Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy

The immune system recognizes and is poised to eliminate cancer but is held in check by inhibitory receptors and ligands. These immune checkpoint pathways, which normally maintain self-tolerance and limit collateral tissue damage during anti-microbial immune responses, can be co-opted by cancer to evade immune destruction. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, and others in early development, can unleash anti-tumor immunity and mediate durable cancer regressions. The complex biology of immune checkpoint pathways still contains many mysteries, and the full activity spectrum of checkpoint-blocking drugs, used alone or in combination, is currently the subject of intense study. The immune system recognizes and is poised to eliminate cancer but is held in check by inhibitory receptors and ligands. These immune checkpoint pathways, which normally maintain self-tolerance and limit collateral tissue damage during anti-microbial immune responses, can be co-opted by cancer to evade immune destruction. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, and others in early development, can unleash anti-tumor immunity and mediate durable cancer regressions. The complex biology of immune checkpoint pathways still contains many mysteries, and the full activity spectrum of checkpoint-blocking drugs, used alone or in combination, is currently the subject of intense study. In the current era in oncology emphasizing personalized therapy, immune checkpoint blockade is distinguished by its “common denominator” approach. Although the vast somatic mutational diversity found in most human cancers creates challenges for therapies targeting individual mutations, it exposes a panoply of new antigens for potential immune recognition. However, cells of the adaptive and innate immune systems that recognize and are poised to attack cancer are held in check by molecular pathways that suppress their activation and effector functions. The seminal observation that blocking the prototypical immune checkpoint receptor cytotoxic T lymphocyte antigen 4 (CTLA-4) could mediate tumor regression in murine models (Leach et al., 1996Leach D.R. Krummel M.F. Allison J.P. Enhancement of antitumor immunity by CTLA-4 blockade.Science. 1996; 271: 1734-1736Crossref PubMed Google Scholar) led to the clinical development and approval of anti-CTLA-4 as a treatment for patients with advanced melanoma (Hodi et al., 2010Hodi F.S. O’Day S.J. McDermott D.F. Weber R.W. Sosman J.A. Haanen J.B. Gonzalez R. Robert C. Schadendorf D. Hassel J.C. et al.Improved survival with ipilimumab in patients with metastatic melanoma.N. Engl. J. Med. 2010; 363: 711-723Crossref PubMed Scopus (3660) Google Scholar). Subsequently, drugs blocking the distinct checkpoints Programmed Death 1 (PD-1) and its major ligand PD-L1 have shown great promise in treating many diverse cancer types, fueling the intensive examination of a growing cohort of unique checkpoint molecules as potential therapeutic targets. This has revealed new treatment options for patients and has revolutionized our approach to cancer therapy. The rapid-fire clinical successes from blocking CTLA-4 and PD-1, the first checkpoint receptors to be discovered, have opened prospects for extending the potential of cancer immunotherapy by inhibiting more recently discovered checkpoint ligands and receptors. It is clear that, despite some commonalities, CTLA-4 and PD-1 have distinct patterns of expression, signaling pathways, and mechanisms of action. Although discovered over 20 years ago, there are still many unanswered questions about their biology, particularly in the context of cancer. The conventional wisdom underlying our vision of how CTLA-4 blockade mediates tumor regression is that it systemically activates T cells that are encountering antigens. CTLA-4 represents the paradigm for regulatory feedback inhibition. Its engagement down-modulates the amplitude of T cell responses, largely by inhibiting co-stimulation by CD28, with which it shares the ligands CD80 (B7.1) and CD86 (B7.2) (Figure 1; Lenschow et al., 1996Lenschow D.J. Walunas T.L. Bluestone J.A. CD28/B7 system of T cell costimulation.Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2009) Google Scholar). As a “master T cell co-stimulator,” CD28 engagement amplifies TCR signaling when the T cell receptor (TCR) is also engaged by cognate peptide-major histocompatibility complex (MHC) (Schwartz, 1992Schwartz R.H. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy.Cell. 1992; 71: 1065-1068Abstract Full Text PDF PubMed Scopus (935) Google Scholar). However, CTLA-4 has a much higher affinity for both CD80 and CD86 compared with CD28 (Linsley et al., 1994Linsley P.S. Greene J.L. Brady W. Bajorath J. Ledbetter J.A. Peach R. Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors.Immunity. 1994; 1: 793-801Abstract Full Text PDF PubMed Scopus (513) Google Scholar), so its expression on activated T cells dampens CD28 co-stimulation by out-competing CD28 binding and, possibly, also via depletion of CD80 and CD86 via “trans-endocytosis” (Qureshi et al., 2011Qureshi O.S. Zheng Y. Nakamura K. Attridge K. Manzotti C. Schmidt E.M. Baker J. Jeffery L.E. Kaur S. Briggs Z. et al.Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4.Science. 2011; 332: 600-603Crossref PubMed Scopus (276) Google Scholar). Because CD80 and CD86 are expressed on antigen-presenting cells (APCs; e.g., dendritic cells and monocytes) but not on non-hematologic tumor cells, CTLA-4’s suppression of anti-tumor immunity has been viewed to reside primarily in secondary lymphoid organs where T cell activation occurs rather than within the tumor microenvironment (TME). Furthermore, CTLA-4 is predominantly expressed on CD4+ “helper” and not CD8+ “killer” T cells. Therefore, heightened CD8 responses in anti-CTLA-4-treated patients likely occur indirectly through increased activation of CD4+ cells. Of note, a few studies suggest that CTLA-4 can act as a direct inhibitory receptor of CD8 T cells (Fallarino et al., 1998Fallarino F. Fields P.E. Gajewski T.F. B7-1 engagement of cytotoxic T lymphocyte antigen 4 inhibits T cell activation in the absence of CD28.J. Exp. Med. 1998; 188: 205-210Crossref PubMed Scopus (109) Google Scholar, Chambers et al., 1998Chambers C.A. Sullivan T.J. Truong T. Allison J.P. Secondary but not primary T cell responses are enhanced in CTLA-4-deficient CD8+ T cells.Eur. J. Immunol. 1998; 28: 3137-3143Crossref PubMed Scopus (90) Google Scholar), although this role in down-modulating anti-tumor CD8 T cell responses remains to be directly demonstrated. The specific signaling pathways by which CTLA-4 inhibits T cell activation are still under investigation, although activation of the phosphatases SHP2 and PP2A appears to be important in counteracting both tyrosine and serine/threonine kinase signals induced by TCR and CD28 (Rudd et al., 2009Rudd C.E. Taylor A. Schneider H. CD28 and CTLA-4 coreceptor expression and signal transduction.Immunol. Rev. 2009; 229: 12-26Crossref PubMed Scopus (236) Google Scholar). CTLA-4 engagement also interferes with the “TCR stop signal,” which maintains the immunological synapse long enough for extended or serial interactions between TCR and its peptide-MHC ligand (Schneider et al., 2006Schneider H. Downey J. Smith A. Zinselmeyer B.H. Rush C. Brewer J.M. Wei B. Hogg N. Garside P. Rudd C.E. Reversal of the TCR stop signal by CTLA-4.Science. 2006; 313: 1972-1975Crossref PubMed Scopus (281) Google Scholar). Naive and resting memory T cells express CD28, but not CTLA-4, on the cell surface, allowing co-stimulation to dominate upon antigen recognition. However, CTLA-4 is rapidly mobilized to the cell surface from intracellular protein stores, allowing feedback inhibition to occur within an hour after antigen engagement. The central role of CTLA-4 in maintaining immune tolerance is dramatically demonstrated by the rapidly lethal systemic immune hyperactivation phenotype of Ctla-4 knockout mice (Tivol et al., 1995Tivol E.A. Borriello F. Schweitzer A.N. Lynch W.P. Bluestone J.A. Sharpe A.H. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4.Immunity. 1995; 3: 541-547Abstract Full Text PDF PubMed Scopus (1538) Google Scholar, Waterhouse et al., 1995Waterhouse P. Penninger J.M. Timms E. Wakeham A. Shahinian A. Lee K.P. Thompson C.B. Griesser H. Mak T.W. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4.Science. 1995; 270: 985-988Crossref PubMed Google Scholar). In humans, anti-CTLA-4 treatment induces expression of activation markers on circulating T cells (Maker et al., 2005Maker A.V. Attia P. Rosenberg S.A. Analysis of the cellular mechanism of antitumor responses and autoimmunity in patients treated with CTLA-4 blockade.J. Immunol. 2005; 175: 7746-7754Crossref PubMed Google Scholar) and a high rate of inflammatory side effects (Phan et al., 2003Phan G.Q. Yang J.C. Sherry R.M. Hwu P. Topalian S.L. Schwartzentruber D.J. Restifo N.P. Haworth L.R. Seipp C.A. Freezer L.J. et al.Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.Proc. Natl. Acad. Sci. USA. 2003; 100: 8372-8377Crossref PubMed Scopus (983) Google Scholar). However, because melanoma patients appear to possess an unusually high proportion of tumor-reactive T cells, anti-tumor responses balance autoimmune toxicity and provide a clinical benefit to roughly 20% of patients with this disease (see below). The PD-1 system of immune modulation bears similarities to CTLA-4 as well as key distinctions (Parry et al., 2005Parry R.V. Chemnitz J.M. Frauwirth K.A. Lanfranco A.R. Braunstein I. Kobayashi S.V. Linsley P.S. Thompson C.B. Riley J.L. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms.Mol. Cell. Biol. 2005; 25: 9543-9553Crossref PubMed Scopus (378) Google Scholar). Similar to CTLA-4, PD-1 is absent on resting naive and memory T cells and is expressed upon TCR engagement. However, in contrast to CTLA-4, PD-1 expression on the surface of activated T cells requires transcriptional activation and is therefore delayed (6–12 hr). Also in contrast to CTLA-4, PD-1 contains a conventional immunoreceptor tyrosine inhibitory motif (ITIM) as well as an immunoreceptor tyrosine switch motif (ITSM). PD-1’s ITIM and ITSM bind the inhibitory phosphatase SHP-2. PD-1 engagement can also activate the inhibitory phosphatase PP2A. PD-1 engagement directly inhibits TCR-mediated effector functions and increases T cell migration within tissues, thereby limiting the time that a T cell has to survey the surface of interacting cells for the presence of cognate peptide-MHC complexes. Therefore, T cells may “pass over” target cells expressing lower levels of peptide-MHC complexes (Honda et al., 2014Honda T. Egen J.G. Lämmermann T. Kastenmüller W. Torabi-Parizi P. Germain R.N. Tuning of antigen sensitivity by T cell receptor-dependent negative feedback controls T cell effector function in inflamed tissues.Immunity. 2014; 40: 235-247Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In contrast to CTLA-4, PD-1 blockade is viewed to work predominantly within the TME, where its ligands are commonly overexpressed by tumor cells as well as infiltrating leukocytes (Keir et al., 2008Keir M.E. Butte M.J. Freeman G.J. Sharpe A.H. PD-1 and its ligands in tolerance and immunity.Annu. Rev. Immunol. 2008; 26: 677-704Crossref PubMed Scopus (1103) Google Scholar). This mechanism is thought to reflect its important physiologic role in restraining collateral tissue damage during T cell responses to infection. In addition, tumor-infiltrating lymphocytes (TILs) commonly express heightened levels of PD-1 and are thought to be “exhausted” because of chronic stimulation by tumor antigens, analogous to the exhausted phenotype seen in murine models of chronic viral infection, which is partially reversible by PD-1 pathway blockade (Barber et al., 2006Barber D.L. Wherry E.J. Masopust D. Zhu B. Allison J.P. Sharpe A.H. Freeman G.J. Ahmed R. Restoring function in exhausted CD8 T cells during chronic viral infection.Nature. 2006; 439: 682-687Crossref PubMed Scopus (1576) Google Scholar). Importantly, the phenotypes of murine knockouts of PD-1 and its two known ligands are very mild, consisting of late-onset organ-specific inflammation, particularly when crossed to autoimmune-prone mouse strains (Nishimura et al., 1999Nishimura H. Nose M. Hiai H. Minato N. Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor.Immunity. 1999; 11: 141-151Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, Nishimura et al., 2001Nishimura H. Okazaki T. Tanaka Y. Nakatani K. Hara M. Matsumori A. Sasayama S. Mizoguchi A. Hiai H. Minato N. Honjo T. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.Science. 2001; 291: 319-322Crossref PubMed Scopus (684) Google Scholar). This contrasts sharply with the Ctla-4 knockout phenotype and highlights the importance of the PD-1 pathway in restricting peripheral tissue inflammation. Furthermore, it is consistent with clinical observations that autoimmune side effects of anti-PD-1 drugs are generally milder and less frequent than with anti-CTLA-4. Despite the conventional wisdom that CTLA-4 acts early in T cell activation in secondary lymphoid tissues whereas PD-1 inhibits execution of effector T cell responses in tissue and tumors, this distinction is not absolute. Beyond its role in dampening activation of effector T cells, CTLA-4 plays a major role in driving the suppressive function of T regulatory (Treg) cells (Wing et al., 2008Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Miyara M. Fehervari Z. Nomura T. Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function.Science. 2008; 322: 271-275Crossref PubMed Scopus (931) Google Scholar, Peggs et al., 2009Peggs K.S. Quezada S.A. Chambers C.A. Korman A.J. Allison J.P. Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies.J. Exp. Med. 2009; 206: 1717-1725Crossref PubMed Scopus (261) Google Scholar). Tregs, which broadly inhibit effector T cell responses, are typically concentrated in tumor tissues and are thought to locally inhibit anti-tumor immunity. Therefore, CTLA-4 blockade may affect intratumoral immune responses by inactivating tumor-infiltrating Treg cells. Recent evidence has demonstrated anti-tumor effects from CTLA-4 blockade even when S1P inhibitors block lymphocyte egress from lymph nodes (Spranger et al., 2014Spranger S. Koblish H.K. Horton B. Scherle P.A. Newton R. Gajewski T.F. Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment.J. Immunother. Cancer. 2014; (Published online February 18, 2015)https://doi.org/10.1186/2051-1426-2-3Crossref Google Scholar), indicating that this checkpoint exerts at least some effects directly in the TME as opposed to secondary lymphoid tissues. Conversely, PD-1 has been shown to play a role in early fate decisions of T cells recognizing antigens presented in the lymph node. In particular, PD-1 engagement limits the initial “burst size” of T cells upon antigen exposure and can partially convert T cell tolerance induction to effector differentiation (Goldberg et al., 2007Goldberg M.V. Maris C.H. Hipkiss E.L. Flies A.S. Zhen L. Tuder R.M. Grosso J.F. Harris T.J. Getnet D. Whartenby K.A. et al.Role of PD-1 and its ligand, B7-H1, in early fate decisions of CD8 T cells.Blood. 2007; 110: 186-192Crossref PubMed Scopus (90) Google Scholar). The receptor-ligand interactions of the PD-1 system appear to be even more complex than the CD28/CTLA-4 system (Figure 1). The two ligands for PD-1 are PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), which share 37% sequence homology and arose via gene duplication (Dong et al., 1999Dong H. Zhu G. Tamada K. Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion.Nat. Med. 1999; 5: 1365-1369Crossref PubMed Scopus (884) Google Scholar, Latchman et al., 2001Latchman Y. Wood C.R. Chernova T. Chaudhary D. Borde M. Chernova I. Iwai Y. Long A.J. Brown J.A. Nunes R. et al.PD-L2 is a second ligand for PD-1 and inhibits T cell activation.Nat. Immunol. 2001; 2: 261-268Crossref PubMed Scopus (930) Google Scholar, Tseng et al., 2001Tseng S.Y. Otsuji M. Gorski K. Huang X. Slansky J.E. Pai S.I. Shalabi A. Shin T. Pardoll D.M. Tsuchiya H. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells.J. Exp. Med. 2001; 193: 839-846Crossref PubMed Scopus (457) Google Scholar). However, their regulation is highly divergent. PD-L1 is induced on activated hematopoietic cells and on epithelial cells by the inflammatory cytokine interferon (IFN)-γ, which is produced by some activated T and natural killer (NK) cells. PD-L2 has much more selective expression on activated dendritic cells and some macrophages. It is induced to a much greater extent by interleukin 4 (IL-4) than by IFN-γ, further emphasizing differences in regulation of expression of PD-L1 and PD-L2. Beyond their role as ligands for PD-1, PD-L1 and PD–L2 appear to have additional partners, indicating additional layers of immune modulation. An unexpected molecular interaction between PD-L1 and CD80 has been discovered (Butte et al., 2007Butte M.J. Keir M.E. Phamduy T.B. Sharpe A.H. Freeman G.J. Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses.Immunity. 2007; 27: 111-122Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, Park et al., 2010Park J.J. Omiya R. Matsumura Y. Sakoda Y. Kuramasu A. Augustine M.M. Yao S. Tsushima F. Narazaki H. Anand S. et al.B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance.Blood. 2010; 116: 1291-1298Crossref PubMed Scopus (81) Google Scholar) whereby CD80 expressed on activated T cells (and possibly APCs) can function as a receptor rather than a ligand, delivering inhibitory signals when engaged by PD-L1. The relevance of this interaction in tumor immune resistance has not yet been determined. Recently, PD-L2 has been shown to bind to repulsive guidance molecule b (RGMb), which itself binds at least three other molecules in cis (neogenin and BMP receptors type I and II) (Xiao et al., 2014Xiao Y. Yu S. Zhu B. Bedoret D. Bu X. Francisco L.M. Hua P. Duke-Cohan J.S. Umetsu D.T. Sharpe A.H. et al.RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance.J. Exp. Med. 2014; 211: 943-959Crossref PubMed Scopus (13) Google Scholar). This interaction appears to be inhibitory independent of PD-1, as demonstrated in a pulmonary tolerance model. Finally, evidence from murine models suggests that PD-L2 and, possibly, PD-L1 may bind to a co-stimulatory T cell receptor (Shin et al., 2003Shin T. Kennedy G. Gorski K. Tsuchiya H. Koseki H. Azuma M. Yagita H. Chen L. Powell J. Pardoll D. Housseau F. Cooperative B7-1/2 (CD80/CD86) and B7-DC costimulation of CD4+ T cells independent of the PD-1 receptor.J. Exp. Med. 2003; 198: 31-38Crossref PubMed Scopus (109) Google Scholar, Shin et al., 2005Shin T. Yoshimura K. Shin T. Crafton E.B. Tsuchiya H. Housseau F. Koseki H. Schulick R.D. Chen L. Pardoll D.M. In vivo costimulatory role of B7-DC in tuning T helper cell 1 and cytotoxic T lymphocyte responses.J. Exp. Med. 2005; 201: 1531-1541Crossref PubMed Scopus (93) Google Scholar), an arrangement reminiscent of the CD80/CD86 ligand pair for the co-stimulatory CD28 and co-inhibitory CTLA-4 receptors. Understanding the roles of these various interactions in cancer is highly relevant for the development of immunomodulatory drugs and the discovery of biomarkers predictive of therapeutic response. A key finding that encouraged the development of drugs blocking the PD-1 pathway for cancer immunotherapy was that PD-1 ligands are upregulated in many human cancers (Dong et al., 2002Dong H. Strome S.E. Salomao D.R. Tamura H. Hirano F. Flies D.B. Roche P.C. Lu J. Zhu G. Tamada K. et al.Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.Nat. Med. 2002; 8: 793-800Crossref PubMed Scopus (0) Google Scholar), whereas PD-1 is highly expressed on tumor-infiltrating lymphocytes (Ahmadzadeh et al., 2009Ahmadzadeh M. Johnson L.A. Heemskerk B. Wunderlich J.R. Dudley M.E. White D.E. Rosenberg S.A. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired.Blood. 2009; 114: 1537-1544Crossref PubMed Scopus (313) Google Scholar, Sfanos et al., 2009Sfanos K.S. Bruno T.C. Meeker A.K. De Marzo A.M. Isaacs W.B. Drake C.G. Human prostate-infiltrating CD8+ T lymphocytes are oligoclonal and PD-1+.Prostate. 2009; 69: 1694-1703Crossref PubMed Scopus (89) Google Scholar). Indeed, PD-L1 appears to be the major ligand expressed in solid tumors, whereas PD-L2 (together with PD-L1) is highly expressed in certain subsets of B cell lymphomas (Ansell et al., 2015Ansell S.M. Lesokhin A.M. Borrello I. Halwani A. Scott E.C. Gutierrez M. Schuster S.J. Millenson M.M. Cattry D. Freeman G.J. et al.PD-1 blockade with nivolumab in relapsed or refractory Hodgkin’s lymphoma.N. Engl. J. Med. 2015; 372: 311-319Crossref PubMed Scopus (32) Google Scholar). Exploration of this phenomenon as a central process by which cancers resist elimination by endogenous tumor-specific T cells revealed two mechanisms for PD-1 ligand upregulation in cancer, known as intrinsic and adaptive immune resistance (Figure 2). These mechanisms are not mutually exclusive and may co-exist in the same TME. Intrinsic resistance refers to the constitutive expression of PD-L1 by tumor cells because of genetic alterations or activation of certain signaling pathways, such as the AKT pathway and STAT3, which are commonly activated in many cancers (Parsa et al., 2007Parsa A.T. Waldron J.S. Panner A. Crane C.A. Parney I.F. Barry J.J. Cachola K.E. Murray J.C. Tihan T. Jensen M.C. et al.Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma.Nat. Med. 2007; 13: 84-88Crossref PubMed Scopus (307) Google Scholar, Marzec et al., 2008Marzec M. Zhang Q. Goradia A. Raghunath P.N. Liu X. Paessler M. Wang H.Y. Wysocka M. Cheng M. Ruggeri B.A. Wasik M.A. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1).Proc. Natl. Acad. Sci. USA. 2008; 105: 20852-20857Crossref PubMed Scopus (116) Google Scholar). Although PD-L1 induction by AKT and STAT3 signaling has been demonstrated in some tumor cell lines, the importance of this intrinsic pathway in PD-L1 expression by tumors in vivo remains to be determined. Genetic alterations in B cell lymphoma subtypes can drive expression of either or both PD-L1 and PD-L2. Primary mediastinal lymphomas commonly display gene fusions between MHC class II transactivator (CIITA) and PD-L1 or PD-L2, placing PD-1 ligands under the transcriptional control of the CIITA promoter, which is highly active in B cell lymphomas (Steidl et al., 2011Steidl C. Shah S.P. Woolcock B.W. Rui L. Kawahara M. Farinha P. Johnson N.A. Zhao Y. Telenius A. Neriah S.B. et al.MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers.Nature. 2011; 471: 377-381Crossref PubMed Scopus (165) Google Scholar). A significant subset of Hodgkin’s lymphoma has amplification of chromosome 9p23-24, where PD-L1 and PD-L2 reside, resulting in overexpression of both ligands. Other cancers, such as a subset of Epstein-Barr virus-induced gastric cancers, also display gene amplification with consequent induction of PD-L1 and PD-L2. The second mechanism, adaptive resistance, refers to the induction of PD-L1 expression on tumor cells in response to specific cytokines, in particular IFN-γ. Because IFN-γ is only produced by activated Th1-type helper CD4 cells, activated CD8 cells, and NK cells, this mechanism represents an adaptation of tumor cells upon “sensing” an inflammatory immune microenvironment that “threatens” the tumor. Indeed, human tumors show significant correlations between PD-L1 expression, levels of T cell infiltration, and IFN-γ in the TME (Taube et al., 2012Taube J.M. Anders R.A. Young G.D. Xu H. Sharma R. McMiller T.L. Chen S. Klein A.P. Pardoll D.M. Topalian S.L. et al.Colocalization of inflammatory response with B7-H1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape.Sci. Transl. Med. 2012; 4: 127ra37Crossref PubMed Scopus (233) Google Scholar, Spranger et al., 2013Spranger S. Spaapen R.M. Zha Y. Williams J. Meng Y. Ha T.T. Gajewski T.F. Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells.Sci. Transl. Med. 2013; 5: 200ra116Crossref PubMed Scopus (144) Google Scholar). Other inhibitory molecules in the TME, such as indoleamine 2′3′ dioxygenase (IDO), which inhibits immunity locally via conversion of tryptophan to kynurenines, are also induced by IFN-γ and coordinately upregulated with PD-L1. The concept of adaptive resistance does not solely apply to induction of PD-L1 on tumor cells. Early studies demonstrated that PD-L1 expression on myeloid cells, including dendritic cells, can significantly impair activation of tumor-specific T cells. Inhibition of T cell responses can be mediated by PD-L1+ suppressive myeloid cells or dendritic cells (DCs) in the TME as well as in tumor-draining lymph nodes (Curiel et al., 2003Curiel T.J. Wei S. Dong H. Alvarez X. Cheng P. Mottram P. Krzysiek R. Knutson K.L. Daniel B. Zimmermann M.C. et al.Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity.Nat. Med. 2003; 9: 562-567Crossref PubMed Scopus (543) Google Scholar). In some tumors, such as microsatellite instability (MSI) colon cancer, myeloid rather than tumor cells are the major cell type expressing PD-L1 (Llosa et al., 2015Llosa N.J. Cruise M. Tam A. Wicks E.C. Hechenbleikner E.M. Taube J.M. Blosser R.L. Fan H. Wang H. Luber B.S. et al.The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints.Cancer Discov. 2015; 5: 43-51Crossref PubMed Scopus (52) Google Scholar). A recent report suggests that PD-L1 expression by infiltrating myeloid cells rather than tumor cells is more predictive of response to PD-1 pathway blockade (Herbst et al., 2014Herbst R.S. Soria J.C. Kowanetz M. Fine G.D. Hamid O. Gordon M.S. Sosman J.A. McDermott D.F. Powderly J.D. Gettinger S.N. et al.Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.Nature. 2014; 515: 563-567Crossref PubMed Scopus (274) Google Scholar). The relative importance of PD-L1 expression on leukocytes in the TME, which would provide “third-party” inhibition, versus direct expression by the tumor cells, remains to be determined. The adaptive resistance mechanism of intratumoral PD-L1 induction, together with the broad therapeutic activity of PD-1 pathway blockade in human cancer, validates one of the most important tenets underlying cancer immunology and immunotherapy, namely, that many cancer patients contain a significant repertoire of tumor-specific T cells capable of killing their tumor save for the adaptive induction of immune checkpoints in the TME. It also implies that PD-L1 expression in the tumor represents a measure of the potential for a patient’s immune system to recognize the tumor. One of the major unanswered questions is this: what are the dominant antigenic targets that T cells recognize when checkpoints are blocked? Circumstantial evidence supports the notion that neoantigens created by the multiple somatic mutations in cancers provide such targets. Indeed, a recent report has demonstrated that melanomas with higher mutational loads were more responsive to anti-CTLA-4 therapy (Snyder et al., 2014Snyder A. Makarov V. Merghoub T. Yuan J. Zaretsky J.M. Desrichard A. Walsh L.A. Postow M.A. Wong P. Ho T.S. et al.Genetic basis for clinical response to CTLA-4 blockade in melanoma.N. Engl. J. Med. 2014; 371: 2189-2199Crossref PubMed Scopus (268) Google Scholar). Also, the tumor types that have been shown to respond to anti-PD-1/PD-L1 therapy tend to be those with higher median mutational loads (i.e., carcinogen-induced cancers such as melanoma, lung, bladder, and head and neck cancers). However, there has been much evidence over the past 20 years that shared self-antigens upregulated in tumors by epigenetic mechanisms (e.g., cancer-testis antigens) are also able to provide selective tumor targeting. The relative importance of mutation-dependent, tumor-specific neoantigens versus tumor-associated self-antigens as T cell targets remains to be determined. Finally, the adaptive resistance mechanism has profound implications for developing synergistic combinatorial cancer immunotherapies. One of the most promising general approaches to immunotherapy utilizes positive drivers of anti-tumor immune responses, such as vaccines, intratumoral injection of immune activators, and co-stimulatory receptor agonists. These modalities with the potential to enhance anti-tumor responses would also be expected to enhance the adaptive induction of checkpoints like PD-1 ligands. This has, in fact, been demonstrated in animal models of vaccination (Fu et al., 2014Fu J. Mal