimulatory molecules and are shown as white crosses on the picture of the mature dendritic cell below.
When the dendritic cell begins to mature, it also starts moving, or migrating toward a lymph node. The lymph nodes contain large numbers of lymphocytes, another type of immune cell. Everyone probably remembers having enlarged lymph nodes in their neck when they had a sore throat. The lymph nodes are where the action is when it comes to the immune system. There are areas in the body that contain large numbers of lymph nodes. The neck, armpits, and groin areas all have clusters of nodes that lie close to the skin.
So the mature dendritic cell has migrated to the lymph node. There it comes in contact with different kinds of lymphocytes. If it has matured properly, the co-stimulatory molecules on its surface will help pass the tumor information along to the cytotoxic T lymphocytes, or CTLs. The CTLs are the body's main defense against tumor cells. When the right CTL comes in contact with the dendritic cell, it will become activated and begin to divide, effectively making an army of cloned soldiers ready to kill any cancerous or pre-cancerous cell having the same altered membrane discovered by the macrophage.
When the CTL soldiers come in contact with cells that have the same surface as the original cancerous cell, they bind to it. They then release a chemical that pokes tiny holes in the membrane of the tumor cell, and the tumor cell spills its guts and dies.
Let's summarize what happens normally in the body after a normal cell turns cancerous. First, a macrophage comes in contact with the tumor cell, which has a different type of membrane that signals the macrophage to eat part of it. The macrophage then digests the eaten tumor cell fragment and starts to turn into a dendritic cell. It then begins to mature, and travels to a nearby lymph node and hands off the tumor cell information to CTLs. The CTLs then divide, circulate throughout the body, and kill any tumor cells they come in contact with.
Above we covered what happens normally in the body when a cell becomes cancerous. This process occurs countless times as cells get genetic mutations and become cancerous. But, if you have cancer, then something must have gone wrong. Did the macrophage fail to recognize the funny cell surface? Did macrophages not become dendritic cells? Or did the T cells not do their job? It is impossible to tell for sure but there are some clues that the problem is with the dendritic cells.
Lately several research groups have been looking at the dendritic cells in and around tumors. What they're finding is that there are dendritic cells there, but they are immature. They don't have the co-stimulatory molecules necessary for the successful hand off of the tumor cell membrane information to the T cells. Moreover, because they are immature, they are much less likely to migrate to the lymph nodes to make the hand off.
To make a football analogy, the dendritic cell is the quarterback and needs to hand off the football to the running back (the T cell). In order to do that, he needs to move toward the running back and hand him the ball without fumbling. When the dendritic cell is immature, it just stands in one place and drops the ball. If that continues to happen, your team never scores and ultimately loses the game.
If you cut up a piece of tumor from kidney cancer or renal cell carcinoma and look at it under the microscope, you'll find millions of dendritic cells many more than in any other type of tumor. Expectedly, the majority of these dendritic cells are immature they don't have co-stimulatory molecules on them. What makes this more interesting is the fact that kidney cancer is the most likely type of cancer to disappear without a trace without any treatment, or spontaneously remiss. What I believe happens when someone has a spontaneous remission is the conditions in and around the tumor change enough to allow at least some of the dendritic cells to mature. This is more likely to induce a remission in renal cell carcinoma simply because of the larger numbers of dendritic cells.
So, what can you do to get dendritic cells to hand off information about your tumor cells to your CTLs? Both animal and human trials of using dendritic cells in the treatment of cancer have shown promising results and give us a direction in which to go.
Mayordomo et al.1 inoculated mice with different types of cancer and allowed the tumors to develop for one to two weeks. Dendritic cells were isolated from the bone marrow of these mice, cultured with some growth factors, and exposed to tumor peptides (information about the tumor cell membranes). These Δprimed' dendritic cells were then injected back into the tumor-bearing mice every four to seven days. Recovery, measured as halting of tumor growth and subsequent regression, was seen 7-10 days after the first injection of dendritic cells. Using this treatment, cure rates of 80% for mice with Lewis lung carcinoma and 90% for mice with sarcoma were achieved.
In a similar study, Nair, et al.2 induced malignant melanoma lung metastases (new tumors that spread from the first tumor) in mice, and then surgically removed the primary tumor. The mice were then treated with dendritic cells which had been Δprimed' in a manner similar to that described above. Of the seven treated animals, four had no visible lung tumors, two had fewer than five remaining tumor nodules, and one mouse had 15 nodules. The number of nodules in control mice, those that did not receive dendritic cell therapy, were too many to count, but comprised approximately three-quarters of the lung by weight.
Hsu et al.3 at Stanford University
Gerald Murphy, M.D.4, and his team at Northwest Hospital
In addition to lymphoma and prostate cancer, the deadly skin cancer malignant melanoma has been treated successfully using dendritic cell therapy. In a recent human study by Nestle et al5, dendritic cells were used to treat sixteen patients with advanced metastatic (the cancer has spread) melanoma. Objective responses were seen in 5 of the 16 patients. There were two complete responses and three partial responses with regression of metastases in several organs, including skin, lung, and pancreas. The participants were followed for 15 months and no cases of autoimmunity a potential side effect of the therapy were found in any of the patients. The authors concluded, vaccination with autologous [derived from the person's own body] dendritic cells generated from peripheral blood is a safe and promising approach in the treatment of metastatic melanoma.
In the studies quoted above, there were little to no side effects. Murphy reports transient hypotension (temporary low blood pressure) as the only side effect seen in his study.
Given all of this compelling evidence that dendritic cells may hold a key position in effective, non-toxic treatments for cancer, we began studying them.
Our research to date has focused mainly on methods of:
- Producing large numbers of dendritic cells from the circulating blood of cancer patients;
- Finding the source of tumor material (antigen) for each type of tumor that will best stimulate the T cells to proliferate and kill tumor cells; and,
- Stimulating the dendritic cells already in and around the tumor to mature, and become better T cell stimulators.
What we have found so far is that we can produce large numbers of dendritic cells from the circulating blood, give them tumor antigen, and mature them. These dendritic cells in culture are able to stimulate large numbers of T cells to become active against tumors. We are now setting out to determine if this is possible in humans.
In order to study the value of dendritic cells and activation of dendritic cells as anti-tumor therapies for patients with metastatic prostate cancer we are currently performing four clinical studies described below.
1. DENDRITIC CELLS TREATED WITH PATIENT'S OWN TUMOR MATERIAL
Before beginning on this protocol, patients must first provide tumor tissue to the laboratory for use with the dendritic cells. Arrangements are made with your surgeon, or urologist prior to surgery for proper collection and transport of the tumor material to the laboratory. At the laboratory, an extract of the tumor tissue will be made, and then filtered to make it sterile.
This study uses the patient's own tumor material and own dendritic cells. Monocytes are first harvested from the circulating blood using a specialized machine. The machine is called an apheresis (Greek for to take out) unit. This type of machine is used at many American Red Cross offices to harvest blood products such as platelets (cell fragments that help blood to clot). The procedure is relatively simple. A needle connected to sterile, one-use tubing is placed into each arm. The tubing is connected to the machine, and approximately one cup of blood is circulated out of one arm, through the machine, and back into the other arm. The machine spins the blood, removes two types of white blood cells, and returns the fluid part of the blood and all of the other blood cells to the patient. Using this machine allows for many more white blood cells to be collected than by simply drawing blood because the other cells are returned to the patient. The procedure takes about two hours.
The white blood cells are then taken to the laboratory where they are grown in a special growth broth that begins to convert them to dendritic cells. After a few days the filtered tumor extract is added to the growth broth. The cells are then allowed to mature for another few days.
The cells are then tested for their maturity and purity. They are then frozen in liquid nitrogen and are ready to be reinfused.
A portion of the cells will then be suspended in sterile saline reinfused intraveneously (by a vein in the arm) into the patient. The patient will receive an infusion once or twice per month.
After the cells are infused, some will migrate to the lymph nodes and some will stay in the circulation. The likelihood is high that many of the dendritic cells will come in contact with T-lymphoctyes (CTL's) and stimulate them to divide and recognize and kill tumor cells. Measuring the serum levels of prostate specific antigen, or PSA can allow your doctor to follow the course of prostate cancer. This test is used to determine if the treatment has been effective.
2. DENDRITIC CELLS TREATED WITH PURIFIED TUMOR ANTIGEN
This procedure is the same as number one with one exception. The agent used to prime or activate the dendritic cells is not an extract of the patient's own tumor. Instead, a known tumor antigen (molecule that has information about the outside of tumor cells) is placed with the dendritic cells while they grow and mature in culture. PSMA, or prostate specific membrane antigen, is used.
PSMA is found primarily on the outside prostate cells. The priming agents used by Murphy, described above, were fragments of the PSMA molecule.
Measurement of the serum levels of PSA will also be used to determine if the treatment has been effective.
3. STIMULATION OF IMMATURE DENDRITIC CELLS TO BECOME MATURE DENDRITIC CELLS.
In many types of tumors the dendritic cells in and around the tumors are immature meaning they are there, they just can not effectively pass along information about the tumor cells to the rest of the immune system, in particular to the CTL's, or T-lymphocytes.
One way to convert immature dendritic cells into mature ones is by using a mixture of growth factors and stimulating factors called cytokines. Many people have heard of some of these cytokines. Interleukin 2, interferon, and tumor necrosis factor are a few. These growth factors are found naturally in the body and can be manufactured using a patient's own white blood cells. The monocytes, when mixed with a medication made from bacterial cell walls, can produce an effective mixture of cytokines. The monocytes are mixed with the medicine and grown in an incubator for 2 days. Then the culture medium, or broth, is collected. It contains a lot of cytokines. We call this mixture MCM, which stands for monocyte-conditioned-medium. When MCM is placed with immature dendritic cells, a majority of them become mature. They can then migrate and effectively convey information about the tumor to lymphocytes, stimulating them to divide at the same time.
Because the MCM is prepared from the patient's own cells, the dosage varies from individual to individual. Because these cytokines are the same molecules that can make you feel poorly and give you a fever when you have the flu, the dosage is increased gradually to make sure the patient doesn't experience those side effects. The MCM is sterile filtered, and then mixed with sterile saline. It is given in the vein the same as the dendritic cells. An increased dose will be given daily until a rise in the patient's body temperature is seen. That dose will then be given daily for two weeks.
The serum PSA test is also used to follow the effects of the treatment on the course of prostate cancer.
4. CELL FRAGMENTS OF DENDRITIC CELLS PRIMED WITH TUMOR CELL ANTIGENS
Researchers in France
This study begins similarly to studies one and two. Monocytes are harvested from the peripheral blood. They are cultured with cytokines and tumor cell antigens are added. The cells are then allowed to mature. Instead of using the dendritic cells the liquid in which the cells are growing is collected. The exosomes are then removed from the liquid. They are sterile filtered and injected into the skin just above the lymph glands in the groin. The theory is that there they interact with the dendritic cells in the skin, which move into the lymph glands and present tumor antigen to the lymphocytes. Which then divide, circulate in the body, and kill the tumor cells they come in contact with.
How Did I Find Out About Dendritic Cell Therapy?
In March 2008 through the knowledge and friendship of Nancy Ehrlich, a friend and colleague I reconnected with Paula Goldberg who is a fellow Real Estate Broker/Manager who works for Prudential Fox & Roach in New Jersey and who I had worked with while a Manager/Regional Manager at Prudential Fox & Roach in the late 90’s. Paula immediately connected me with Dr. Raymond Chang. At this time Paula had been a patient (non-small cell lung cancer) of Dr. Chang for several months, and a believer in his approach to treating cancer. Paula told me about Dendritic Cell Therapy early on although at this point she hadn’t had the treatment herself and that wouldn’t come until July 2008. Paula was introduced to a young lady who after failing several traditional cancer treatments for a brain tumor came to be treated by Dr. Chang, ultimately traveled to Germany for Dendritic Cell Therapy and has had her cancer STOP (they can see it on her scans but it hasn’t grown and appears to be encapsulated) for almost 3 years now.
Paula, as noted, had DC in July 2008 but in December found out that her cancer has come back for the third time and is back on a traditional form of chemotherapy which is incompatible with ongoing DC treatments. Please keep her in your prayers too.
Dr. Chang has been guiding my care ever since, with the express consent of Kristi (my wife and a doctor) as well as my brother Jay who both accompanied me, along with a healthy dose of skepticism, on my very first appointment, since the very beginning. It took Kristi and Jay about 45 minutes into that first appointment in early March 2008. They didn’t even crack a smile or really ask a question of Dr. Chang and I did most of the talking although Dr. Chang was really directing his comments to Kristi, a fellow medical professional. What broke the ice was question about a drug that had been FDA approved for small-cell lung cancer (my initial diagnosis) in the U.S. only two days prior and brought to my attention by a friend and colleague, Roger Farber. When I asked about the drug, manufactured by Celgene Dr. Chang immediately reached up on his bookshelf, pulled down a binder that said Celgene on the cover, opened to page 257 and explained/showed to us that I would not be eligible for this particular chemo treatment because of the two heart surgeries (stents) I had placed in my left main artery (LAD) in 2007.
Dr. Chang knows most of what’s out there when it comes to cancer and cancer treatments and if you know someone who wants to do EVERYTHING possible to stay alive and as healthy as possible during cancer treatments AND will work with traditional Medical and Radiation Oncologists I would recommend making an appointment ASAP.
Dr. Chang’s approach is to wage guerilla warfare on cancer. If it won’t kill you why not try it?
Dr. Raymond Chang MD FACP - Medical Director, Meridian Medical Group
Hailed by New York Magazine as early as 1999 as one of the "New Healers"; Dr. Chang has also been interviewed on the Today Show (click for video download), cited by Reuters, Foxnews, WebMD, and Mamm magazines for his pioneering work in alternative medicine.
Dr. Chang who was originally from Hong Kong, is an internationally respected physician-acupuncturist uniquely trained in traditional Chinese medicine as well as contemporary Western medicine, and he is an acknowledged pioneer-specialist in the field of alternative and complementary therapy programs for cancer, infertility and other difficult conditions.
Dr. Chang received both his B.A. (Magna Cum Laude) and M.D. degrees from Brown University where he was elected Phi Beta Kappa as well as Sigma Xi. He subsequently completed post-doctoral training at Waterbury and New York Hospitals, and was a diplomate of the American Board of Internal Medicine before joining Memorial Sloan-Kettering Cancer to direct its acute care department. He was on the faculty of Medicine at Cornell Medical College and was an Associate Attending in the Dept. of Medicine at Memorial Hospital as well as an Associate Member of the Sloan-Kettering Institute till 1997. In the mid-90's, he developed what is acknowledged to be nationally one of the most complete programs in alternative medicine education at Cornell Medical College. In 1995, he was elected Fellow of the American College of Physicians and was the first visiting professor of the Taiwan Cancer Foundation at the National Taiwan University. He was visiting professor at Shanghai Medical College's Obstetrics/Gynecology Hospital in the summer of 1998. Currently, he attends at the New York - Presbyterian and Beth Israel Hospitals in New York City and is on the faculty of the Albert Einstein School of Medicine.
Aside from his unique background in traditional Chinese medicine, Dr. Chang has been a strong proponent for an evidence-based and complementary approach to alternative medicine. In the research arena, Dr, Chang is an expert in the use of immunopotentiating medicinal herbs and fungi for cancer, hepatitis and HIV. Dr. Chang lectures frequently in the U.S. as well as in Asia on alternative therapies and topics related to the regulatory issues of botanicals and supplements - see select lectures by Dr. Chang, and he has also contributed to multiple textbooks, and recently published a book on fertility and on integrating Western and Chinese medicines, as well as many related articles both in Chinese and in English. He is a member of a number of academic medical societies and is also the current President of the Institute of East-West Medicine and served as editorial committee member of the international journal "Complementary and Alternative Medicine", as well as advisory board member of the PDQ Database of the National Cancer Institute and the Global Forum on Indigenous and Traditional Health Systems. \ USA - Meridian Medical offers consultation in the use of DC Therapy for cancer and offers logistic planning and clinical assistance in US patients interested in private treatment in affiliated European clinics. Meridian is experienced in the use of cytokines and immune potentiating herbal extracts as adjuvants in DC Therapy. For further infomation, please email Dr. Raymond Chang.
Europe - The Institute of Tumor Therapy in Duderstadt under the direction of Dr. Thomas Nesselhut in collaboration with Professor JH Peters of the U. of Gottingen in Germany have pioneered the clinical use of DC Therapy in cancer and has treated over 1000 patients to date. For further info: US patients please email Dr. Raymond Chang, all other patients please email Dr. Dagmar Marx.
Duderstadt, Germany
From Wikipedia, the free encyclopedia
Duderstadt is a city in southern Lower Saxony, Germany, located in the district of Göttingen. It is the center and capital of the northern part of the Eichsfeld ("Untereichsfeld"). In earlier times it was the private wealth of the Roman Catholic archbishop of Mainz.
The earliest documentary mention of Duderstadt was in 929 AD, and the city celebrates its anniversaries counting from that year.
Architecture
The city contains many historical buildings in the carcass style, most notably along the Market Street, which stretches from the St. Cyriakuskirche (Catholic, built 1250 - 1490), also called "Oberkirche" (upper church), down to the St. Servatiuskirche (Protestant, built 1370 - 1520), also called "Unterkirche" (nether church).
Old town gate Westerturm
Built in 1343, the Westerturm is one of at least eight gate towers and peels of the city's fortress wall, it burned down in 1424 and was rebuilt over the course of 12 years. The Westerturm has a very distinctive twisted roof. Though not the only tower in Germany with a twisted roof, its execution was particularly successful. There are a number of folk tales to explain the twist: one tale claims that an unoiled weather vane caused the wind to twist the roof. Another claims that when the devil was driving the men of Duderstadt to drink, the women drove him away, but not before he grabbed the tower and twisted it while passing over the wall, making his escape.
Construction of the Rathaus (city government building) began in 1302, with additional wings and components added until 1674. It was completely restored in the 1980s and most recently in 2002. At particular hours, a carillon plays from one of the towers as a bust of the "Anreis" (also: "Anreischke") comes out and nods.
The "Anreischke"
The city Innenstadt is surrounded by a 3km long earthen wall which was constructed by a traveling master fortress builder named Andreas. The city council (Rat) of Duderstadt contracted with Andreas in 1506 to build the wall. Since the surrounding farmers were also to be protected by the wall, the Rat conscripted them to work on the construction of the wall. Andreas was reportedly a cruel taskmaster, and the farmers detested him. They soon also blamed the citizens of Duderstadt for their woes, and took to calling all of them "Anreischke", after Andreas, which was pronounced "Anreis" in the "platt" (low) German spoken by the farmers.
Main road Marktstraße
The citizens of Duderstadt, in turn, had a wooden bust of Andreas constructed and attached to a clockwork. Every 2 hours since then, the wooden "Anreischke" would come out and nod to the farmers coming to market, to remind them of the detested Andreas, and of their dependency on the city.
That wooden Anreischke nods until this day from the Rathaus tower at 9am, 11, 1pm, 3, 5 and 7.
Otto Bock
A Duderstadt-headquartered company is the Otto Bock corporation, named after its founder. Otto Bock produces state-of-the-art prosthetic limbs and other health products. Though now an international company, it is still headquartered in Duderstadt. 2003 revenue was 355.5 million euros. Otto Bock was a significant contributor to economic growth in the region in the postwar period.
January 04, 2009
Vision!!!
On 12/24 I lost 95% of the vision in my left eye and all color. I woke up on Christmas morning with about 50% back and it has been changing somewhat since. As of today I have about 80% of my vision back in the left eye (right was always OK) with perfect color!!!! Finished my 5 Cyberkife radiation treatments this morning. Next Cyberknife visit is 1/29 @ 10:00AM for follow up on the eye and to discuss going after the tumor (primary) in my chest.
I leave for Germany on 1/10 for Dendritic Cell Therapy! First treatment will be in New York on 1/22.
Life is good!!!
Thanks for all of the prayers!!!
Dan
January 03, 2009
Jaunuary 3, 2009 Benefit for Dan Gallagher - How did we get here?
First I want to thank EACH and EVERY person who has offered their support of Kristi and me. We are TRULY grateful. Personally, when my brothers picked up on my mother’s suggestion for this type of event I wasn’t so sure it was a good idea or fit for me. After all, I’m the guy who tries to help others out, hold the family reunions, chip in where I can and has an excellent life, with a beautiful wife and children.
We’ll here’s how we got here tonight and I have been truly HUMBLED by the outpouring of support from SO many people including a few I barely even knew before. Thanks!
On February 27, 2007 I had a CAT (CT) scan of my chest because I was having chronic chest pain and even though chest X-rays and visit’s to my cardiologist didn’t pick anything up this CT showed several tumors in my body including one between my left lung and esophagus. Ultimately in the days following I had MANY more tests and a fine needle biopsy of one of the tumors in my left ribs and it was determined by the pathologist at St. Luke’s Hospital and confirmed by a pathologist at the University of Pennsylvania that I had small-cell lung cancer (the smoker’s cancer, even though I never smoked and in fact have been an adamant non-smoker my entire life, There was skepticism from ALL of the doctors we met with but we had two pathology reports telling us that it was small-cell lung cancer.
I was given one year to live, based on statistics, by some of the doctors I saw but some said don’t read too much into this because I was generally healthy and should be able to get better results (live longer). Typically only 1% of patients with this cancer are alive at 5 years. I was determined to be one of this 1%. Of course that’s me!
Ultimately I failed the initial chemo treatment which surprised my doctors. During this time my sister-in-law, Pat Shand, connected with a member of her church in Falls Church, VA who has a son, Dr. Richard Gralla, who is the head of the Monter Cancer Center at Long Island Jewish Hospital in NY who specializes in treating non-smokers with lung cancer. After meeting with him and having the most thorough exam I have EVER had he told us that he did not believe that I had small-cell lung cancer. He recommended another biopsy of a tumor under my chin which was performed on July 2nd, 2008. He was right; I had Adenoid Cystic Carcinoma (ACC) which is a REALLY rare form of glandular cancer that is typically very slow growing. Dr. Gralla also consulted with my medical oncologist at the University of Pennsylvania, Dr. Kenneth Algazy, and recommended treatment with Tarceva which is a chemotherapeutic drug taken orally with minimal side effects. Unfortunately I failed this drug too although the cancer wasn’t growing so fast.
In September we switched to a combination of chemo know as CAV which I feel darn near killed me but didn’t stop the cancer and during a 6 week period saw significant growth in some of the existing tumors and a few new ones. We stopped this treatment after two cycles and went back on Tarceva and the medicines which Dr. Raymond Chang had me on since March, 2008 and which I now take every day. 108 pills per day!
Here in lies the financial challenge that really got us here tonight. The unreimbursed cost of these medicines and supplements have been costing us $5,000 – 6,000 per month since March and although Kristi and I had some resources we ultimately struggled to keep up with this expense even though we made and continue to make efforts to “downsize” our lives. In November, 2008 we determined that Dendritic Cell Therapy is an opportunity to do one more of those EVERYTHING I can do to beat cancer things and that we had to figure out how to pay for it. The cost for one year of DC is approximately $45,000 with the initial cost (next week) of about $12,000 (fluctuates with the dollar/euro conversion rate) including the trip to Germany, the initial consultation/blood work and first monthly treatment. Provided my tests (CT scans) show that we have significantly slowed and ultimately stopped the cancer the typical plan is to have four consecutive monthly injections which are given in New York by one of the doctors from the clinic in Germany to all U.S. based patients (they fly your serum over once a month) typically followed by four additional treatments every other month for 8 months or one year total of treatments. A CT will typically be performed every 60 days or so to determine if the treatment is working.
In addition to DC treatments I am on two chemotherapy (oral regimens) and the side effect is peripheral neuropathy which can damage the nerves in your feet (I have this and it is why I move slow or awkwardly but this should go away some day) and potentially the hands which I don’t have. I’m also on a program known as Copper Chelating Therapy which, using pharmaceuticals, lowers the copper level in your body to about 20% of the normal level as the goal. The idea is that this will starve the blood supply to the tumors and cause them to commit suicide. This is based on a trial from the University of Michigan which Dr. Chang got me on on a compassionate use basis. We are waging guerilla warfare on cancer after all!!!
In early December I had radiation to my left ribs to reduce the pain and my need for significant amounts of pain medication and the radiation worked quite well. However towards the end of this treatment I developed impaired vision in my left eye, ultimately losing 95% of the vision and all color. I started CyberKnife radiation (very precise) on December 23 and had a second treatment (ultimately 5 total treatments) and my doctor told me on the morning of December 24 that I may never regain the vision in the left eye. Probably the most challenging day I have had in my life. Well, miracles and lots of prayers do work. On Christmas morning I woke up with 50% of the vision back and it has ultimately recovered to 95+%. Thanks to my “brother” Lakshmi I have NEVER had to drive to any appointment and always had him beside me if needed. No Drama!!!
Even though I have cancer I consider myself to be TRULY blessed. I am more than willing to deal with cancer than have ANY child have to deal with this insidious disease. In fact, I pray to Saint Padre’ Pio each night to “bring it on” as I can handle it and to ask Jesus to spare a child or children on my behalf from having to deal with cancer. I also pray to Pope John Paul, II to ask Jesus for a miracle and complete cure. JP2 4ME
Should I die from cancer prematurely I will know that I did EVERYTHING in my power, thanks in part to your assistance tonight, to beat cancer!
Dan
December 23, 2008
Update
I'm starting Cyberknife Radiation TODAY and I'm at the center now!!!
The squeaky wheel I guess!!!
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