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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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RT, anti-CTLA4, and anti-PD-L1 have distinct effects on the TCR repertoire, Tregs, and T cell exhaustiona) Heat map of changes in the frequency of immune cells or their ratios from B16-F10 tumors. Black hatches indicate treatment. Bar plots show bootstrap importance scores (mean +/− SE) that assess changes in immune parameters predicted by treatment type (read row-wise). Higher values (yellow) represent stronger association. b) T cell subsets and their ratios. c) Frequency distribution (dashed line is 0.5%) and d) boxplot of diversity index (0: clonal, 1: fully diverse) for most frequent TCR clonotypes found in TILs of unirradiated B16-F10 tumors after RT and/or anti-CTLA4. Boxplot summarizes data for mice treated with anti-CTLA4 (NoRT) or RT +/− anti-CTLA4 (+RT). e) Representative contour plots and f) ratios examining PD-1+Eomes+ splenic CD8 T cells from mice with B16-F10 tumors for Ki67+GzmB+ (reinvigorated) or Ki67−GzmB− (exhausted) subsets. g) TCR clonal frequency in post-treatment blood vs. TILs (top row) or vs. pre-treatment blood (bottom row). Quadrant boundaries are top 5% quantiles from the control. Clones below detection in pre-treatment blood are assigned upper bounds (blue). h) Maximum clonal frequency in post-treatment blood (dot) of the most frequent TCR clonotypes found in TILs. i) Distances to cluster centroids for the average CDR3 amino acid features of the five most frequent clones in pre- or post-treatment blood from mice treated with (red) or without (orange) RT. Membership into two clusters (circles and squares) determined by k-means.
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Figure 3: RT, anti-CTLA4, and anti-PD-L1 have distinct effects on the TCR repertoire, Tregs, and T cell exhaustiona) Heat map of changes in the frequency of immune cells or their ratios from B16-F10 tumors. Black hatches indicate treatment. Bar plots show bootstrap importance scores (mean +/− SE) that assess changes in immune parameters predicted by treatment type (read row-wise). Higher values (yellow) represent stronger association. b) T cell subsets and their ratios. c) Frequency distribution (dashed line is 0.5%) and d) boxplot of diversity index (0: clonal, 1: fully diverse) for most frequent TCR clonotypes found in TILs of unirradiated B16-F10 tumors after RT and/or anti-CTLA4. Boxplot summarizes data for mice treated with anti-CTLA4 (NoRT) or RT +/− anti-CTLA4 (+RT). e) Representative contour plots and f) ratios examining PD-1+Eomes+ splenic CD8 T cells from mice with B16-F10 tumors for Ki67+GzmB+ (reinvigorated) or Ki67−GzmB− (exhausted) subsets. g) TCR clonal frequency in post-treatment blood vs. TILs (top row) or vs. pre-treatment blood (bottom row). Quadrant boundaries are top 5% quantiles from the control. Clones below detection in pre-treatment blood are assigned upper bounds (blue). h) Maximum clonal frequency in post-treatment blood (dot) of the most frequent TCR clonotypes found in TILs. i) Distances to cluster centroids for the average CDR3 amino acid features of the five most frequent clones in pre- or post-treatment blood from mice treated with (red) or without (orange) RT. Membership into two clusters (circles and squares) determined by k-means.

Mentions: Notably, RT is needed to achieve high CR rates as dual checkpoint blockade proved inferior to dual checkpoint blockade plus RT (Fig. 2d), a requirement additionally seen in a pancreatic cancer model (Extended Data Fig. 3j). The superiority of triple therapy in multiple cancer types suggests non-redundant mechanisms for each treatment. To examine this notion, we assessed treatment-related changes in TILs from unirradiated tumors. RF modeling of immune cell profiles confirmed that anti-CTLA4 predominantly caused a decrease in Tregs, anti-PD-L1 strongly increased CD8 TIL frequency, and the blockade of both increased the CD8/Treg ratio (Fig. 3a–b, Extended Data Fig. 4a). In contrast, RT caused only a modest increase in CD8 TILs; however, TCR sequencing revealed that this was accompanied by increased diversity of TCR clonotypes, which could be observed even in the presence of CTLA4 blockade (Fig. 3c–3d). Thus, within the tumor microenvironment, CTLA4 blockade primarily decreases Tregs, PD-L1 blockade predominantly reinvigorates exhausted CD8 TILs, and RT diversifies the TCR repertoire of TILs from unirradiated tumors.


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)

RT, anti-CTLA4, and anti-PD-L1 have distinct effects on the TCR repertoire, Tregs, and T cell exhaustiona) Heat map of changes in the frequency of immune cells or their ratios from B16-F10 tumors. Black hatches indicate treatment. Bar plots show bootstrap importance scores (mean +/− SE) that assess changes in immune parameters predicted by treatment type (read row-wise). Higher values (yellow) represent stronger association. b) T cell subsets and their ratios. c) Frequency distribution (dashed line is 0.5%) and d) boxplot of diversity index (0: clonal, 1: fully diverse) for most frequent TCR clonotypes found in TILs of unirradiated B16-F10 tumors after RT and/or anti-CTLA4. Boxplot summarizes data for mice treated with anti-CTLA4 (NoRT) or RT +/− anti-CTLA4 (+RT). e) Representative contour plots and f) ratios examining PD-1+Eomes+ splenic CD8 T cells from mice with B16-F10 tumors for Ki67+GzmB+ (reinvigorated) or Ki67−GzmB− (exhausted) subsets. g) TCR clonal frequency in post-treatment blood vs. TILs (top row) or vs. pre-treatment blood (bottom row). Quadrant boundaries are top 5% quantiles from the control. Clones below detection in pre-treatment blood are assigned upper bounds (blue). h) Maximum clonal frequency in post-treatment blood (dot) of the most frequent TCR clonotypes found in TILs. i) Distances to cluster centroids for the average CDR3 amino acid features of the five most frequent clones in pre- or post-treatment blood from mice treated with (red) or without (orange) RT. Membership into two clusters (circles and squares) determined by k-means.
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Related In: Results  -  Collection

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Figure 3: RT, anti-CTLA4, and anti-PD-L1 have distinct effects on the TCR repertoire, Tregs, and T cell exhaustiona) Heat map of changes in the frequency of immune cells or their ratios from B16-F10 tumors. Black hatches indicate treatment. Bar plots show bootstrap importance scores (mean +/− SE) that assess changes in immune parameters predicted by treatment type (read row-wise). Higher values (yellow) represent stronger association. b) T cell subsets and their ratios. c) Frequency distribution (dashed line is 0.5%) and d) boxplot of diversity index (0: clonal, 1: fully diverse) for most frequent TCR clonotypes found in TILs of unirradiated B16-F10 tumors after RT and/or anti-CTLA4. Boxplot summarizes data for mice treated with anti-CTLA4 (NoRT) or RT +/− anti-CTLA4 (+RT). e) Representative contour plots and f) ratios examining PD-1+Eomes+ splenic CD8 T cells from mice with B16-F10 tumors for Ki67+GzmB+ (reinvigorated) or Ki67−GzmB− (exhausted) subsets. g) TCR clonal frequency in post-treatment blood vs. TILs (top row) or vs. pre-treatment blood (bottom row). Quadrant boundaries are top 5% quantiles from the control. Clones below detection in pre-treatment blood are assigned upper bounds (blue). h) Maximum clonal frequency in post-treatment blood (dot) of the most frequent TCR clonotypes found in TILs. i) Distances to cluster centroids for the average CDR3 amino acid features of the five most frequent clones in pre- or post-treatment blood from mice treated with (red) or without (orange) RT. Membership into two clusters (circles and squares) determined by k-means.
Mentions: Notably, RT is needed to achieve high CR rates as dual checkpoint blockade proved inferior to dual checkpoint blockade plus RT (Fig. 2d), a requirement additionally seen in a pancreatic cancer model (Extended Data Fig. 3j). The superiority of triple therapy in multiple cancer types suggests non-redundant mechanisms for each treatment. To examine this notion, we assessed treatment-related changes in TILs from unirradiated tumors. RF modeling of immune cell profiles confirmed that anti-CTLA4 predominantly caused a decrease in Tregs, anti-PD-L1 strongly increased CD8 TIL frequency, and the blockade of both increased the CD8/Treg ratio (Fig. 3a–b, Extended Data Fig. 4a). In contrast, RT caused only a modest increase in CD8 TILs; however, TCR sequencing revealed that this was accompanied by increased diversity of TCR clonotypes, which could be observed even in the presence of CTLA4 blockade (Fig. 3c–3d). Thus, within the tumor microenvironment, CTLA4 blockade primarily decreases Tregs, PD-L1 blockade predominantly reinvigorates exhausted CD8 TILs, and RT diversifies the TCR repertoire of TILs from unirradiated tumors.

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