- Email: [email protected]
Immunodominance and tumor escape
seminars in CANCER BIOLOGY, Vol. 12, 2002: pp. 25–31 doi:10.1006/scbi.2001.0401, available online at http://www.idealibrary.com on
Immunodominance and tumor escape H. Schreiber a,∗ , T. H. Wu a , J. Nachman b and W. M. Kast c
are required to cause a fully malignant phenotype. We 2 and subsequently others 3–6 have shown that unique antigens recognized by the host are indeed tumor specific because they represent somatic tumorspecific mutations. It still may appear surprising that there are so many TSA because the chance that a point mutation will also generate a mutant peptide that acts as a major antigen is relatively small. Presumably, the number of point mutations present in a single cancer cell is so large that multiple TSA exist. Selection of antigen loss variants using T cell clones showed that the loss of one antigen did not affect the expression of the remaining antigens. 7 Further experiments showed that the frequency of loss of an individual antigen could be as high as 1 in 104 cloned cancer cells 8 Despite this high frequency, tumor cells failed to survive in vitro when exposed to a combination of cytolytic T cell clones directed against two or more independent antigens. Simultaneous attack against multiple independent TSA does not occur in vivo, presumably because responses are limited to a single or very few epitopes on malignant cells, a phenomenon referred to as immunodominance. 9,10
Cancers in mouse and man express multiple tumor-specific as well as tumor-associated antigens. Immunodominance in the host response to these antigens can result in successive selection of heritable antigen loss variants. Immunodominance may also prevent the development of responses to new tumor-specific antigens that may arise during tumor progression. Some tumor-specific antigens are retained during tumor progression possibly because they are essential for survival of the malignant phenotype. Immunodominance may allow cancer cells to escape even after loss of a single MHC Class I allele because cross-presentation of the retained antigen by this allele that must be expressed on the surrounding antigen presenting cells sustains the immunodominant response. This prevents effective responses to secondary antigens that may remain as potential targets. Immunization with in vitro selected cancer cell variants that lack the immunodominant antigen can break the immunodominance and prevent escape of cancers from host immunity. Key words: CD8+ T cells / cross-presentation / MHC Class I loss / multiplicity / Tumor-specific antigens / variants c 2002 Academic Press
Immunodominance
Multiplicity of tumor-specific antigens expressed on a single cancer cell
Immunodominance was first shown for viruses expressing multiple epitopes. 11,12 Shortly thereafter, remarkable immunodominance was observed in the response of mice to minor histocompatibility antigens; 13 mice challenged with more than 40 minor histocompatibility antigens responded to a single or very few antigens, which is observed for tumor antigens as well. 7 One important factor in determining whether an antigen becomes immunodominant over others seems to be the rapidity by which it induces a response in relation to other antigens. Thus, immunodominance or ‘priority’ is given to the ‘first’ response (see below). As long as the parental tumor cells expressing
Multiple TSA are expressed on single murine and human cancer cells. 1 It is now generally accepted that multiple different, somatic, tumor-specific mutations
From the a Department of Pathology and b the Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA and c The Cancer Immunology Program, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL 60153, USA. *Corresponding author. Department of Pathology, The University of Chicago, 5841 S. Maryland Ave., MC 3008, Chicago, IL 60637. E-mail: [email protected] c 2002 Academic Press
1044–579X / 02 / 010025+ 07 / $35.00 / 0
25
H. Schreiber et al.
Figure 1. Upper panel: Immunodominance and priority of the first response in response to a dominant (A) and recessive (B) TSA. An immunodominant antigen A prevents the response to an immuno-recessive antigen B when injected at the same site or when present on the same cell as B. B alone is capable of inducing a response, however this response is slower and lower initially. When the dominant and recessive antigens are injected at separate site, both responses are induced. These findings predict the escape of B variants when inoculated with immunodominant, parental A containing cells at the same injection site, and the rejection of B variants when inoculated at a separate site as observed experimentally (see Figure 2). Lower panel: Exposure to one antigen before the other determines which one will become immunodominant. This finding of a ‘priority of the first response’ is not only relevant to the failure of the host to respond to a new TSA appearing subsequently as a result of tumor progression but is also relevant to explaining the recessiveness of an antigen coexpressed on the original cancer cell but capable of inducing only a slower response (such is observed for the response to the B antigen in the upper panel).
the dominant antigen are present at the same site of tumor growth, immunodominance protects tumor variants that have lost the immunodominant antigen by forcing immune attack on the dominant antigen on the parental tumor and away from the immunorecessive antigen on the variant. The problems associated with local immunodominance can be modeled with parental tumors and their antigen loss variants (Figure 1, upper panel). These tumor cell lines can also be used to demonstrate the effect of local immunodominance in immune
escape (Figure 2). Immunodominant T and B cell responses against a particular antigen or epitope are commonly multiclonal; though the receptors are not identical, they recognize the same epitope. Virtually monoclonal antibody responses to a complex antigen are sometimes observed, e.g. the antibody response of BALB/c mice to phosphorylcholine of the rough pneumococcal bacterium R36A, 14 but such responses are the exception rather than the rule. 15 Cytolytic T cell responses against unique TSA are usually focussed on a single immunodominant 26
Immunodominance and tumor escape
inoculum. Similarly, new hierarchies emerge when an immunodominant antigen is lost following immune selection but multiple other antigens remain on the tumor cell. This process of reestablishment of new hierarchies continues with each additional loss of a TSA leading to outgrowth of new antigen loss variants. Thus, there is a ‘pecking order’ in the response to multiple TSA expressed by a single malignant cell 9 and a stepwise immunological selection of antigenic variants during tumor growth. 19 Unfortunately, by the time the host responds effectively to the next immunodominant antigen, too many new variant cancer cells have grown that have already lost this antigen. Therefore, these new variants survive the next round of killing. Theoretically, the same scenario of events should repeat, so that cancer cell with progressively fewer target antigens would emerge. However, hosts bearing the escape tumors often fail to mount an immune response to antigens remaining on the variants because of either a systemic tumor-induced T cell defect, 20,21 a local, possibly stromal, barrier 22,23 in the established tumor, or a priority of the first response, as discussed below.
Figure 2. Immune escape due to local immunodominance: the less malignant parental AB tumor ‘helps’ the escape of the more malignant antigen loss progressor variant B which kills the host (upper panel). The effect is due to local immunodominance of the A antigen preventing an anti B T cell response until it is too late and the variant tumor is well established (see Figure 1). Such problem does not arise in the experimental control setting (lower panel) when parental and variant tumor cells are injected at different sites. In real life, however, the variant evolves from the parental tumor at the same site (upper panel).
epitope/peptide but involve multiple different TCR clonotypes. 16 Occasionally, however, a single or a restricted number of Vβ regions are used by the responding T cells. 16 Such restrictions have then been used in cancer models to identify immunodominant TSA T cell responses. Another indicator of immunodominance is the selective T cell reactivity to one of multiple independent antigens contained in the immunizing tumor cell inoculum. 7 Such reactivity can be proven by demonstrating selective lysis by the responding T cells of a cancer cell variant that expresses only this antigen and lacks other T cellrecognized antigens. 7 Finally, with the identification of the genetic origins of some of the tumor-specific T cell recognized peptides, immunodominance can also be demonstrated in the response to molecularly defined mutant peptide epitopes. 17
‘Priority of the first response’: antigens inducing a faster response become immunodominant over antigens appearing later and/or inducing a slower response In theory, with tumor progression 24,25 tumor cells will acquire more and more ‘mutations’ (translocations, point mutations, etc) and express more and more epitopes which could be antigenic and probably immunogenic if the response to them was not down regulated. Importantly it has been found that an existent response to an antigen already present on the tumor cell will prevent a response to these newly arising antigens, even though such second antigens may be stronger than the original antigen and could become an important target if an effective response to these antigens could be induced. The ‘priority of the first response’ is suggested by experiments in mice: when mice are immunized repeatedly with an antigen A, later addition of an antigen B to the immunogen may not elicit any anti-B CD8+ T cell response in vivo (Figure 1, lower panel). Vice versa, repeated immunization with B will prevent a CD8+ T cell response to antigen A if A is added later to the immunogen. 10,26
‘Pecking order’ and sequential antigen loss during escape It has been reported that if five immunodominant epitopes all having the same MHC Class I restriction element were mixed as antigen, a new pattern of dominance of only two of the epitopes was established 18 Thus, novel hierarchies emerge when multiple dominant epitopes are given in a single 27
H. Schreiber et al.
Figure 3. Loss of one of three MHC Class I alleles is sufficient to prevent responses of the host to tumor antigens presented by the two other alleles. Cross-presentation of the A antigen despite loss of the presenting MHC-Class I allele from the tumor leads to continued induction of the immunodominant anti-A response by the host. 29 This persistent response can no longer kill the escape variant and prevents the elimination of the variant by killer T cells that recognize the remaining potential antigenic targets.
Cross-presentation and single allelic MHC class I allele loss: escape due to persistent immunodominance
stimulate the immunodominant response. The UVinduced cancer 6130 may be an example of such a cancer. We have found that the 6130 progressor variant lost the MHC Class I K allele of the k haplotype while retaining the D allele. 29 Despite the Kk loss, the variant cell cancer continued to induce a CD8+ tumor-specific T cell response to this antigen; however the antigen no longer is a target with variant cancer cells though the antigen is crosspresented by APC of the host. Thus, the host is locked into a futile immunodominant response that prevents effective new responses to other antigens that may be expressed (Figure 3). At present, we do not know whether the common occurrence of a single MHC Class I allelic loss results from a
In common human malignancies, complete MHC Class I antigen loss occurs in a small but significant fraction (approximately 10% of cancers), while loss of expression of a single MHC Class I allele is observed in the large majority (>80%) of certain human malignancies. 27,28 It is certainly tempting to suggest that: (i) an MHC class I allele was lost because it presented an essential immunodominant target antigen and (ii) cross-presentation of this antigen by surrounding dendritic cells or other APC occurs that must express the presenting allele continues to 28
Immunodominance and tumor escape
injected at a subcutaneous site separate from the immunodominant antigen, were effectively rejected. Further experiments showed that immunization with variants (that retained the immunorecessive antigen but lost the immunodominant antigen) at separate sites prevented the outgrowth of antigen loss escape variants from the tumor inoculum that contained a mixture of less malignant parental AB cells and more malignant escape variant B cells. 10 Vaccination with the parental tumor cells was ineffective. Similarly, vaccination with a recessive peptide antigen or another form of a defined antigen can prevent the outgrowth of cancer cells that express dominant antigens not used for immunization. For example, it has been shown that immunization with the E7 RAHYNIVTF peptide antigen can prevent the outgrowth of HPV16 transformed tumor cells even though the antigen is not immunodominant on these cancer cells. 33,34 Thus, vaccination with individual tumor antigens at separate sites rather than with multiple antigens at one site may be needed to prevent tumor escape and recurrence of cancer.
cytolytic T cell response to immunodominant antigen restricted by the allele lost by the cancer cell, but if this is indeed the case, then loss of a single MHC Class I allele restricting an immunodominant antigen may be a common mechanism for cancers to escape immune destruction.
Vaccination with in vitro-selected variants or immunorecessive antigens may prevent tumor escape Recently, it has been proposed that immunodominance in response to a mixture of multiple defined synthetic peptides can be broken by immunizing the host with dendritic cells loaded with these peptides. 18 The mixture of peptides was used for priming phase, whereas spleen cells loaded with the individual peptides were used as stimulators for the generation of CTL in vitro. It is still unclear whether dendritic cell vaccination can prevent or reverse immunodominance during the course of an ongoing immune stimulation by complex antigens such as tumor cells which express multiple epitopes. This approach is particularly attractive because DC loaded with multiple antigens from tumor cells could be used without having to know the identity of the individual antigens 30–32 and without having to isolate cancer cell variants expressing the immunorecessive antigens for immunization, as outlined below. 10 Therefore, it will be important to show that vaccination using DC can break immunodominance and prevent tumor escape. Immunodominance in the response to multiple tumor antigens expressed on a single cell can be broken by inducing a cytolytic T cell response with variants lacking the immunodominant antigen. 10 For example, Figure 1 (upper panel) shows that when antigen A is dominant over antigen B, immunization with parental AB tumor cells induces an anti-A response whereas the B antigen fails to induce a cytolytic anti-B response even when B variant tumor cells are added to the same inoculum. However, when mice were primed with the parental AB cancer cells and the B variant at separate subcutaneous sites, an anti-B response was induced. Thus, an immunodominant response can prevent induction of CTL responses to immunorecessive antigens when it occurs at the same site. As would then be expected, mice exposed to parental AB cells as well as variant B cells in the same inoculum failed to reject the variant cancer cells. As would be expected further, the B variant cancer cells, when
Conclusions A single cancer cell expresses multiple independent target antigens and an immune response to these antigens can eradicate cancer completely. However, immunodominance favors escape by several mechanisms. First, immunodominance of a single dominant antigen is likely to favor the sequential selection of antigen loss variants during tumor progression. Secondly, the priority of the first response is likely to prevent the recognition of new antigens that may arise due to the mutational changes that cause tumor progression. Third, when the immunodominant antigen is retained, possibly because it is essential for maintaining cancer cell survival, but the presenting MHC Class I molecule is lost, cross-presentation of the retained antigen may propagate the immunodominant response. This traps the host to mount a futile response to an antigen that no longer serves as a target on the cancer cell and prevents the host from mounting a response to secondary antigens retained by the variant. The detrimental effects of immunodominance can be prevented by immunizations with in vitro-selected variants that lack the immunodominant antigens. Such immunizations induce CD8+ T cell responses to secondary antigens and prevent immune escape. It still needs to be established whether tumor antigen 29
H. Schreiber et al.
loaded dendritic cells can break immunodominance and prevent immune escape.
12. Doherty PC, Biddison WE, Bennink JR, Knowles BB (1978) Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype. J Exp Med 148:534–543 13. Wettstein PJ, Bailey DW (1982) Immunodominance in the immune response to “multiple” histocompatibility antigens. Immunogenetics 16:47–58 14. Lee W, Cosenza H, Kohler H (1974) Clonal restriction of the immune response to phosphorylcholine. Nature 247:55–57 15. Claflin JL, Davie JM (1974) Clonal nature of the immune response to phosphorylcholine. IV. Idiotypic uniformity of binding site-associated antigenic determinants among mouse antiphosphorylcholine antibodies. J Exp Med 140:673–686 16. Seung S, Urban JL, Schreiber H (1993) DNA sequence analysis of T-cell receptor genes reveals an oligoclonal T-cell response to a tumor with multiple target antigens. Cancer Res 53:840–845 17. Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H (1997) The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68. J Exp Med 185:695–705 18. Sandberg JK, Grufman P, Wolpert EZ, Franksson L, Chambers BJ, Karre K (1998) Superdominance among immunodominant H-2Kb-restricted epitopes and reversal by dendritic cell-mediated antigen delivery. J Immunol 160:3163–3169 19. Urban JL, Kripke ML, Schreiber H (1986) Stepwise immunologic selection of antigenic variants during tumor growth. J Immunol 137:3036–3041 20. Mullen CA, Urban JL, Van Waes C, Rowley DA, Schreiber H (1985) Multiple cancers. Tumor burden permits the outgrowth of other cancers. J Exp Med 162:1665–1682 21. Mizoguchi H, O’Shea JJ, Longo DL, Loeffler CM, McVicar DW, Ochoa AC (1992) Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795–1798 22. Singh S, Ross SR, Acena M, Rowley DA, Schreiber H (1992) Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells. J Exp Med 175:139–146 23. Wick M, Dubey P, Koeppen H, Siegel CT, Fields PE, Fitch FW, Chen L, Bluestone JA, Schreiber H (1997) Antigenic cancer cells can grow progressively in immune hosts without evidence for T cell exhaustion or systemic anergy. J Exp Med 186:229–237 24. Nowell PC (1976) The clonal evolution of tumor cell populations. Science 194:23–29 25. Klein G, Klein E (1985) Evolution of tumours and the impact of molecular oncology. Nature 315:190–195 26. Rowley DA, Stach RM (1993) A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens. J Exp Med 178:835–840 27. Ruiz-Cabello F, Garrido F (1998) HLA and cancer: from research to clinical impact. Immunol Today 19:539–542 28. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S (2000) Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv Immunol 74:181–273 29. Seung S, Urban JL, Schreiber H (1993) A tumor escape variant that has lost one major histocompatibility complex class I restriction element induces specific CD8+ T cells to an antigen that no longer serves as a target. J Exp Med 178:933–940
Acknowledgements This work was supported by National Institutes of Health grants PO1-CA74182, RO1-CA-37516, RO1.CA/AI 78399 and RO1-CA-22677 and by University of Chicago Cancer Center Core grant CA-14599. We thank Dr Donald A. Rowley for review of the manuscript. The authors also gratefully acknowledge support by a gift from the Passis family.
References 1. Wortzel RD, Philipps C, Schreiber H (1983) Multiple tumourspecific antigens expressed on a single tumour cell. Nature 304:165–167 2. Monach PA, Meredith SC, Siegel CT, Schreiber H (1995) A unique tumor antigen produced by a single amino acid substitution. Immunity 2:45–59 3. Coulie PG, Lehmann F, Lethe B, Herman J, Lurquin C, Andrawiss M, Boon T (1995) A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA 92:7976–7980 4. Wölfel T, Hauer M, Schneider J, Serrano M, Wölfel C, Klehmann-Hieb E, De Plaen E, Hankeln T, Meyer zum Buschenfelde KH, Beach D (1995) A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269:1281–1284 5. Robbins PF, El-Gamil M, Li YF, Kawakami Y, Loftus D, Appella E, Rosenberg SA (1996) A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes. J Exp Med 183:1185–1192 6. Mandruzzato S, Brasseur F, Andry G, Boon T, van der Bruggen P (1997) A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma. J Exp Med 186:785–793 7. Wortzel RD, Urban JL, Philipps C, Fitch FW, Schreiber H (1983) Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones. J Immunol 130:2461–2466 8. Wortzel RD, Urban JL, Schreiber H (1984) Malignant growth in the normal host after variant selection in vitro with cytolytic T-cell lines. Proc Natl Acad Sci USA 81:2186–2190 9. Urban JL, Van Waes C, Schreiber H (1984) Pecking order among tumor-specific antigens. Eur J Immunol 14:181–187 10. Van Waes C, Monach PA, Urban JL, Wortzel RD, Schreiber H (1996) Immunodominance deters the response to other tumor antigens thereby favoring escape: prevention by vaccination with tumor variants selected with cloned cytolytic T cells in vitro. Tissue Antigens 47:399–407 11. Zinkernagel RM, Althage A, Cooper S, Kreeb G, Klein PA, Sefton B, Flaherty L, Stimpfling J, Shreffler D, Klein J (1978) Ir-genes in H-2 regulate generation of anti-viral cytotoxic T cells. Mapping to K or D and dominance of unresponsiveness. J Exp Med 148:592–606
30
Immunodominance and tumor escape
30. Zitvogel L, Mayordomo JI, Tjandrawan T, DeLeo AB, Clarke MR, Lotze MT, Storkus WJ (1996) Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Med 183:87–97 31. Fields RC, Shimizu K, Mule JJ (1998) Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc Natl Acad Sci USA 95:9482–9487 32. Ranieri E, Kierstead LS, Zarour H, Kirkwood JM, Lotze MT, Whiteside T, Storkus WJ (2000) Dendritic cell/peptide cancer
vaccines: clinical responsiveness and epitope spreading. Immunol Invest 29:121–125 33. Melief CJ, Kast WM (1994) Prospects for T cell immunotherapy of tumours by vaccination with immunodominant and subdominant peptides. Ciba Found Symp 187:97–104 34. Feltkamp MC, Vreugdenhil GR, Vierboom MP, Ras E, van der Burg SH, ter Schegget J, Melief CJ, Kast WM (1995) Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors. Eur J Immunol 25:2638–2642
31
Immunodominance and tumor escape H. Schreiber a,∗ , T. H. Wu a , J. Nachman b and W. M. Kast c
are required to cause a fully malignant phenotype. We 2 and subsequently others 3–6 have shown that unique antigens recognized by the host are indeed tumor specific because they represent somatic tumorspecific mutations. It still may appear surprising that there are so many TSA because the chance that a point mutation will also generate a mutant peptide that acts as a major antigen is relatively small. Presumably, the number of point mutations present in a single cancer cell is so large that multiple TSA exist. Selection of antigen loss variants using T cell clones showed that the loss of one antigen did not affect the expression of the remaining antigens. 7 Further experiments showed that the frequency of loss of an individual antigen could be as high as 1 in 104 cloned cancer cells 8 Despite this high frequency, tumor cells failed to survive in vitro when exposed to a combination of cytolytic T cell clones directed against two or more independent antigens. Simultaneous attack against multiple independent TSA does not occur in vivo, presumably because responses are limited to a single or very few epitopes on malignant cells, a phenomenon referred to as immunodominance. 9,10
Cancers in mouse and man express multiple tumor-specific as well as tumor-associated antigens. Immunodominance in the host response to these antigens can result in successive selection of heritable antigen loss variants. Immunodominance may also prevent the development of responses to new tumor-specific antigens that may arise during tumor progression. Some tumor-specific antigens are retained during tumor progression possibly because they are essential for survival of the malignant phenotype. Immunodominance may allow cancer cells to escape even after loss of a single MHC Class I allele because cross-presentation of the retained antigen by this allele that must be expressed on the surrounding antigen presenting cells sustains the immunodominant response. This prevents effective responses to secondary antigens that may remain as potential targets. Immunization with in vitro selected cancer cell variants that lack the immunodominant antigen can break the immunodominance and prevent escape of cancers from host immunity. Key words: CD8+ T cells / cross-presentation / MHC Class I loss / multiplicity / Tumor-specific antigens / variants c 2002 Academic Press
Immunodominance
Multiplicity of tumor-specific antigens expressed on a single cancer cell
Immunodominance was first shown for viruses expressing multiple epitopes. 11,12 Shortly thereafter, remarkable immunodominance was observed in the response of mice to minor histocompatibility antigens; 13 mice challenged with more than 40 minor histocompatibility antigens responded to a single or very few antigens, which is observed for tumor antigens as well. 7 One important factor in determining whether an antigen becomes immunodominant over others seems to be the rapidity by which it induces a response in relation to other antigens. Thus, immunodominance or ‘priority’ is given to the ‘first’ response (see below). As long as the parental tumor cells expressing
Multiple TSA are expressed on single murine and human cancer cells. 1 It is now generally accepted that multiple different, somatic, tumor-specific mutations
From the a Department of Pathology and b the Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA and c The Cancer Immunology Program, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL 60153, USA. *Corresponding author. Department of Pathology, The University of Chicago, 5841 S. Maryland Ave., MC 3008, Chicago, IL 60637. E-mail: [email protected] c 2002 Academic Press
1044–579X / 02 / 010025+ 07 / $35.00 / 0
25
H. Schreiber et al.
Figure 1. Upper panel: Immunodominance and priority of the first response in response to a dominant (A) and recessive (B) TSA. An immunodominant antigen A prevents the response to an immuno-recessive antigen B when injected at the same site or when present on the same cell as B. B alone is capable of inducing a response, however this response is slower and lower initially. When the dominant and recessive antigens are injected at separate site, both responses are induced. These findings predict the escape of B variants when inoculated with immunodominant, parental A containing cells at the same injection site, and the rejection of B variants when inoculated at a separate site as observed experimentally (see Figure 2). Lower panel: Exposure to one antigen before the other determines which one will become immunodominant. This finding of a ‘priority of the first response’ is not only relevant to the failure of the host to respond to a new TSA appearing subsequently as a result of tumor progression but is also relevant to explaining the recessiveness of an antigen coexpressed on the original cancer cell but capable of inducing only a slower response (such is observed for the response to the B antigen in the upper panel).
the dominant antigen are present at the same site of tumor growth, immunodominance protects tumor variants that have lost the immunodominant antigen by forcing immune attack on the dominant antigen on the parental tumor and away from the immunorecessive antigen on the variant. The problems associated with local immunodominance can be modeled with parental tumors and their antigen loss variants (Figure 1, upper panel). These tumor cell lines can also be used to demonstrate the effect of local immunodominance in immune
escape (Figure 2). Immunodominant T and B cell responses against a particular antigen or epitope are commonly multiclonal; though the receptors are not identical, they recognize the same epitope. Virtually monoclonal antibody responses to a complex antigen are sometimes observed, e.g. the antibody response of BALB/c mice to phosphorylcholine of the rough pneumococcal bacterium R36A, 14 but such responses are the exception rather than the rule. 15 Cytolytic T cell responses against unique TSA are usually focussed on a single immunodominant 26
Immunodominance and tumor escape
inoculum. Similarly, new hierarchies emerge when an immunodominant antigen is lost following immune selection but multiple other antigens remain on the tumor cell. This process of reestablishment of new hierarchies continues with each additional loss of a TSA leading to outgrowth of new antigen loss variants. Thus, there is a ‘pecking order’ in the response to multiple TSA expressed by a single malignant cell 9 and a stepwise immunological selection of antigenic variants during tumor growth. 19 Unfortunately, by the time the host responds effectively to the next immunodominant antigen, too many new variant cancer cells have grown that have already lost this antigen. Therefore, these new variants survive the next round of killing. Theoretically, the same scenario of events should repeat, so that cancer cell with progressively fewer target antigens would emerge. However, hosts bearing the escape tumors often fail to mount an immune response to antigens remaining on the variants because of either a systemic tumor-induced T cell defect, 20,21 a local, possibly stromal, barrier 22,23 in the established tumor, or a priority of the first response, as discussed below.
Figure 2. Immune escape due to local immunodominance: the less malignant parental AB tumor ‘helps’ the escape of the more malignant antigen loss progressor variant B which kills the host (upper panel). The effect is due to local immunodominance of the A antigen preventing an anti B T cell response until it is too late and the variant tumor is well established (see Figure 1). Such problem does not arise in the experimental control setting (lower panel) when parental and variant tumor cells are injected at different sites. In real life, however, the variant evolves from the parental tumor at the same site (upper panel).
epitope/peptide but involve multiple different TCR clonotypes. 16 Occasionally, however, a single or a restricted number of Vβ regions are used by the responding T cells. 16 Such restrictions have then been used in cancer models to identify immunodominant TSA T cell responses. Another indicator of immunodominance is the selective T cell reactivity to one of multiple independent antigens contained in the immunizing tumor cell inoculum. 7 Such reactivity can be proven by demonstrating selective lysis by the responding T cells of a cancer cell variant that expresses only this antigen and lacks other T cellrecognized antigens. 7 Finally, with the identification of the genetic origins of some of the tumor-specific T cell recognized peptides, immunodominance can also be demonstrated in the response to molecularly defined mutant peptide epitopes. 17
‘Priority of the first response’: antigens inducing a faster response become immunodominant over antigens appearing later and/or inducing a slower response In theory, with tumor progression 24,25 tumor cells will acquire more and more ‘mutations’ (translocations, point mutations, etc) and express more and more epitopes which could be antigenic and probably immunogenic if the response to them was not down regulated. Importantly it has been found that an existent response to an antigen already present on the tumor cell will prevent a response to these newly arising antigens, even though such second antigens may be stronger than the original antigen and could become an important target if an effective response to these antigens could be induced. The ‘priority of the first response’ is suggested by experiments in mice: when mice are immunized repeatedly with an antigen A, later addition of an antigen B to the immunogen may not elicit any anti-B CD8+ T cell response in vivo (Figure 1, lower panel). Vice versa, repeated immunization with B will prevent a CD8+ T cell response to antigen A if A is added later to the immunogen. 10,26
‘Pecking order’ and sequential antigen loss during escape It has been reported that if five immunodominant epitopes all having the same MHC Class I restriction element were mixed as antigen, a new pattern of dominance of only two of the epitopes was established 18 Thus, novel hierarchies emerge when multiple dominant epitopes are given in a single 27
H. Schreiber et al.
Figure 3. Loss of one of three MHC Class I alleles is sufficient to prevent responses of the host to tumor antigens presented by the two other alleles. Cross-presentation of the A antigen despite loss of the presenting MHC-Class I allele from the tumor leads to continued induction of the immunodominant anti-A response by the host. 29 This persistent response can no longer kill the escape variant and prevents the elimination of the variant by killer T cells that recognize the remaining potential antigenic targets.
Cross-presentation and single allelic MHC class I allele loss: escape due to persistent immunodominance
stimulate the immunodominant response. The UVinduced cancer 6130 may be an example of such a cancer. We have found that the 6130 progressor variant lost the MHC Class I K allele of the k haplotype while retaining the D allele. 29 Despite the Kk loss, the variant cell cancer continued to induce a CD8+ tumor-specific T cell response to this antigen; however the antigen no longer is a target with variant cancer cells though the antigen is crosspresented by APC of the host. Thus, the host is locked into a futile immunodominant response that prevents effective new responses to other antigens that may be expressed (Figure 3). At present, we do not know whether the common occurrence of a single MHC Class I allelic loss results from a
In common human malignancies, complete MHC Class I antigen loss occurs in a small but significant fraction (approximately 10% of cancers), while loss of expression of a single MHC Class I allele is observed in the large majority (>80%) of certain human malignancies. 27,28 It is certainly tempting to suggest that: (i) an MHC class I allele was lost because it presented an essential immunodominant target antigen and (ii) cross-presentation of this antigen by surrounding dendritic cells or other APC occurs that must express the presenting allele continues to 28
Immunodominance and tumor escape
injected at a subcutaneous site separate from the immunodominant antigen, were effectively rejected. Further experiments showed that immunization with variants (that retained the immunorecessive antigen but lost the immunodominant antigen) at separate sites prevented the outgrowth of antigen loss escape variants from the tumor inoculum that contained a mixture of less malignant parental AB cells and more malignant escape variant B cells. 10 Vaccination with the parental tumor cells was ineffective. Similarly, vaccination with a recessive peptide antigen or another form of a defined antigen can prevent the outgrowth of cancer cells that express dominant antigens not used for immunization. For example, it has been shown that immunization with the E7 RAHYNIVTF peptide antigen can prevent the outgrowth of HPV16 transformed tumor cells even though the antigen is not immunodominant on these cancer cells. 33,34 Thus, vaccination with individual tumor antigens at separate sites rather than with multiple antigens at one site may be needed to prevent tumor escape and recurrence of cancer.
cytolytic T cell response to immunodominant antigen restricted by the allele lost by the cancer cell, but if this is indeed the case, then loss of a single MHC Class I allele restricting an immunodominant antigen may be a common mechanism for cancers to escape immune destruction.
Vaccination with in vitro-selected variants or immunorecessive antigens may prevent tumor escape Recently, it has been proposed that immunodominance in response to a mixture of multiple defined synthetic peptides can be broken by immunizing the host with dendritic cells loaded with these peptides. 18 The mixture of peptides was used for priming phase, whereas spleen cells loaded with the individual peptides were used as stimulators for the generation of CTL in vitro. It is still unclear whether dendritic cell vaccination can prevent or reverse immunodominance during the course of an ongoing immune stimulation by complex antigens such as tumor cells which express multiple epitopes. This approach is particularly attractive because DC loaded with multiple antigens from tumor cells could be used without having to know the identity of the individual antigens 30–32 and without having to isolate cancer cell variants expressing the immunorecessive antigens for immunization, as outlined below. 10 Therefore, it will be important to show that vaccination using DC can break immunodominance and prevent tumor escape. Immunodominance in the response to multiple tumor antigens expressed on a single cell can be broken by inducing a cytolytic T cell response with variants lacking the immunodominant antigen. 10 For example, Figure 1 (upper panel) shows that when antigen A is dominant over antigen B, immunization with parental AB tumor cells induces an anti-A response whereas the B antigen fails to induce a cytolytic anti-B response even when B variant tumor cells are added to the same inoculum. However, when mice were primed with the parental AB cancer cells and the B variant at separate subcutaneous sites, an anti-B response was induced. Thus, an immunodominant response can prevent induction of CTL responses to immunorecessive antigens when it occurs at the same site. As would then be expected, mice exposed to parental AB cells as well as variant B cells in the same inoculum failed to reject the variant cancer cells. As would be expected further, the B variant cancer cells, when
Conclusions A single cancer cell expresses multiple independent target antigens and an immune response to these antigens can eradicate cancer completely. However, immunodominance favors escape by several mechanisms. First, immunodominance of a single dominant antigen is likely to favor the sequential selection of antigen loss variants during tumor progression. Secondly, the priority of the first response is likely to prevent the recognition of new antigens that may arise due to the mutational changes that cause tumor progression. Third, when the immunodominant antigen is retained, possibly because it is essential for maintaining cancer cell survival, but the presenting MHC Class I molecule is lost, cross-presentation of the retained antigen may propagate the immunodominant response. This traps the host to mount a futile response to an antigen that no longer serves as a target on the cancer cell and prevents the host from mounting a response to secondary antigens retained by the variant. The detrimental effects of immunodominance can be prevented by immunizations with in vitro-selected variants that lack the immunodominant antigens. Such immunizations induce CD8+ T cell responses to secondary antigens and prevent immune escape. It still needs to be established whether tumor antigen 29
H. Schreiber et al.
loaded dendritic cells can break immunodominance and prevent immune escape.
12. Doherty PC, Biddison WE, Bennink JR, Knowles BB (1978) Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype. J Exp Med 148:534–543 13. Wettstein PJ, Bailey DW (1982) Immunodominance in the immune response to “multiple” histocompatibility antigens. Immunogenetics 16:47–58 14. Lee W, Cosenza H, Kohler H (1974) Clonal restriction of the immune response to phosphorylcholine. Nature 247:55–57 15. Claflin JL, Davie JM (1974) Clonal nature of the immune response to phosphorylcholine. IV. Idiotypic uniformity of binding site-associated antigenic determinants among mouse antiphosphorylcholine antibodies. J Exp Med 140:673–686 16. Seung S, Urban JL, Schreiber H (1993) DNA sequence analysis of T-cell receptor genes reveals an oligoclonal T-cell response to a tumor with multiple target antigens. Cancer Res 53:840–845 17. Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H (1997) The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68. J Exp Med 185:695–705 18. Sandberg JK, Grufman P, Wolpert EZ, Franksson L, Chambers BJ, Karre K (1998) Superdominance among immunodominant H-2Kb-restricted epitopes and reversal by dendritic cell-mediated antigen delivery. J Immunol 160:3163–3169 19. Urban JL, Kripke ML, Schreiber H (1986) Stepwise immunologic selection of antigenic variants during tumor growth. J Immunol 137:3036–3041 20. Mullen CA, Urban JL, Van Waes C, Rowley DA, Schreiber H (1985) Multiple cancers. Tumor burden permits the outgrowth of other cancers. J Exp Med 162:1665–1682 21. Mizoguchi H, O’Shea JJ, Longo DL, Loeffler CM, McVicar DW, Ochoa AC (1992) Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795–1798 22. Singh S, Ross SR, Acena M, Rowley DA, Schreiber H (1992) Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells. J Exp Med 175:139–146 23. Wick M, Dubey P, Koeppen H, Siegel CT, Fields PE, Fitch FW, Chen L, Bluestone JA, Schreiber H (1997) Antigenic cancer cells can grow progressively in immune hosts without evidence for T cell exhaustion or systemic anergy. J Exp Med 186:229–237 24. Nowell PC (1976) The clonal evolution of tumor cell populations. Science 194:23–29 25. Klein G, Klein E (1985) Evolution of tumours and the impact of molecular oncology. Nature 315:190–195 26. Rowley DA, Stach RM (1993) A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens. J Exp Med 178:835–840 27. Ruiz-Cabello F, Garrido F (1998) HLA and cancer: from research to clinical impact. Immunol Today 19:539–542 28. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S (2000) Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv Immunol 74:181–273 29. Seung S, Urban JL, Schreiber H (1993) A tumor escape variant that has lost one major histocompatibility complex class I restriction element induces specific CD8+ T cells to an antigen that no longer serves as a target. J Exp Med 178:933–940
Acknowledgements This work was supported by National Institutes of Health grants PO1-CA74182, RO1-CA-37516, RO1.CA/AI 78399 and RO1-CA-22677 and by University of Chicago Cancer Center Core grant CA-14599. We thank Dr Donald A. Rowley for review of the manuscript. The authors also gratefully acknowledge support by a gift from the Passis family.
References 1. Wortzel RD, Philipps C, Schreiber H (1983) Multiple tumourspecific antigens expressed on a single tumour cell. Nature 304:165–167 2. Monach PA, Meredith SC, Siegel CT, Schreiber H (1995) A unique tumor antigen produced by a single amino acid substitution. Immunity 2:45–59 3. Coulie PG, Lehmann F, Lethe B, Herman J, Lurquin C, Andrawiss M, Boon T (1995) A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA 92:7976–7980 4. Wölfel T, Hauer M, Schneider J, Serrano M, Wölfel C, Klehmann-Hieb E, De Plaen E, Hankeln T, Meyer zum Buschenfelde KH, Beach D (1995) A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269:1281–1284 5. Robbins PF, El-Gamil M, Li YF, Kawakami Y, Loftus D, Appella E, Rosenberg SA (1996) A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes. J Exp Med 183:1185–1192 6. Mandruzzato S, Brasseur F, Andry G, Boon T, van der Bruggen P (1997) A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma. J Exp Med 186:785–793 7. Wortzel RD, Urban JL, Philipps C, Fitch FW, Schreiber H (1983) Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones. J Immunol 130:2461–2466 8. Wortzel RD, Urban JL, Schreiber H (1984) Malignant growth in the normal host after variant selection in vitro with cytolytic T-cell lines. Proc Natl Acad Sci USA 81:2186–2190 9. Urban JL, Van Waes C, Schreiber H (1984) Pecking order among tumor-specific antigens. Eur J Immunol 14:181–187 10. Van Waes C, Monach PA, Urban JL, Wortzel RD, Schreiber H (1996) Immunodominance deters the response to other tumor antigens thereby favoring escape: prevention by vaccination with tumor variants selected with cloned cytolytic T cells in vitro. Tissue Antigens 47:399–407 11. Zinkernagel RM, Althage A, Cooper S, Kreeb G, Klein PA, Sefton B, Flaherty L, Stimpfling J, Shreffler D, Klein J (1978) Ir-genes in H-2 regulate generation of anti-viral cytotoxic T cells. Mapping to K or D and dominance of unresponsiveness. J Exp Med 148:592–606
30
Immunodominance and tumor escape
30. Zitvogel L, Mayordomo JI, Tjandrawan T, DeLeo AB, Clarke MR, Lotze MT, Storkus WJ (1996) Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Med 183:87–97 31. Fields RC, Shimizu K, Mule JJ (1998) Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc Natl Acad Sci USA 95:9482–9487 32. Ranieri E, Kierstead LS, Zarour H, Kirkwood JM, Lotze MT, Whiteside T, Storkus WJ (2000) Dendritic cell/peptide cancer
vaccines: clinical responsiveness and epitope spreading. Immunol Invest 29:121–125 33. Melief CJ, Kast WM (1994) Prospects for T cell immunotherapy of tumours by vaccination with immunodominant and subdominant peptides. Ciba Found Symp 187:97–104 34. Feltkamp MC, Vreugdenhil GR, Vierboom MP, Ras E, van der Burg SH, ter Schegget J, Melief CJ, Kast WM (1995) Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors. Eur J Immunol 25:2638–2642
31