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Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer.

Twyman-Saint Victor C, Rech AJ, Maity A, Rengan R, Pauken KE, Stelekati E, Benci JL, Xu B, Dada H, Odorizzi PM, Herati RS, Mansfield KD, Patsch D, Amaravadi RK, Schuchter LM, Ishwaran H, Mick R, Pryma DA, Xu X, Feldman MD, Gangadhar TC, Hahn SM, Wherry EJ, Vonderheide RH, Minn AJ - Nature (2015)

Bottom Line: Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies.Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio.Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

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Addition of PD-L1 blockade reinvigorates exhausted T cells and improves response to RT + anti-CTLA4a) Representative contour plot of CD8 TILs from B16-F10 or Res 499 tumors after RT and anti-CTLA4 (C4) +/− anti-PD-L1 (P1) examined for PD-1 and Eomes (top row), followed by examination of the PD-1+Eomes+ subset for Ki67 and GzmB (bottom row). Schema shows exhaustion and reinvigoration markers. b) Proportion of PD-1+Eomes+ CD8 T cells that are either Ki67−GzmB− or Ki67+GzmB+. c) Changes in T cell subsets and their ratio from Res 499 tumors. d) Survival of mice with B16-F10 tumors (n=18 for RT+C4, n=5 for others). Shown are overall p-values.
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Figure 2: Addition of PD-L1 blockade reinvigorates exhausted T cells and improves response to RT + anti-CTLA4a) Representative contour plot of CD8 TILs from B16-F10 or Res 499 tumors after RT and anti-CTLA4 (C4) +/− anti-PD-L1 (P1) examined for PD-1 and Eomes (top row), followed by examination of the PD-1+Eomes+ subset for Ki67 and GzmB (bottom row). Schema shows exhaustion and reinvigoration markers. b) Proportion of PD-1+Eomes+ CD8 T cells that are either Ki67−GzmB− or Ki67+GzmB+. c) Changes in T cell subsets and their ratio from Res 499 tumors. d) Survival of mice with B16-F10 tumors (n=18 for RT+C4, n=5 for others). Shown are overall p-values.

Mentions: Elevated levels of PD-L1 can promote T cell exhaustion, a state characterized by dysfunction in T cell proliferation and effector function13. Exhausted T cells co-express the PD-L1 receptor PD-1 and the transcription factor Eomes14. Reversal of exhaustion, known as reinvigoration, is marked by an increase in the proliferation marker Ki67 and the cytotoxic protein GzmB within the exhausted T cell pool. In both untreated parental and resistant tumors, approximately 20% of CD8 TILs co-expressed PD-1 and Eomes, and only a minority of these cells were Ki67+GzmB+, indicating that a significant fraction was exhausted (Fig. 2a–b). In B16-F10 tumors, RT + anti-CTLA4 markedly increased both the proportion of PD-1+Eomes+ CD8 T cells and the proportion that were Ki67+GzmB+ within this subset. In contrast, in resistant tumors the average proportion of PD-1+Eomes+ T cells that were Ki67+GzmB+ only marginally increased after RT + anti-CTLA4; however, addition of anti-PD-L1 increased this to levels observed in parental tumors treated with only RT + anti-CTLA4. The frequency of CD8+CD44+ TILs and the CD8/Treg ratio also increased (Fig. 2c), and these were strongly correlated with the proportion of PD-1+Eomes+ CD8 TILs that were Ki67+GzmB+ (Extended Data Fig. 3a). Importantly, addition of anti-PD-L1 improved responses of resistant Res 499 tumors after RT + anti-CTLA4 (Extended Data Fig. 3b–c). For treatment naïve tumors, responses were even more dramatic as the addition of either anti-PD-L1 or anti-PD-1 to RT + anti-CTLA4 markedly improved survival and increased CRs to 80% (Fig. 2d, Extended Data Fig. 3d–f). On average, 58% of mice with CRs after adding anti-PD-L1 or anti-PD-1 were alive 90+ days after tumor rechallenge, and similar improvements were observed with Res 237 breast cancer tumors after addition of PD-L1 blockade (Extended Data Fig. 3g–i). Thus, elevated PD-L1 on tumor cells results in persistent T cell exhaustion that impairs the CD8/Treg ratio. Addition of PD-L1 blockade inhibits resistance and results in long-term immunity.


Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer.

Twyman-Saint Victor C, Rech AJ, Maity A, Rengan R, Pauken KE, Stelekati E, Benci JL, Xu B, Dada H, Odorizzi PM, Herati RS, Mansfield KD, Patsch D, Amaravadi RK, Schuchter LM, Ishwaran H, Mick R, Pryma DA, Xu X, Feldman MD, Gangadhar TC, Hahn SM, Wherry EJ, Vonderheide RH, Minn AJ - Nature (2015)

Addition of PD-L1 blockade reinvigorates exhausted T cells and improves response to RT + anti-CTLA4a) Representative contour plot of CD8 TILs from B16-F10 or Res 499 tumors after RT and anti-CTLA4 (C4) +/− anti-PD-L1 (P1) examined for PD-1 and Eomes (top row), followed by examination of the PD-1+Eomes+ subset for Ki67 and GzmB (bottom row). Schema shows exhaustion and reinvigoration markers. b) Proportion of PD-1+Eomes+ CD8 T cells that are either Ki67−GzmB− or Ki67+GzmB+. c) Changes in T cell subsets and their ratio from Res 499 tumors. d) Survival of mice with B16-F10 tumors (n=18 for RT+C4, n=5 for others). Shown are overall p-values.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401634&req=5

Figure 2: Addition of PD-L1 blockade reinvigorates exhausted T cells and improves response to RT + anti-CTLA4a) Representative contour plot of CD8 TILs from B16-F10 or Res 499 tumors after RT and anti-CTLA4 (C4) +/− anti-PD-L1 (P1) examined for PD-1 and Eomes (top row), followed by examination of the PD-1+Eomes+ subset for Ki67 and GzmB (bottom row). Schema shows exhaustion and reinvigoration markers. b) Proportion of PD-1+Eomes+ CD8 T cells that are either Ki67−GzmB− or Ki67+GzmB+. c) Changes in T cell subsets and their ratio from Res 499 tumors. d) Survival of mice with B16-F10 tumors (n=18 for RT+C4, n=5 for others). Shown are overall p-values.
Mentions: Elevated levels of PD-L1 can promote T cell exhaustion, a state characterized by dysfunction in T cell proliferation and effector function13. Exhausted T cells co-express the PD-L1 receptor PD-1 and the transcription factor Eomes14. Reversal of exhaustion, known as reinvigoration, is marked by an increase in the proliferation marker Ki67 and the cytotoxic protein GzmB within the exhausted T cell pool. In both untreated parental and resistant tumors, approximately 20% of CD8 TILs co-expressed PD-1 and Eomes, and only a minority of these cells were Ki67+GzmB+, indicating that a significant fraction was exhausted (Fig. 2a–b). In B16-F10 tumors, RT + anti-CTLA4 markedly increased both the proportion of PD-1+Eomes+ CD8 T cells and the proportion that were Ki67+GzmB+ within this subset. In contrast, in resistant tumors the average proportion of PD-1+Eomes+ T cells that were Ki67+GzmB+ only marginally increased after RT + anti-CTLA4; however, addition of anti-PD-L1 increased this to levels observed in parental tumors treated with only RT + anti-CTLA4. The frequency of CD8+CD44+ TILs and the CD8/Treg ratio also increased (Fig. 2c), and these were strongly correlated with the proportion of PD-1+Eomes+ CD8 TILs that were Ki67+GzmB+ (Extended Data Fig. 3a). Importantly, addition of anti-PD-L1 improved responses of resistant Res 499 tumors after RT + anti-CTLA4 (Extended Data Fig. 3b–c). For treatment naïve tumors, responses were even more dramatic as the addition of either anti-PD-L1 or anti-PD-1 to RT + anti-CTLA4 markedly improved survival and increased CRs to 80% (Fig. 2d, Extended Data Fig. 3d–f). On average, 58% of mice with CRs after adding anti-PD-L1 or anti-PD-1 were alive 90+ days after tumor rechallenge, and similar improvements were observed with Res 237 breast cancer tumors after addition of PD-L1 blockade (Extended Data Fig. 3g–i). Thus, elevated PD-L1 on tumor cells results in persistent T cell exhaustion that impairs the CD8/Treg ratio. Addition of PD-L1 blockade inhibits resistance and results in long-term immunity.

Bottom Line: Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies.Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio.Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Show MeSH
Related in: MedlinePlus