- Email: [email protected]
New perspectives on immunobiology and immunotherapy of melanoma
TRENDS I M M U N O L O G Y TO D AY
Peter Schneider ([email protected]) is at the Institute for Legal Medicine, University of Mainz, D-55131 Mainz, Germany; Reinhard WŸrzner ([email protected]) is at the Institute for Hygiene, University of Innsbruck, A-6020 Innsbruck, Austria.
03 Rose, M. and Geserick, G. (1969) Acta Biol.
07 Qu, X.D., Yang, Z., Shen, L. et al. (1998)
Med. Ger. 48, 553Ð557
Nucleic Acids Res. 26, 4068Ð4077
04 Schneider, P.M. and Rittner, C. (1997) in
08 Barba, G., Rittner, C. and Schneider P.M.
Complement Ð A Practical Approach (Dodds, A.
(1993) J. Clin. Invest. 91, 1681Ð1686
and Sim, R.B., eds), pp. 165Ð198, Oxford
09 Rowe, J.A., Moulds, J.M., Newbold, C.I.
University Press
and Miller, L.H. (1997) Nature 388,
05 Mauff, G. and WŸrzner, R. (1997) in Handbook
292Ð295
References
of Experimental Immunology (Weir, D.M., ed.),
10 Carroll, M.C. (1998) Nat. Genet. 19, 3Ð4
01 Alper, C.A. and Propp, R.P. (1968) J. Clin.
pp. 77.1Ð77.11, Blackwell Press
11 Prodeus, A.P., Goerg, S., Shen, L.M. et al.
Invest. 47, 2181Ð2191
06 Schneider, P.M., Rittner, C., Mauff, G. and
02 Azen, E.A. and Smithis, O. (1968) Science 162,
Immunity (in press)
WŸrzner, R., eds (1998) Exp. Clin.
905Ð907
12 Botto, M., DellÕAgnola, C., Bygrave, A.E. et al.
Immunogenet. 15, 197
(1998) Nat. Genet. 19, 56Ð59
New perspectives on immunobiology and immunotherapy of melanoma Frank O. Nestle, Günter Burg and Reinhard Dummer Melanoma is a malignant skin tumor of melanocytic origin. A recent
T
he past decades of melanoma epidemiology have been dominated by a continuously rising incidence and mortality. Consequently, tremendous efforts have been made in various countries to stop this trend by means of primary and secondary prevention programs, and we can now see Ôa light at the end of the tunnel of the melanoma epidemicÕ (R.M. MacKie, Glasgow)1. Incidences of melanoma are still rising in the USA, Germany [a lifetime risk of 1:100 (C. Garbe, TŸbingen)], Switzerland (G. SchŸler, ZŸrich) and southern Europe; however, Australia and northern Europe (including Scotland) have experienced a stabilization Ð a trend that is more pronounced in young people and females. It may be that public education campaigns, which increase awareness of the risk factors responsible for melanoma development, have helped to bring about this stabilization. UV light is the most relevant environmental risk factor for melanoma development. B. Gilchrest (Boston, MA) discussed molecular evidence for a relationship between UV exposure and melanoma genesis. Melanocytes are protected from UV-induced apoptosis by
meeting* discussed the mechanisms
nisms. These findings might explain why intermittent exposure to high doses of UV light carries the highest risk for melanoma development: during intermittent exposure to UV, DNA damage accumulates while DNA repair mechanisms are not yet active.
of immune activation and immune escape during an anti-melanomaspecific response, and new concepts for immunotherapeutic intervention. the expression of high levels of Bcl2. By contrast, keratinocytes undergo apoptosis after exposure to high doses of UV light. UVinduced photodamage leads to the accumulation of thymidine dimers, which appear to stimulate melanogenesis (tanning) and other photoprotective responses such as enhanced DNA repair capacity. In parallel, UV light induces mutations in potential oncogenes and tumor suppressor genes or alters the expression of gene products such as epidermal growth factor receptor and p16 (G. Kraehn, Ulm). Further potentially UV-related genetic hot spots for mutations during the malignant transformation of a normal melanocyte are located on chromosome 1p, 9p, 6q and the long arm of chromosome 11 (R.A. Herbst, Hannover) and are under current investigation. UV-related mutations are counteracted by UV-inducible DNA repair mecha-
PII: S0167-5699(98)01373-5
*The Alpine Melanoma Meeting was a satellite symposium of the International Investigative Dermatology Society Meeting at Davos, Switzerland, on 4Ð6 May 1998.
Host immune response to melanoma antigens Insights into the host immune response against melanoma antigens has set the stage for new approaches to melanoma immunotherapy. Generally, cytotoxic T lymphocytes (CTLs) are believed to be the major effector cells for an anti-melanoma-specific immune response, and in the past years, melanoma-specific CTL clones have served as tools to discover new antigens. The expression pattern of the majority of these antigens may be followed in situ at the protein level. In an extensive immunohistochemical study, 44% of primary melanomas expressed MAGE-3, 88% expressed MelanA/MART-1 and 94% expressed tyrosinase (G. Hofbauer, ZŸrich)2. In a high percentage of melanomas, expression of melanoma antigens is heterogeneous with frequent loss variants. Fur0167-5699/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.
J A N U A R Y Vo l . 2 0
No.1
1 9 9 9 5
TRENDS I M M U N O L O G Y T O D AY
thermore, detection of message by the polymerase chain reaction (PCR) might not correlate with protein expression (G. Spagnoli, Basel). It will be important to determine the impact of antigen loss on melanoma survival and the response to immunotherapy. Recently, new melanoma antigens such as NYESO-1 have been described by analysis of recombinant cDNA expression libraries (SEREX) of human tumors with autologous serum3. The analysis of the humoral immune responses against melanoma may inaugurate a third phase in human cancer serology4. Nonspecific anti-melanoma effector mechanisms might be mediated by natural killer (NK) cells and macrophages. The emergence of a new family of killer-cell inhibitory/activatory receptors (KIRs) expressed on NK cells and T cells raises a number of questions about the activation and de-activation of the antimelanoma immune response. In vivo, there is heterogeneous expression of these receptors on NK T cells (CD561, CD31) with strong variations between individuals probably reflecting multiple clonally distributed NK-Tcell subpopulations (D. Speiser, Lausanne). Tcell clones specific for the nonapeptide TRP-2 can differentially express functionally active KIRs, which suggests that they have different functions during the effector phase of the immune response (C. Noppen, Basel).
Mechanisms of immune escape Understanding the mechanisms that lead to immune escape might help to develop successful anticancer therapies. During an effective CTL response, either mounted by a successful immune response during tumor development or induced during immunotherapy, immune escape can occur at several levels of tumor antigen presentation. Escape variants of melanoma cells can be selected during the natural course of tumor growth in an immunocompetent host or during immunotherapy. First, melanoma cells might lose the relevant tumor antigen (antigen loss variant). Second, defects in the antigen processing machinery might interfere with the generation of immunogenic peptides in the cytosol (e.g. proteasomal defects) or the loading of peptides into the endoplasmic reticulum [e.g. defects in the transporter associated with antigen processing (TAP)]. Third, downregulation or ab-
J A N U A R Y 6
Vo l . 2 0
sence of the relevant HLA allele might prevent presentation of the peptideÐmajor histocompatibility complex (MHC) on the melanoma cell surface. All these abnormalities were detected in situ in metastasis from non-responders to a dendritic cell (DC) vaccination protocol (F.O. Nestle, ZŸrich). In melanoma cell lines, TAP deficiency can be reconstituted by addition of interferon g (IFN-g). On a molecular level, mutations in the b2-microglobulin gene are observed in melanoma cell lines (S. Ferrone, Valhalla, NY). Interestingly, in most cases, the mutation interferes with translation of the protein but not transcription at the message level. Induction of apoptosis in Fas1 CTLs by expression of death ligands such as Fas ligand (FasL) on melanoma cells was recently suggested as another way to escape a tumor-specific immune response. DCs might interfere with this process by providing survival signals to T cells, however, tumor cells might also kill DCs through Fas-mediated mechanisms (M. Lotze, Pittsburgh, PA). Melanoma cells can express FasL in situ but no Fas1 CTL clones were shown to be killed by FasL1 melanoma cells (G. Parmiani, Milan). Another method of tumor-cell escape from apoptosis mediated by FasL1 CTLs is the reduced expression of Fas and the increased expression of a specific inhibitor of receptor-mediated cell death (FLIP, FLICE inhibitory protein) during cutaneous melanoma development (P. Wehrli, R. Bullani and L. French, Lausanne). Finally, melanoma cells might induce apoptosis in activated T cells by shedding HLA class I antigens (S. Ferrone).
New horizons in immunotherapy At the turn of the century, hopes of gene therapy curing cancer or infectious diseases are far from being realized. At the same time, there is increasing pressure to demonstrate that immunological concepts developed in vitro and in animal models are also applicable to humans. Therefore, translational research that brings the concepts of immunology into a therapeutic context for human benefit is needed. The field of melanoma immunotherapy is a frontrunner in this regard. To date, only one immunotherapy protocol has made an impact on melanoma sur-
1 9 9 9 No.1
vival: high dose IFN-a used in an adjuvant setting (i.e. free of primary tumor or lymph node metastasis at the time of treatment) in high-risk melanoma patients (J. Kirkwoood, Pittsburgh, PA). However, no treatment has had a major impact on metastatic disease (A. Hauschild, Kiel). There are several ways that might increase the efficiency of current immunotherapy approaches. Co-administration of tumor necrosis factor a (TNF-a) and IFN-g during isolated limb perfusion in melanoma patients has been shown to act through the reduced activation of the integrin aVb3 and the disruption of the tumor vasculature (F. Lejeune, Lausanne)5. A further possibility, to increase immunogenicity, is to transfect melanoma cells with genes that encode cytokine or costimulatory molecules. In a few patients, transfection of melanoma cells with interleukin 7 (IL-7) or IL-12 increases CTL frequencies without any major clinical responses (D. Schadendorf, Heidelberg)6. Transfection of melanoma cells with IL-2 induces positive delayed-type hypersensitivity (DTH) responses and the development of vitiligo in 3 out of 15 patients (S. Schreiber and G. Stingl, Vienna). Furthermore, IL-12-transfected fibroblasts can lead to regression of certain tumors (M. Lotze). The injection of cutaneous melanoma metastases with a canary pox virus carrying human cytokine transgenes resulted in inflammatory infiltrates dominated by lymphocytes in the case of IL-2 transgenes, and by macrophages and neutrophils in the case of granulocyteÐmacrophage colony-stimulating factor (GM-CSF) transgenes. This observation suggests that gene transfer strategies can orchestrate a cellular immune response independent from the vector system (R. Dummer, ZŸrich; F. Lejeune). Overwhelming evidence suggests that local dendritic antigen-presenting cells (APCs) are crucial for the induction of a specific antitumor response. Stimulation of the CD40ÐCD40 ligand (CD40L) pathway might lead to a ÔconditionedÕ or ÔlicensedÕ APC, such as a DC, which can directly prime a CTL in the absence of a T helper (Th) cell (R. Offringa, Leiden). This finding is of major importance for the understanding of CTL activation and has been independently confirmed by several research groups7Ð10. Therefore, mouse models might be used to analyze these signals further, for example, by
TRENDS I M M U N O L O G Y TO D AY
transfection of melanoma cells with CD40L to target CD401 lesional APCs (A. Schneeberger, Vienna). Current peptide-based immunotherapy approaches were not successful in inducing durable clinical responses or CTL responses. To overcome unresponsiveness, superpotent peptide analogs, which are recognized by tumor infiltrating lymphocytes (TILs) more efficiently than the natural peptides, may be used (D. Valmori, Lausanne). A recent study in humans has administered such immunodominant peptides with success, but clinical response was dependent on co-administration of IL-2 (Ref. 11). Another innovative approach to increase the immunogenicity of a given peptide is to deliver the antigen in the context of its natural adjuvant Ð a DC. In a recent pilot study, DCs pulsed with peptide or tumor lysate were injected in uninvolved inguinal lymph nodes in 16 patients with metastatic melanoma. Five out of 16 patients responded to therapy with one additional minor response. Clincal responses were correlated with a positive DTH response and peptide-specific CTL responses12. In this study, keyhole limpet hemocyanin (KLH) was used as helper antigen. Addition of KLH induced a Th1-type cytokine response in vitro and in vivo, which might be beneficial for the generation of melanoma peptide-specific CTLs (M. Gilliet, ZŸrich). Finally, fusion of APCs such as B cells or DCs with melanoma cells might represent a novel approach to induce potent anti-melanoma-specific immune responses and is currently tested in a Phase I/II trial (U. Trefzer and W. Sterry, Berlin).
Monitoring the CTL response and markers for disease progression A primary endpoint to judge success of immunotherapy is the measurement of a successfully generated CTL response. In humans, analysis of CTL frequencies by conventional chromium release assays is cumbersome and often not feasible. A new approach might be the use of tetrameric MHC molecules to detect an increase in frequency of peptide-specific CTLs (P. Romero, Lausanne; E. Cerundolo and R. Dunbar, Oxford). HLA-A2/MelanA tetramers were used to detect peptide-specific TILs in lymphnode suspensions of melanoma patients. Another important endpoint for success
of immunotherapy is provided by serum markers that predict response to therapy. Recently, a variety of such markers became available for melanoma. Serum levels of soluble S100 protein, the melanoma inhibitory activity (MIA) protein and 5-S-cysteinyldopa (Ref. 13) are markers for the tumor load of individual patients, which allow monitoring of therapeutic interventions (J. Meyer, ZŸrich; E. Schultz, Erlangen; P. Kaskel, Ulm; E. Stockfleth, Kiel; A. Bosserhof, Regensburg). These markers also hold promise to identify occult metastases during follow-up of high-risk patients.
have provided a new basis for immunotherapeutic approaches. Characterization of the pathways involved in the early phases of natural and acquired immunity, such as CD40ÐCD40L interactions, the involvement of DCs and the role of KIRs, will have a major impact on the road to successful anticancer immunotherapy.
We apologize to all speakers whose contributions could not be cited here because of space limitations. Abstracts of this meeting are published in J. Invest. Dermatol. (1998) 110, 710Ð723. F.O.N. is supported by grants from the Cancer League ZŸrich and the Swiss Cancer League, and R.D. is supported by the
Pitfalls of peptide-based vaccination strategies
Swiss National Science Foundation. The conference
Currently, most readouts during immunotherapy are focused on the induction of a positive CTL response and a beneficial clinical response. Little attention is given to the possibility of inducing a negative CTL response and thereby inducing progression of disease, for example, by peptide vaccination (R. Offringa, Leiden). Vaccination with synthetic peptides from the human adenovirus type 5 E1A-region enhanced rather than inhibited the growth of Ad5E1A-expressing tumors. Injection of large amounts of free peptide might reach the systemic circulation and bind to MHC class I1 cells where presentation to effector T cells takes place in the absence of costimulatory signals. Delivery of the peptide in the context of professional APCs such as DCs could overcome the negative effects of CTL generation and lead to the induction of an effective immune response in this model. These data indicate that injection of free peptides also have negative effects on the development of a peptide-specific immune response and that a safeguard might be the delivery of peptides in an immunologically relevant context, e.g. pulsed on professional APCs.
Society of Investigative Dermatology.
was a satellite symposium of the International
Frank Nestle ([email protected]), GŸnter Burg and Reinhard Dummer are at the Dept of Dermatology, University of Zurich Medical School, Gloriastrasse 31, 8091 Zurich, Switzerland. References 01 MacKie, R.M., Hole, D., Hunter, A. et al. (1997) Br. Med. J. 315, 1117Ð1121 02 Hofbauer, G.F.L., Schaefer, C., Noppen, C. et al. (1997) Am. J. Pathol. 151, 1549Ð1553 03 Sahin, U., TŸreci, …. and Pfreundschuh, M. (1998) Curr. Opin. Immunol. 9, 709Ð716 04 Old, L.J. and Chen, Y.T. (1998) J. Exp. Med. 187, 1163Ð1167 05 RŸegg, C., Yilmaz, A., Bieler, F. et al. (1998) Nat. Med. 4, 408Ð414 06 Sun, Y., Jurgovsky, K., Mšller, P. et al. (1998) Gene Ther. 5, 481Ð490 07 Lanzavechia, A. (1998) Nature 393, 413Ð414 08 Schoenberger, S.P., Toes, R.E.M., van der Hoort, E.I.H., Offringa, R. and Melief, C.J.M. (1998) Nature 393, 480Ð483 09 Ridge, J.P., di Rosa, F. and Matzinger, P. (1998) Nature 393, 474Ð477 10 Bennett, S.R.M., Carbonne, F.R., Karamelis, F. et al. (1998) Nature 393, 478Ð480
Concluding remarks
11 Rosenberg, S., Yang, J.C., Schwartzentruber,
Melanoma is the prototype of a tumor where concepts of immunology and immunointervention can be translated into a successful clinical therapy. Recent developments in understanding the generation of and the escape from a melanoma-specific immune response
D.J. et al. (1998) Nat. Med. 4, 321Ð327 12 Nestle, F.O., Alijagic, S., Gilliet, M. et al. (1998) Nat. Med. 4, 328Ð332 13 Wimmer, I., Meyer, J.C., Seiffert, B., Dummer, R., Flace, A. and Burg, G. (1997) Cancer Res. 57, 5073Ð5076
J A N U A R Y Vo l . 2 0
No.1
1 9 9 9 7
Peter Schneider ([email protected]) is at the Institute for Legal Medicine, University of Mainz, D-55131 Mainz, Germany; Reinhard WŸrzner ([email protected]) is at the Institute for Hygiene, University of Innsbruck, A-6020 Innsbruck, Austria.
03 Rose, M. and Geserick, G. (1969) Acta Biol.
07 Qu, X.D., Yang, Z., Shen, L. et al. (1998)
Med. Ger. 48, 553Ð557
Nucleic Acids Res. 26, 4068Ð4077
04 Schneider, P.M. and Rittner, C. (1997) in
08 Barba, G., Rittner, C. and Schneider P.M.
Complement Ð A Practical Approach (Dodds, A.
(1993) J. Clin. Invest. 91, 1681Ð1686
and Sim, R.B., eds), pp. 165Ð198, Oxford
09 Rowe, J.A., Moulds, J.M., Newbold, C.I.
University Press
and Miller, L.H. (1997) Nature 388,
05 Mauff, G. and WŸrzner, R. (1997) in Handbook
292Ð295
References
of Experimental Immunology (Weir, D.M., ed.),
10 Carroll, M.C. (1998) Nat. Genet. 19, 3Ð4
01 Alper, C.A. and Propp, R.P. (1968) J. Clin.
pp. 77.1Ð77.11, Blackwell Press
11 Prodeus, A.P., Goerg, S., Shen, L.M. et al.
Invest. 47, 2181Ð2191
06 Schneider, P.M., Rittner, C., Mauff, G. and
02 Azen, E.A. and Smithis, O. (1968) Science 162,
Immunity (in press)
WŸrzner, R., eds (1998) Exp. Clin.
905Ð907
12 Botto, M., DellÕAgnola, C., Bygrave, A.E. et al.
Immunogenet. 15, 197
(1998) Nat. Genet. 19, 56Ð59
New perspectives on immunobiology and immunotherapy of melanoma Frank O. Nestle, Günter Burg and Reinhard Dummer Melanoma is a malignant skin tumor of melanocytic origin. A recent
T
he past decades of melanoma epidemiology have been dominated by a continuously rising incidence and mortality. Consequently, tremendous efforts have been made in various countries to stop this trend by means of primary and secondary prevention programs, and we can now see Ôa light at the end of the tunnel of the melanoma epidemicÕ (R.M. MacKie, Glasgow)1. Incidences of melanoma are still rising in the USA, Germany [a lifetime risk of 1:100 (C. Garbe, TŸbingen)], Switzerland (G. SchŸler, ZŸrich) and southern Europe; however, Australia and northern Europe (including Scotland) have experienced a stabilization Ð a trend that is more pronounced in young people and females. It may be that public education campaigns, which increase awareness of the risk factors responsible for melanoma development, have helped to bring about this stabilization. UV light is the most relevant environmental risk factor for melanoma development. B. Gilchrest (Boston, MA) discussed molecular evidence for a relationship between UV exposure and melanoma genesis. Melanocytes are protected from UV-induced apoptosis by
meeting* discussed the mechanisms
nisms. These findings might explain why intermittent exposure to high doses of UV light carries the highest risk for melanoma development: during intermittent exposure to UV, DNA damage accumulates while DNA repair mechanisms are not yet active.
of immune activation and immune escape during an anti-melanomaspecific response, and new concepts for immunotherapeutic intervention. the expression of high levels of Bcl2. By contrast, keratinocytes undergo apoptosis after exposure to high doses of UV light. UVinduced photodamage leads to the accumulation of thymidine dimers, which appear to stimulate melanogenesis (tanning) and other photoprotective responses such as enhanced DNA repair capacity. In parallel, UV light induces mutations in potential oncogenes and tumor suppressor genes or alters the expression of gene products such as epidermal growth factor receptor and p16 (G. Kraehn, Ulm). Further potentially UV-related genetic hot spots for mutations during the malignant transformation of a normal melanocyte are located on chromosome 1p, 9p, 6q and the long arm of chromosome 11 (R.A. Herbst, Hannover) and are under current investigation. UV-related mutations are counteracted by UV-inducible DNA repair mecha-
PII: S0167-5699(98)01373-5
*The Alpine Melanoma Meeting was a satellite symposium of the International Investigative Dermatology Society Meeting at Davos, Switzerland, on 4Ð6 May 1998.
Host immune response to melanoma antigens Insights into the host immune response against melanoma antigens has set the stage for new approaches to melanoma immunotherapy. Generally, cytotoxic T lymphocytes (CTLs) are believed to be the major effector cells for an anti-melanoma-specific immune response, and in the past years, melanoma-specific CTL clones have served as tools to discover new antigens. The expression pattern of the majority of these antigens may be followed in situ at the protein level. In an extensive immunohistochemical study, 44% of primary melanomas expressed MAGE-3, 88% expressed MelanA/MART-1 and 94% expressed tyrosinase (G. Hofbauer, ZŸrich)2. In a high percentage of melanomas, expression of melanoma antigens is heterogeneous with frequent loss variants. Fur0167-5699/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.
J A N U A R Y Vo l . 2 0
No.1
1 9 9 9 5
TRENDS I M M U N O L O G Y T O D AY
thermore, detection of message by the polymerase chain reaction (PCR) might not correlate with protein expression (G. Spagnoli, Basel). It will be important to determine the impact of antigen loss on melanoma survival and the response to immunotherapy. Recently, new melanoma antigens such as NYESO-1 have been described by analysis of recombinant cDNA expression libraries (SEREX) of human tumors with autologous serum3. The analysis of the humoral immune responses against melanoma may inaugurate a third phase in human cancer serology4. Nonspecific anti-melanoma effector mechanisms might be mediated by natural killer (NK) cells and macrophages. The emergence of a new family of killer-cell inhibitory/activatory receptors (KIRs) expressed on NK cells and T cells raises a number of questions about the activation and de-activation of the antimelanoma immune response. In vivo, there is heterogeneous expression of these receptors on NK T cells (CD561, CD31) with strong variations between individuals probably reflecting multiple clonally distributed NK-Tcell subpopulations (D. Speiser, Lausanne). Tcell clones specific for the nonapeptide TRP-2 can differentially express functionally active KIRs, which suggests that they have different functions during the effector phase of the immune response (C. Noppen, Basel).
Mechanisms of immune escape Understanding the mechanisms that lead to immune escape might help to develop successful anticancer therapies. During an effective CTL response, either mounted by a successful immune response during tumor development or induced during immunotherapy, immune escape can occur at several levels of tumor antigen presentation. Escape variants of melanoma cells can be selected during the natural course of tumor growth in an immunocompetent host or during immunotherapy. First, melanoma cells might lose the relevant tumor antigen (antigen loss variant). Second, defects in the antigen processing machinery might interfere with the generation of immunogenic peptides in the cytosol (e.g. proteasomal defects) or the loading of peptides into the endoplasmic reticulum [e.g. defects in the transporter associated with antigen processing (TAP)]. Third, downregulation or ab-
J A N U A R Y 6
Vo l . 2 0
sence of the relevant HLA allele might prevent presentation of the peptideÐmajor histocompatibility complex (MHC) on the melanoma cell surface. All these abnormalities were detected in situ in metastasis from non-responders to a dendritic cell (DC) vaccination protocol (F.O. Nestle, ZŸrich). In melanoma cell lines, TAP deficiency can be reconstituted by addition of interferon g (IFN-g). On a molecular level, mutations in the b2-microglobulin gene are observed in melanoma cell lines (S. Ferrone, Valhalla, NY). Interestingly, in most cases, the mutation interferes with translation of the protein but not transcription at the message level. Induction of apoptosis in Fas1 CTLs by expression of death ligands such as Fas ligand (FasL) on melanoma cells was recently suggested as another way to escape a tumor-specific immune response. DCs might interfere with this process by providing survival signals to T cells, however, tumor cells might also kill DCs through Fas-mediated mechanisms (M. Lotze, Pittsburgh, PA). Melanoma cells can express FasL in situ but no Fas1 CTL clones were shown to be killed by FasL1 melanoma cells (G. Parmiani, Milan). Another method of tumor-cell escape from apoptosis mediated by FasL1 CTLs is the reduced expression of Fas and the increased expression of a specific inhibitor of receptor-mediated cell death (FLIP, FLICE inhibitory protein) during cutaneous melanoma development (P. Wehrli, R. Bullani and L. French, Lausanne). Finally, melanoma cells might induce apoptosis in activated T cells by shedding HLA class I antigens (S. Ferrone).
New horizons in immunotherapy At the turn of the century, hopes of gene therapy curing cancer or infectious diseases are far from being realized. At the same time, there is increasing pressure to demonstrate that immunological concepts developed in vitro and in animal models are also applicable to humans. Therefore, translational research that brings the concepts of immunology into a therapeutic context for human benefit is needed. The field of melanoma immunotherapy is a frontrunner in this regard. To date, only one immunotherapy protocol has made an impact on melanoma sur-
1 9 9 9 No.1
vival: high dose IFN-a used in an adjuvant setting (i.e. free of primary tumor or lymph node metastasis at the time of treatment) in high-risk melanoma patients (J. Kirkwoood, Pittsburgh, PA). However, no treatment has had a major impact on metastatic disease (A. Hauschild, Kiel). There are several ways that might increase the efficiency of current immunotherapy approaches. Co-administration of tumor necrosis factor a (TNF-a) and IFN-g during isolated limb perfusion in melanoma patients has been shown to act through the reduced activation of the integrin aVb3 and the disruption of the tumor vasculature (F. Lejeune, Lausanne)5. A further possibility, to increase immunogenicity, is to transfect melanoma cells with genes that encode cytokine or costimulatory molecules. In a few patients, transfection of melanoma cells with interleukin 7 (IL-7) or IL-12 increases CTL frequencies without any major clinical responses (D. Schadendorf, Heidelberg)6. Transfection of melanoma cells with IL-2 induces positive delayed-type hypersensitivity (DTH) responses and the development of vitiligo in 3 out of 15 patients (S. Schreiber and G. Stingl, Vienna). Furthermore, IL-12-transfected fibroblasts can lead to regression of certain tumors (M. Lotze). The injection of cutaneous melanoma metastases with a canary pox virus carrying human cytokine transgenes resulted in inflammatory infiltrates dominated by lymphocytes in the case of IL-2 transgenes, and by macrophages and neutrophils in the case of granulocyteÐmacrophage colony-stimulating factor (GM-CSF) transgenes. This observation suggests that gene transfer strategies can orchestrate a cellular immune response independent from the vector system (R. Dummer, ZŸrich; F. Lejeune). Overwhelming evidence suggests that local dendritic antigen-presenting cells (APCs) are crucial for the induction of a specific antitumor response. Stimulation of the CD40ÐCD40 ligand (CD40L) pathway might lead to a ÔconditionedÕ or ÔlicensedÕ APC, such as a DC, which can directly prime a CTL in the absence of a T helper (Th) cell (R. Offringa, Leiden). This finding is of major importance for the understanding of CTL activation and has been independently confirmed by several research groups7Ð10. Therefore, mouse models might be used to analyze these signals further, for example, by
TRENDS I M M U N O L O G Y TO D AY
transfection of melanoma cells with CD40L to target CD401 lesional APCs (A. Schneeberger, Vienna). Current peptide-based immunotherapy approaches were not successful in inducing durable clinical responses or CTL responses. To overcome unresponsiveness, superpotent peptide analogs, which are recognized by tumor infiltrating lymphocytes (TILs) more efficiently than the natural peptides, may be used (D. Valmori, Lausanne). A recent study in humans has administered such immunodominant peptides with success, but clinical response was dependent on co-administration of IL-2 (Ref. 11). Another innovative approach to increase the immunogenicity of a given peptide is to deliver the antigen in the context of its natural adjuvant Ð a DC. In a recent pilot study, DCs pulsed with peptide or tumor lysate were injected in uninvolved inguinal lymph nodes in 16 patients with metastatic melanoma. Five out of 16 patients responded to therapy with one additional minor response. Clincal responses were correlated with a positive DTH response and peptide-specific CTL responses12. In this study, keyhole limpet hemocyanin (KLH) was used as helper antigen. Addition of KLH induced a Th1-type cytokine response in vitro and in vivo, which might be beneficial for the generation of melanoma peptide-specific CTLs (M. Gilliet, ZŸrich). Finally, fusion of APCs such as B cells or DCs with melanoma cells might represent a novel approach to induce potent anti-melanoma-specific immune responses and is currently tested in a Phase I/II trial (U. Trefzer and W. Sterry, Berlin).
Monitoring the CTL response and markers for disease progression A primary endpoint to judge success of immunotherapy is the measurement of a successfully generated CTL response. In humans, analysis of CTL frequencies by conventional chromium release assays is cumbersome and often not feasible. A new approach might be the use of tetrameric MHC molecules to detect an increase in frequency of peptide-specific CTLs (P. Romero, Lausanne; E. Cerundolo and R. Dunbar, Oxford). HLA-A2/MelanA tetramers were used to detect peptide-specific TILs in lymphnode suspensions of melanoma patients. Another important endpoint for success
of immunotherapy is provided by serum markers that predict response to therapy. Recently, a variety of such markers became available for melanoma. Serum levels of soluble S100 protein, the melanoma inhibitory activity (MIA) protein and 5-S-cysteinyldopa (Ref. 13) are markers for the tumor load of individual patients, which allow monitoring of therapeutic interventions (J. Meyer, ZŸrich; E. Schultz, Erlangen; P. Kaskel, Ulm; E. Stockfleth, Kiel; A. Bosserhof, Regensburg). These markers also hold promise to identify occult metastases during follow-up of high-risk patients.
have provided a new basis for immunotherapeutic approaches. Characterization of the pathways involved in the early phases of natural and acquired immunity, such as CD40ÐCD40L interactions, the involvement of DCs and the role of KIRs, will have a major impact on the road to successful anticancer immunotherapy.
We apologize to all speakers whose contributions could not be cited here because of space limitations. Abstracts of this meeting are published in J. Invest. Dermatol. (1998) 110, 710Ð723. F.O.N. is supported by grants from the Cancer League ZŸrich and the Swiss Cancer League, and R.D. is supported by the
Pitfalls of peptide-based vaccination strategies
Swiss National Science Foundation. The conference
Currently, most readouts during immunotherapy are focused on the induction of a positive CTL response and a beneficial clinical response. Little attention is given to the possibility of inducing a negative CTL response and thereby inducing progression of disease, for example, by peptide vaccination (R. Offringa, Leiden). Vaccination with synthetic peptides from the human adenovirus type 5 E1A-region enhanced rather than inhibited the growth of Ad5E1A-expressing tumors. Injection of large amounts of free peptide might reach the systemic circulation and bind to MHC class I1 cells where presentation to effector T cells takes place in the absence of costimulatory signals. Delivery of the peptide in the context of professional APCs such as DCs could overcome the negative effects of CTL generation and lead to the induction of an effective immune response in this model. These data indicate that injection of free peptides also have negative effects on the development of a peptide-specific immune response and that a safeguard might be the delivery of peptides in an immunologically relevant context, e.g. pulsed on professional APCs.
Society of Investigative Dermatology.
was a satellite symposium of the International
Frank Nestle ([email protected]), GŸnter Burg and Reinhard Dummer are at the Dept of Dermatology, University of Zurich Medical School, Gloriastrasse 31, 8091 Zurich, Switzerland. References 01 MacKie, R.M., Hole, D., Hunter, A. et al. (1997) Br. Med. J. 315, 1117Ð1121 02 Hofbauer, G.F.L., Schaefer, C., Noppen, C. et al. (1997) Am. J. Pathol. 151, 1549Ð1553 03 Sahin, U., TŸreci, …. and Pfreundschuh, M. (1998) Curr. Opin. Immunol. 9, 709Ð716 04 Old, L.J. and Chen, Y.T. (1998) J. Exp. Med. 187, 1163Ð1167 05 RŸegg, C., Yilmaz, A., Bieler, F. et al. (1998) Nat. Med. 4, 408Ð414 06 Sun, Y., Jurgovsky, K., Mšller, P. et al. (1998) Gene Ther. 5, 481Ð490 07 Lanzavechia, A. (1998) Nature 393, 413Ð414 08 Schoenberger, S.P., Toes, R.E.M., van der Hoort, E.I.H., Offringa, R. and Melief, C.J.M. (1998) Nature 393, 480Ð483 09 Ridge, J.P., di Rosa, F. and Matzinger, P. (1998) Nature 393, 474Ð477 10 Bennett, S.R.M., Carbonne, F.R., Karamelis, F. et al. (1998) Nature 393, 478Ð480
Concluding remarks
11 Rosenberg, S., Yang, J.C., Schwartzentruber,
Melanoma is the prototype of a tumor where concepts of immunology and immunointervention can be translated into a successful clinical therapy. Recent developments in understanding the generation of and the escape from a melanoma-specific immune response
D.J. et al. (1998) Nat. Med. 4, 321Ð327 12 Nestle, F.O., Alijagic, S., Gilliet, M. et al. (1998) Nat. Med. 4, 328Ð332 13 Wimmer, I., Meyer, J.C., Seiffert, B., Dummer, R., Flace, A. and Burg, G. (1997) Cancer Res. 57, 5073Ð5076
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