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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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TCR clonotypes associated with RT are not observed in random clones from post-treatment blood and have distinct CDR3 featuresa) Boxplot of the bootstrap variance explained by multivariable RF regression model for effect of RT, anti-CTLA4, and/or anti-PD-L1 on immune variables from TILs. b) K-means clustering (k=2) was used on the average CDR3 amino acid features of randomly sampled clones from post-treatment blood after anti-CTLA4, anti-PD-L1, and/or RT. Membership into each cluster was determined and the p-value for separation into treatment groups with and without RT was calculated. Boxplot shows log10 p-values from 1000 random iterations. Comparison to the p-value from the observed data (red dotted line) gives a simulated p < 0.001. c) Log10 p-values for separation into treatment groups with and without RT vs. cut-off value used to select the most frequent clones. The 0.05 significance level is indicated (red dotted line). d) Average % occupancy in the CDR3 of the most frequent T cell clonotypes after RT +/− checkpoint blockade (+RT, red line) or checkpoint blockade alone (NoRT, orange line) by contiguous short amino acid sequences of length three (3-tuples) belonging to e) subsets with distinct treatment-related amino acid properties. These properties are characterized by Atchley factors, which measure 1) PAH: accessibility, polarity, and hydrophobicity, 2) PSS: propensity for secondary structure, 3) MS: molecular size, 4) CC: codon composition, and 5) EC: electrostatic charge. Shown (right) are the average values of each Atchley factor for amino acids that comprise the 3-tuples from the indicated subset (red) compared to all unselected 3-tuples (blue). Boxplots (left) show the proportion of 3-tuples from each of these subsets that are found in the CDR3s of the five most frequent clones after treatment. Compared to pre-treatment samples (Pre-tx), subset 6 is associated with RT +/− checkpoint blockade (+RT) or checkpoint blockade alone (NoRT). Subset 1 is primarily associated with checkpoint blockade alone, and subset 16 is primarily associated with RT +/− checkpoint blockade.
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Figure 8: TCR clonotypes associated with RT are not observed in random clones from post-treatment blood and have distinct CDR3 featuresa) Boxplot of the bootstrap variance explained by multivariable RF regression model for effect of RT, anti-CTLA4, and/or anti-PD-L1 on immune variables from TILs. b) K-means clustering (k=2) was used on the average CDR3 amino acid features of randomly sampled clones from post-treatment blood after anti-CTLA4, anti-PD-L1, and/or RT. Membership into each cluster was determined and the p-value for separation into treatment groups with and without RT was calculated. Boxplot shows log10 p-values from 1000 random iterations. Comparison to the p-value from the observed data (red dotted line) gives a simulated p < 0.001. c) Log10 p-values for separation into treatment groups with and without RT vs. cut-off value used to select the most frequent clones. The 0.05 significance level is indicated (red dotted line). d) Average % occupancy in the CDR3 of the most frequent T cell clonotypes after RT +/− checkpoint blockade (+RT, red line) or checkpoint blockade alone (NoRT, orange line) by contiguous short amino acid sequences of length three (3-tuples) belonging to e) subsets with distinct treatment-related amino acid properties. These properties are characterized by Atchley factors, which measure 1) PAH: accessibility, polarity, and hydrophobicity, 2) PSS: propensity for secondary structure, 3) MS: molecular size, 4) CC: codon composition, and 5) EC: electrostatic charge. Shown (right) are the average values of each Atchley factor for amino acids that comprise the 3-tuples from the indicated subset (red) compared to all unselected 3-tuples (blue). Boxplots (left) show the proportion of 3-tuples from each of these subsets that are found in the CDR3s of the five most frequent clones after treatment. Compared to pre-treatment samples (Pre-tx), subset 6 is associated with RT +/− checkpoint blockade (+RT) or checkpoint blockade alone (NoRT). Subset 1 is primarily associated with checkpoint blockade alone, and subset 16 is primarily associated with RT +/− checkpoint blockade.

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)

TCR clonotypes associated with RT are not observed in random clones from post-treatment blood and have distinct CDR3 featuresa) Boxplot of the bootstrap variance explained by multivariable RF regression model for effect of RT, anti-CTLA4, and/or anti-PD-L1 on immune variables from TILs. b) K-means clustering (k=2) was used on the average CDR3 amino acid features of randomly sampled clones from post-treatment blood after anti-CTLA4, anti-PD-L1, and/or RT. Membership into each cluster was determined and the p-value for separation into treatment groups with and without RT was calculated. Boxplot shows log10 p-values from 1000 random iterations. Comparison to the p-value from the observed data (red dotted line) gives a simulated p < 0.001. c) Log10 p-values for separation into treatment groups with and without RT vs. cut-off value used to select the most frequent clones. The 0.05 significance level is indicated (red dotted line). d) Average % occupancy in the CDR3 of the most frequent T cell clonotypes after RT +/− checkpoint blockade (+RT, red line) or checkpoint blockade alone (NoRT, orange line) by contiguous short amino acid sequences of length three (3-tuples) belonging to e) subsets with distinct treatment-related amino acid properties. These properties are characterized by Atchley factors, which measure 1) PAH: accessibility, polarity, and hydrophobicity, 2) PSS: propensity for secondary structure, 3) MS: molecular size, 4) CC: codon composition, and 5) EC: electrostatic charge. Shown (right) are the average values of each Atchley factor for amino acids that comprise the 3-tuples from the indicated subset (red) compared to all unselected 3-tuples (blue). Boxplots (left) show the proportion of 3-tuples from each of these subsets that are found in the CDR3s of the five most frequent clones after treatment. Compared to pre-treatment samples (Pre-tx), subset 6 is associated with RT +/− checkpoint blockade (+RT) or checkpoint blockade alone (NoRT). Subset 1 is primarily associated with checkpoint blockade alone, and subset 16 is primarily associated with RT +/− checkpoint blockade.
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Figure 8: TCR clonotypes associated with RT are not observed in random clones from post-treatment blood and have distinct CDR3 featuresa) Boxplot of the bootstrap variance explained by multivariable RF regression model for effect of RT, anti-CTLA4, and/or anti-PD-L1 on immune variables from TILs. b) K-means clustering (k=2) was used on the average CDR3 amino acid features of randomly sampled clones from post-treatment blood after anti-CTLA4, anti-PD-L1, and/or RT. Membership into each cluster was determined and the p-value for separation into treatment groups with and without RT was calculated. Boxplot shows log10 p-values from 1000 random iterations. Comparison to the p-value from the observed data (red dotted line) gives a simulated p < 0.001. c) Log10 p-values for separation into treatment groups with and without RT vs. cut-off value used to select the most frequent clones. The 0.05 significance level is indicated (red dotted line). d) Average % occupancy in the CDR3 of the most frequent T cell clonotypes after RT +/− checkpoint blockade (+RT, red line) or checkpoint blockade alone (NoRT, orange line) by contiguous short amino acid sequences of length three (3-tuples) belonging to e) subsets with distinct treatment-related amino acid properties. These properties are characterized by Atchley factors, which measure 1) PAH: accessibility, polarity, and hydrophobicity, 2) PSS: propensity for secondary structure, 3) MS: molecular size, 4) CC: codon composition, and 5) EC: electrostatic charge. Shown (right) are the average values of each Atchley factor for amino acids that comprise the 3-tuples from the indicated subset (red) compared to all unselected 3-tuples (blue). Boxplots (left) show the proportion of 3-tuples from each of these subsets that are found in the CDR3s of the five most frequent clones after treatment. Compared to pre-treatment samples (Pre-tx), subset 6 is associated with RT +/− checkpoint blockade (+RT) or checkpoint blockade alone (NoRT). Subset 1 is primarily associated with checkpoint blockade alone, and subset 16 is primarily associated with RT +/− checkpoint blockade.
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