- Email: [email protected]
New Clones on the Block
Immunity
Previews New Clones on the Block Nandini Acharya1,2 and Ana C. Anderson1,2,* 1Evergrande
Center for Immunologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115, USA Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA 02115, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2019.09.018 2Ann
Although immune checkpoint blockade (ICB) has yielded striking clinical responses in subsets of cancer patients, the mechanism of action is still unclear. In a recent issue of Nature Medicine, Yost et al., 2019 report that the T cell clones that dominate the intra-tumoral T cell landscape after ICB are distinct from those prior to treatment, a phenomenon referred to by the authors as ‘‘clonal replacement.’’ In tumors, a subset of T cells expresses checkpoint receptors including PD1. These receptors act as an ‘‘off switch’’ to dampen anti-tumor T cell functions, resulting in a T cell phenotype commonly referred to as exhausted or dysfunctional. Immune checkpoint blockade (ICB) disrupts checkpoint receptor signaling and promotes immune-mediated elimination of tumor cells. Several ICB approaches are FDA approved for treating various types of cancers, including melanoma and non-small-cell lung carcinoma; however, widespread use of ICB to treat cancers is limited by low response rates and the development of resistance in some cancer patients. Achieving a clear understanding of how ICB works would enable the design of more effective and durable therapies. Although it was initially thought that ICB functions by directly rejuvenating dysfunctional T cells present in the tumor microenvironment (TME), several studies have shown that ICB can promote only limited transcriptional re-wiring of dysfunctional T cells (Pauken et al., 2016) due to their fixed chromatin landscape (Pauken et al., 2016; Sen et al., 2016). In pre-clinical cancer models, stem-like TCF-1+ CD8+ T cells that are not dysfunctional and express either few or no checkpoint receptors are critical for the efficacy of ICB (Kurtulus et al., 2019; Miller et al., 2019; Siddiqui et al., 2019). Similar observations have been reported in melanoma patients (Sade-Feldman et al., 2018). In the August issue of Nature Medicine, Yost et al. report that ICB leads to accumulation of T cell clones in tumors of patients with basal cell carcinoma (BCC) (n = 11) or squamous cell carcinoma (SCC) (n = 4) that are distinct from pre-treatment T cell clones, a phenomenon referred to by the
authors as ‘‘clonal replacement’’ (Yost et al., 2019) (Figure 1). Their findings add another layer to our understanding of how ICB affects the intra-tumoral T cell response. The authors generated single-cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (scTCR-seq) libraries from site-matched primary tumors of 11 patients with advanced BCC before and after anti-PD1 treatment to enable the pairing of TCR clones with T cell phenotypes. Based on the scRNA-seq profiles, 19 clusters of immune and stromal cells were identified. Immune cells from different patients clustered together, indicating conservation in the immune compartment across tumor samples. In contrast, the malignant cells showed patient-specific copy number variations and clustered together based on the patient they were derived from and on the BCC subtype. Thus, patient-specific malignant pathways affected the gene expression pattern of the tumor cells but did not have much influence on the immune cells. The authors then focused on understanding the clonality of the T cell responses to ICB. 33,106 tumor-infiltrating lymphocytes (TILs) were re-clustered. Nine distinct T cell clusters consisting of T cells from different patients pre- and post-treatment were identified. CD8+ T cell clusters were comprised of naive, memory, effector-memory, exhausted, and intermediate exhausted-activated cells. Preferential increase in the frequency of the exhausted and intermediate activated-exhausted CD8+ T cells was observed after treatment with antiPD1. Analysis of the scRNA-seq data using diffusion maps to identify cellular differentiation trajectories separated the
606 Immunity 51, October 15, 2019 ª 2019 Elsevier Inc.
CD8+ T cell clusters into two diffusion components. The first component separated activated from exhausted T cells and highly correlated with PDCD1 (encodes PD1) and HAVCR2 (encodes Tim3) expression. The second component separated naive and memory T cells from the activated and exhausted T cells and highly correlated with IFNG and TNF expression. Next, comparison of the T cell clonal landscape pre- and post-treatment revealed that the highest degree of TCR clonality (number of expanded TCR clones) was observed among T cells with dysfunctional or exhausted phenotype after treatment with anti-PD1. Yost et al. then characterized individual T cell clones by averaging exhaustion and activation scores for all the cells expressing a given TCR clone. They found that the largest clones displayed high expression of the T cell exhaustion gene signature. Comparison of the clonotype (based on TRB sequences) with the T cell phenotype (based on gene expression pattern) preand post-treatment revealed that pretreatment exhausted clones did not revert to non-exhausted phenotype post-treatment, further supporting the notion that exhausted T cells cannot be reinvigorated by ICB treatment. Tcf7 (encodes TCF-1), a transcription factor downstream of the canonical Wingless/Integration 1 (Wnt) signaling pathway, has been shown to be critical for the efficacy of ICB therapy (Kurtulus et al., 2019; Miller et al., 2019; Siddiqui et al., 2019; Sade-Feldman et al., 2018). A higher frequency of TCF7+ CD8+ cells has been reported in the tumor specimens of patients who respond to ICB than in tumor specimens of patients who do not respond (Sade-Feldman et al., 2018).
Immunity
Previews
Figure 1. Newly Incoming Players Dominate the Field Post-Immune Checkpoint Blockade Immune checkpoint blockade does not rejuvenate pre-treatment exhausted T cell clones (#1 and #2) but rather leads to accumulation of novel T cell clones (#3–#6) into the tumor, a phenomenon referred to by Yost et al. as clonal replacement.
TCF-1+ CD8+ T cells have stem-like properties suggesting that such cells could be the precursors that lead to clonal expansion following ICB therapy. However, the authors could not detect an intra-tumoral Tcf7+ precursor for the majority of the post-treatment expanded TCR clones, suggesting that the post-treatment clones are derived from TCF-1+ precursors present at extra-tumoral sites. Of note, most of the CD8+ T cell clones that were detected post-treatment were novel in that they were not detected in pre-treatment site-matched primary tumors, consistent with previous observations in melanoma patients treated with anti-PD1 (Sade-Feldman et al., 2018). Interestingly, the distribution of posttreatment novel clones was strikingly different among the various T cell phenotypes observed; 84% of exhausted T cells were derived from novel clonotypes compared to only 40% of naive, activated, memory, or effector-memory CD8+ T cells. Together, these results indicate that the effect of ICB is mediated by novel T cell clones or clonal replacement rather than by rejuvenation of pre-existing dysfunctional T cells. These results raise the important question: what is the source of the novel T cell clones? 11.8% of the novel TCR clonotypes were detected in the peripheral blood before treatment with anti-PD1, suggesting that these cells could be derived from the periphery. However, studies in murine models of colon cancer and melanoma have shown that periph-
eral recruitment of T cells is dispensable for positive response to anti-PD1 and anti-CTLA4 plus anti-PDL1 (Garris et al., 2018; Spranger et al., 2014; Chow et al., 2019). Thus, it will be important to examine if blocking T cell migration from the periphery precludes clonal replacement. If inhibiting T cell migration into the TME reduces clonal replacement, it will be important to identify the cues in the TME post-ICB that favor T cell recruitment. Additionally, although the authors examined the peripheral blood for the presence of the precursors of the novel TCR clones, analysis of the tumor-draining lymph nodes would provide a key missing piece of information for identifying the peripheral sources of the novel clones. Another possibility is that novel clones are expanded from rare precursors that were present, but not detectable, at baseline in the TME. A further question highlighted by this study is what is the dominant site of action of ICB? Does it work peripherally (e.g., in the tumor-draining lymph node or blood), or does it alter the TME, allowing for the emergence of novel clones? Or are both mechanisms in play? The authors speculate that the expanded T cell clones are due to tumor antigen stimulation, thus explaining their higher proportions in the dysfunctional T cell subset. This prompts the question: what is the antigen specificity of the novel T cell clones? Are they reactive to neoantigens? The authors show that the cells grouped by clonotype were more
likely to share a gene expression pattern than cells that were grouped randomly, indicating that antigen specificity may determine their fate. A related question is whether TCR affinity contributes to clonal replacement. Further, although the highest number of novel T cell clones was found among exhausted cells, a significant proportion was found among activated T cells, raising the question of which of these comprises the anti-tumor response. Overall, this study adds a dimension to our understanding of how ICB affects the intra-tumoral T cell response. It further corroborates the notion that exhausted T cells are not the direct targets of ICB and opens a number of questions. Aside from the source and specificity of the novel T cell clones, the critical question yet to be answered is whether the degree of clonal replacement positively correlates with clinical response to ICB. Indeed, if this were the case, the detection of novel T cell clones in the blood could serve as a biomarker. Notwithstanding these considerations, the new clones on the block are here. ACKNOWLEDGMENTS Work in the authors’ laboratory is supported by grants from the National Institutes of Health (P01AI073748, R01CA229400, R01CA187975). DECLARATION OF INTERESTS A.C.A. is a member of the scientific advisory board for Tizona Therapeutics, Compass Therapeutics, Zumutor Biologics, and Astellas Global Pharma Development Inc., which have interests in cancer immunotherapy. A.C.A. is an inventor on a provisional patent application related to Tcf7. REFERENCES Chow, M.T., Ozga, A.J., Servis, R.L., Frederick, D.T., Lo, J.A., Fisher, D.E., Freeman, G.J., Boland, G.M., and Luster, A.D. (2019). Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy. Immunity 50, 1498–1512.e5. Garris, C.S., Arlauckas, S.P., Kohler, R.H., Trefny, M.P., Garren, S., Piot, C., Engblom, C., Pfirschke, C., Siwicki, M., Gungabeesoon, J., et al. (2018). Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-gamma and IL-12. Immunity 49, 1148–1161.e7. Kurtulus, S., Madi, A., Escobar, G., Klapholz, M., Nyman, J., Christian, E., Pawlak, M., Dionne, D., Xia, J., Rozenblatt-Rosen, O., et al. (2019). Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1 CD8+ TumorInfiltrating T Cells. Immunity 50, 181–194.e6.
Immunity 51, October 15, 2019 607
Immunity
Previews Miller, B.C., Sen, D.R., Al Abosy, R., Bi, K., Virkud, Y.V., LaFleur, M.W., Yates, K.B., Lako, A., Felt, K., Naik, G.S., et al. (2019). Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol. 20, 326–336. Pauken, K.E., Sammons, M.A., Odorizzi, P.M., Manne, S., Godec, J., Khan, O., Drake, A.M., Chen, Z., Sen, D.R., Kurachi, M., et al. (2016). Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 354, 1160–1165. Sade-Feldman, M., Yizhak, K., Bjorgaard, S.L., Ray, J.P., de Boer, C.G., Jenkins, R.W., Lieb, D.J., Chen, J.H., Frederick, D.T., Barzily-Rokni, M., et al. (2018). Defining T Cell States
608 Immunity 51, October 15, 2019
Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 175, 998– 1013.e20. Sen, D.R., Kaminski, J., Barnitz, R.A., Kurachi, M., Gerdemann, U., Yates, K.B., Tsao, H.W., Godec, J., LaFleur, M.W., Brown, F.D., et al. (2016). The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169. Siddiqui, I., Schaeuble, K., Chennupati, V., Fuertes Marraco, S.A., Calderon-Copete, S., Pais Ferreira, D., Carmona, S.J., Scarpellino, L., Gfeller, D., Pradervand, S., et al. (2019). Intratumoral Tcf1+PD-1+CD8+ T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint
Blockade Immunotherapy. Immunity 50, 195– 211.e10. Spranger, S., Koblish, H.K., Horton, B., Scherle, P.A., Newton, R., and Gajewski, T.F. (2014). 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 2, 3. Yost, K.E., Satpathy, A.T., Wells, D.K., Qi, Y., Wang, C., Kageyama, R., McNamara, K.L., Granja, J.M., Sarin, K.Y., Brown, R.A., et al. (2019). Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat. Med. 25, 1251–1259.
Previews New Clones on the Block Nandini Acharya1,2 and Ana C. Anderson1,2,* 1Evergrande
Center for Immunologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115, USA Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA 02115, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2019.09.018 2Ann
Although immune checkpoint blockade (ICB) has yielded striking clinical responses in subsets of cancer patients, the mechanism of action is still unclear. In a recent issue of Nature Medicine, Yost et al., 2019 report that the T cell clones that dominate the intra-tumoral T cell landscape after ICB are distinct from those prior to treatment, a phenomenon referred to by the authors as ‘‘clonal replacement.’’ In tumors, a subset of T cells expresses checkpoint receptors including PD1. These receptors act as an ‘‘off switch’’ to dampen anti-tumor T cell functions, resulting in a T cell phenotype commonly referred to as exhausted or dysfunctional. Immune checkpoint blockade (ICB) disrupts checkpoint receptor signaling and promotes immune-mediated elimination of tumor cells. Several ICB approaches are FDA approved for treating various types of cancers, including melanoma and non-small-cell lung carcinoma; however, widespread use of ICB to treat cancers is limited by low response rates and the development of resistance in some cancer patients. Achieving a clear understanding of how ICB works would enable the design of more effective and durable therapies. Although it was initially thought that ICB functions by directly rejuvenating dysfunctional T cells present in the tumor microenvironment (TME), several studies have shown that ICB can promote only limited transcriptional re-wiring of dysfunctional T cells (Pauken et al., 2016) due to their fixed chromatin landscape (Pauken et al., 2016; Sen et al., 2016). In pre-clinical cancer models, stem-like TCF-1+ CD8+ T cells that are not dysfunctional and express either few or no checkpoint receptors are critical for the efficacy of ICB (Kurtulus et al., 2019; Miller et al., 2019; Siddiqui et al., 2019). Similar observations have been reported in melanoma patients (Sade-Feldman et al., 2018). In the August issue of Nature Medicine, Yost et al. report that ICB leads to accumulation of T cell clones in tumors of patients with basal cell carcinoma (BCC) (n = 11) or squamous cell carcinoma (SCC) (n = 4) that are distinct from pre-treatment T cell clones, a phenomenon referred to by the
authors as ‘‘clonal replacement’’ (Yost et al., 2019) (Figure 1). Their findings add another layer to our understanding of how ICB affects the intra-tumoral T cell response. The authors generated single-cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (scTCR-seq) libraries from site-matched primary tumors of 11 patients with advanced BCC before and after anti-PD1 treatment to enable the pairing of TCR clones with T cell phenotypes. Based on the scRNA-seq profiles, 19 clusters of immune and stromal cells were identified. Immune cells from different patients clustered together, indicating conservation in the immune compartment across tumor samples. In contrast, the malignant cells showed patient-specific copy number variations and clustered together based on the patient they were derived from and on the BCC subtype. Thus, patient-specific malignant pathways affected the gene expression pattern of the tumor cells but did not have much influence on the immune cells. The authors then focused on understanding the clonality of the T cell responses to ICB. 33,106 tumor-infiltrating lymphocytes (TILs) were re-clustered. Nine distinct T cell clusters consisting of T cells from different patients pre- and post-treatment were identified. CD8+ T cell clusters were comprised of naive, memory, effector-memory, exhausted, and intermediate exhausted-activated cells. Preferential increase in the frequency of the exhausted and intermediate activated-exhausted CD8+ T cells was observed after treatment with antiPD1. Analysis of the scRNA-seq data using diffusion maps to identify cellular differentiation trajectories separated the
606 Immunity 51, October 15, 2019 ª 2019 Elsevier Inc.
CD8+ T cell clusters into two diffusion components. The first component separated activated from exhausted T cells and highly correlated with PDCD1 (encodes PD1) and HAVCR2 (encodes Tim3) expression. The second component separated naive and memory T cells from the activated and exhausted T cells and highly correlated with IFNG and TNF expression. Next, comparison of the T cell clonal landscape pre- and post-treatment revealed that the highest degree of TCR clonality (number of expanded TCR clones) was observed among T cells with dysfunctional or exhausted phenotype after treatment with anti-PD1. Yost et al. then characterized individual T cell clones by averaging exhaustion and activation scores for all the cells expressing a given TCR clone. They found that the largest clones displayed high expression of the T cell exhaustion gene signature. Comparison of the clonotype (based on TRB sequences) with the T cell phenotype (based on gene expression pattern) preand post-treatment revealed that pretreatment exhausted clones did not revert to non-exhausted phenotype post-treatment, further supporting the notion that exhausted T cells cannot be reinvigorated by ICB treatment. Tcf7 (encodes TCF-1), a transcription factor downstream of the canonical Wingless/Integration 1 (Wnt) signaling pathway, has been shown to be critical for the efficacy of ICB therapy (Kurtulus et al., 2019; Miller et al., 2019; Siddiqui et al., 2019; Sade-Feldman et al., 2018). A higher frequency of TCF7+ CD8+ cells has been reported in the tumor specimens of patients who respond to ICB than in tumor specimens of patients who do not respond (Sade-Feldman et al., 2018).
Immunity
Previews
Figure 1. Newly Incoming Players Dominate the Field Post-Immune Checkpoint Blockade Immune checkpoint blockade does not rejuvenate pre-treatment exhausted T cell clones (#1 and #2) but rather leads to accumulation of novel T cell clones (#3–#6) into the tumor, a phenomenon referred to by Yost et al. as clonal replacement.
TCF-1+ CD8+ T cells have stem-like properties suggesting that such cells could be the precursors that lead to clonal expansion following ICB therapy. However, the authors could not detect an intra-tumoral Tcf7+ precursor for the majority of the post-treatment expanded TCR clones, suggesting that the post-treatment clones are derived from TCF-1+ precursors present at extra-tumoral sites. Of note, most of the CD8+ T cell clones that were detected post-treatment were novel in that they were not detected in pre-treatment site-matched primary tumors, consistent with previous observations in melanoma patients treated with anti-PD1 (Sade-Feldman et al., 2018). Interestingly, the distribution of posttreatment novel clones was strikingly different among the various T cell phenotypes observed; 84% of exhausted T cells were derived from novel clonotypes compared to only 40% of naive, activated, memory, or effector-memory CD8+ T cells. Together, these results indicate that the effect of ICB is mediated by novel T cell clones or clonal replacement rather than by rejuvenation of pre-existing dysfunctional T cells. These results raise the important question: what is the source of the novel T cell clones? 11.8% of the novel TCR clonotypes were detected in the peripheral blood before treatment with anti-PD1, suggesting that these cells could be derived from the periphery. However, studies in murine models of colon cancer and melanoma have shown that periph-
eral recruitment of T cells is dispensable for positive response to anti-PD1 and anti-CTLA4 plus anti-PDL1 (Garris et al., 2018; Spranger et al., 2014; Chow et al., 2019). Thus, it will be important to examine if blocking T cell migration from the periphery precludes clonal replacement. If inhibiting T cell migration into the TME reduces clonal replacement, it will be important to identify the cues in the TME post-ICB that favor T cell recruitment. Additionally, although the authors examined the peripheral blood for the presence of the precursors of the novel TCR clones, analysis of the tumor-draining lymph nodes would provide a key missing piece of information for identifying the peripheral sources of the novel clones. Another possibility is that novel clones are expanded from rare precursors that were present, but not detectable, at baseline in the TME. A further question highlighted by this study is what is the dominant site of action of ICB? Does it work peripherally (e.g., in the tumor-draining lymph node or blood), or does it alter the TME, allowing for the emergence of novel clones? Or are both mechanisms in play? The authors speculate that the expanded T cell clones are due to tumor antigen stimulation, thus explaining their higher proportions in the dysfunctional T cell subset. This prompts the question: what is the antigen specificity of the novel T cell clones? Are they reactive to neoantigens? The authors show that the cells grouped by clonotype were more
likely to share a gene expression pattern than cells that were grouped randomly, indicating that antigen specificity may determine their fate. A related question is whether TCR affinity contributes to clonal replacement. Further, although the highest number of novel T cell clones was found among exhausted cells, a significant proportion was found among activated T cells, raising the question of which of these comprises the anti-tumor response. Overall, this study adds a dimension to our understanding of how ICB affects the intra-tumoral T cell response. It further corroborates the notion that exhausted T cells are not the direct targets of ICB and opens a number of questions. Aside from the source and specificity of the novel T cell clones, the critical question yet to be answered is whether the degree of clonal replacement positively correlates with clinical response to ICB. Indeed, if this were the case, the detection of novel T cell clones in the blood could serve as a biomarker. Notwithstanding these considerations, the new clones on the block are here. ACKNOWLEDGMENTS Work in the authors’ laboratory is supported by grants from the National Institutes of Health (P01AI073748, R01CA229400, R01CA187975). DECLARATION OF INTERESTS A.C.A. is a member of the scientific advisory board for Tizona Therapeutics, Compass Therapeutics, Zumutor Biologics, and Astellas Global Pharma Development Inc., which have interests in cancer immunotherapy. A.C.A. is an inventor on a provisional patent application related to Tcf7. REFERENCES Chow, M.T., Ozga, A.J., Servis, R.L., Frederick, D.T., Lo, J.A., Fisher, D.E., Freeman, G.J., Boland, G.M., and Luster, A.D. (2019). Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy. Immunity 50, 1498–1512.e5. Garris, C.S., Arlauckas, S.P., Kohler, R.H., Trefny, M.P., Garren, S., Piot, C., Engblom, C., Pfirschke, C., Siwicki, M., Gungabeesoon, J., et al. (2018). Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-gamma and IL-12. Immunity 49, 1148–1161.e7. Kurtulus, S., Madi, A., Escobar, G., Klapholz, M., Nyman, J., Christian, E., Pawlak, M., Dionne, D., Xia, J., Rozenblatt-Rosen, O., et al. (2019). Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1 CD8+ TumorInfiltrating T Cells. Immunity 50, 181–194.e6.
Immunity 51, October 15, 2019 607
Immunity
Previews Miller, B.C., Sen, D.R., Al Abosy, R., Bi, K., Virkud, Y.V., LaFleur, M.W., Yates, K.B., Lako, A., Felt, K., Naik, G.S., et al. (2019). Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol. 20, 326–336. Pauken, K.E., Sammons, M.A., Odorizzi, P.M., Manne, S., Godec, J., Khan, O., Drake, A.M., Chen, Z., Sen, D.R., Kurachi, M., et al. (2016). Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 354, 1160–1165. Sade-Feldman, M., Yizhak, K., Bjorgaard, S.L., Ray, J.P., de Boer, C.G., Jenkins, R.W., Lieb, D.J., Chen, J.H., Frederick, D.T., Barzily-Rokni, M., et al. (2018). Defining T Cell States
608 Immunity 51, October 15, 2019
Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 175, 998– 1013.e20. Sen, D.R., Kaminski, J., Barnitz, R.A., Kurachi, M., Gerdemann, U., Yates, K.B., Tsao, H.W., Godec, J., LaFleur, M.W., Brown, F.D., et al. (2016). The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169. Siddiqui, I., Schaeuble, K., Chennupati, V., Fuertes Marraco, S.A., Calderon-Copete, S., Pais Ferreira, D., Carmona, S.J., Scarpellino, L., Gfeller, D., Pradervand, S., et al. (2019). Intratumoral Tcf1+PD-1+CD8+ T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint
Blockade Immunotherapy. Immunity 50, 195– 211.e10. Spranger, S., Koblish, H.K., Horton, B., Scherle, P.A., Newton, R., and Gajewski, T.F. (2014). 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 2, 3. Yost, K.E., Satpathy, A.T., Wells, D.K., Qi, Y., Wang, C., Kageyama, R., McNamara, K.L., Granja, J.M., Sarin, K.Y., Brown, R.A., et al. (2019). Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat. Med. 25, 1251–1259.