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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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Tumor cells resistant to RT + anti-CTLA4 upregulate PD-L1 but not other candidate inhibitory receptor pathwaysa) Unirradiated tumor growth (left: normalized, right: raw values) for mice implanted with Res 177 (n=21), Res 499 (n=25), and B16-F10 (n=18) melanoma cells and treated with RT + anti-CTLA4. For normalization, volumes were divided by average of untreated controls (V/Vcont) to account for differences in growth between untreated tumor types. The p-values are for comparisons with B16-F10 tumors. b) Corresponding tumor volumes of unirradiated or irradiated index tumors at day 21 (blue line is mean). c) Clonogenic survival for Res 499 and B16-F10 cells (n=2). d) Selection of immune variables that robustly predict resistance to RT + anti-CTLA4 using minimal depth (MD). A variable was selected if its MD was less than a threshold value for significance. Shown are bootstrap distributions of MD values (left) and % bootstrap models for which the MD for the indicated variable was significant (right). Bootstrap mean +/− SD for the out-of-bag prediction error rate is listed on top. e) Volcano plot of differentially expressed genes from resistant tumors. Horizontal black line is 5% false-discovery rate and dotted green line is fold-change cut-off. Ligands for select inhibitory receptors are indicated. See SI Table 1. f) Unirradiated tumor volumes (day 26–29) and g) survival after RT + anti-CTLA4 for mice with bilateral tumors from TSA breast cancer cells (n=25) or from the Res 237 subline selected to be resistant (n=21). h) Expression of candidate T cell inhibitory receptor ligands on B16-F10 and Res 499. Interferon-gamma (IFNg) responsiveness was tested. i) Boxplots show distribution of % positive CD8+CD44+ T cells for the indicated inhibitory receptor compared to IgG control. j) PD-L1 surface expression for CRISPR PD-L1 homozygous knockout Res 499 and wild type control cells. IFNg was used to induce PD-L1 and confirm abrogated response.
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Figure 6: Tumor cells resistant to RT + anti-CTLA4 upregulate PD-L1 but not other candidate inhibitory receptor pathwaysa) Unirradiated tumor growth (left: normalized, right: raw values) for mice implanted with Res 177 (n=21), Res 499 (n=25), and B16-F10 (n=18) melanoma cells and treated with RT + anti-CTLA4. For normalization, volumes were divided by average of untreated controls (V/Vcont) to account for differences in growth between untreated tumor types. The p-values are for comparisons with B16-F10 tumors. b) Corresponding tumor volumes of unirradiated or irradiated index tumors at day 21 (blue line is mean). c) Clonogenic survival for Res 499 and B16-F10 cells (n=2). d) Selection of immune variables that robustly predict resistance to RT + anti-CTLA4 using minimal depth (MD). A variable was selected if its MD was less than a threshold value for significance. Shown are bootstrap distributions of MD values (left) and % bootstrap models for which the MD for the indicated variable was significant (right). Bootstrap mean +/− SD for the out-of-bag prediction error rate is listed on top. e) Volcano plot of differentially expressed genes from resistant tumors. Horizontal black line is 5% false-discovery rate and dotted green line is fold-change cut-off. Ligands for select inhibitory receptors are indicated. See SI Table 1. f) Unirradiated tumor volumes (day 26–29) and g) survival after RT + anti-CTLA4 for mice with bilateral tumors from TSA breast cancer cells (n=25) or from the Res 237 subline selected to be resistant (n=21). h) Expression of candidate T cell inhibitory receptor ligands on B16-F10 and Res 499. Interferon-gamma (IFNg) responsiveness was tested. i) Boxplots show distribution of % positive CD8+CD44+ T cells for the indicated inhibitory receptor compared to IgG control. j) PD-L1 surface expression for CRISPR PD-L1 homozygous knockout Res 499 and wild type control cells. IFNg was used to induce PD-L1 and confirm abrogated response.

Mentions: Although responses were observed, the majority of patients in our trial did not respond. To understand the contribution of RT to immune checkpoint blockade and to discover mechanisms of resistance, we utilized the B16-F10 melanoma mouse model. Mice with bilateral flank tumors received anti-CTLA4, irradiation of one tumor (index) using a micro-irradiator, or both treatments delivered concurrently (Fig. 1d). The best responses in both tumors occurred with RT + anti-CTLA4. RT given before or concurrently with CTLA4 blockade yielded similar results (Extended Data Fig. 1c). Complete responses (CRs) were CD8 T cell-dependent, and mice with CRs also exhibited CD8 T cell-dependent immunity to tumor re-challenge (Extended Data Fig. 1d–e). However, similar to our clinical trial, only approximately 17% of mice responded. To better understand determinants of response, we derived cell lines from unirradiated tumors that relapsed after RT + anti-CTLA4 (Res 499 and Res 177). Resistance was confirmed in vivo and was not due to intrinsic RT resistance (Extended Data Fig. 2a–c). Random forest (RF) machine learning analysis8,9 of tumor infiltrating lymphocytes (TILs) demonstrated that the top predictor of resistance, as measured by variable importance scores and selection, was the CD8+CD44+ to Treg (CD8/Treg) ratio (Fig. 1e, Extended Data Fig. 2d). In resistant tumors, the CD8/Treg ratio failed to increase after RT + anti-CTLA4 as it did in sensitive tumors because CD8+CD44+ T cells did not significantly expand despite reduction in Tregs (Fig. 1f). Other immune variables associated with resistance were also related to the failure to accumulate CD8 TILs.


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)

Tumor cells resistant to RT + anti-CTLA4 upregulate PD-L1 but not other candidate inhibitory receptor pathwaysa) Unirradiated tumor growth (left: normalized, right: raw values) for mice implanted with Res 177 (n=21), Res 499 (n=25), and B16-F10 (n=18) melanoma cells and treated with RT + anti-CTLA4. For normalization, volumes were divided by average of untreated controls (V/Vcont) to account for differences in growth between untreated tumor types. The p-values are for comparisons with B16-F10 tumors. b) Corresponding tumor volumes of unirradiated or irradiated index tumors at day 21 (blue line is mean). c) Clonogenic survival for Res 499 and B16-F10 cells (n=2). d) Selection of immune variables that robustly predict resistance to RT + anti-CTLA4 using minimal depth (MD). A variable was selected if its MD was less than a threshold value for significance. Shown are bootstrap distributions of MD values (left) and % bootstrap models for which the MD for the indicated variable was significant (right). Bootstrap mean +/− SD for the out-of-bag prediction error rate is listed on top. e) Volcano plot of differentially expressed genes from resistant tumors. Horizontal black line is 5% false-discovery rate and dotted green line is fold-change cut-off. Ligands for select inhibitory receptors are indicated. See SI Table 1. f) Unirradiated tumor volumes (day 26–29) and g) survival after RT + anti-CTLA4 for mice with bilateral tumors from TSA breast cancer cells (n=25) or from the Res 237 subline selected to be resistant (n=21). h) Expression of candidate T cell inhibitory receptor ligands on B16-F10 and Res 499. Interferon-gamma (IFNg) responsiveness was tested. i) Boxplots show distribution of % positive CD8+CD44+ T cells for the indicated inhibitory receptor compared to IgG control. j) PD-L1 surface expression for CRISPR PD-L1 homozygous knockout Res 499 and wild type control cells. IFNg was used to induce PD-L1 and confirm abrogated response.
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Figure 6: Tumor cells resistant to RT + anti-CTLA4 upregulate PD-L1 but not other candidate inhibitory receptor pathwaysa) Unirradiated tumor growth (left: normalized, right: raw values) for mice implanted with Res 177 (n=21), Res 499 (n=25), and B16-F10 (n=18) melanoma cells and treated with RT + anti-CTLA4. For normalization, volumes were divided by average of untreated controls (V/Vcont) to account for differences in growth between untreated tumor types. The p-values are for comparisons with B16-F10 tumors. b) Corresponding tumor volumes of unirradiated or irradiated index tumors at day 21 (blue line is mean). c) Clonogenic survival for Res 499 and B16-F10 cells (n=2). d) Selection of immune variables that robustly predict resistance to RT + anti-CTLA4 using minimal depth (MD). A variable was selected if its MD was less than a threshold value for significance. Shown are bootstrap distributions of MD values (left) and % bootstrap models for which the MD for the indicated variable was significant (right). Bootstrap mean +/− SD for the out-of-bag prediction error rate is listed on top. e) Volcano plot of differentially expressed genes from resistant tumors. Horizontal black line is 5% false-discovery rate and dotted green line is fold-change cut-off. Ligands for select inhibitory receptors are indicated. See SI Table 1. f) Unirradiated tumor volumes (day 26–29) and g) survival after RT + anti-CTLA4 for mice with bilateral tumors from TSA breast cancer cells (n=25) or from the Res 237 subline selected to be resistant (n=21). h) Expression of candidate T cell inhibitory receptor ligands on B16-F10 and Res 499. Interferon-gamma (IFNg) responsiveness was tested. i) Boxplots show distribution of % positive CD8+CD44+ T cells for the indicated inhibitory receptor compared to IgG control. j) PD-L1 surface expression for CRISPR PD-L1 homozygous knockout Res 499 and wild type control cells. IFNg was used to induce PD-L1 and confirm abrogated response.
Mentions: Although responses were observed, the majority of patients in our trial did not respond. To understand the contribution of RT to immune checkpoint blockade and to discover mechanisms of resistance, we utilized the B16-F10 melanoma mouse model. Mice with bilateral flank tumors received anti-CTLA4, irradiation of one tumor (index) using a micro-irradiator, or both treatments delivered concurrently (Fig. 1d). The best responses in both tumors occurred with RT + anti-CTLA4. RT given before or concurrently with CTLA4 blockade yielded similar results (Extended Data Fig. 1c). Complete responses (CRs) were CD8 T cell-dependent, and mice with CRs also exhibited CD8 T cell-dependent immunity to tumor re-challenge (Extended Data Fig. 1d–e). However, similar to our clinical trial, only approximately 17% of mice responded. To better understand determinants of response, we derived cell lines from unirradiated tumors that relapsed after RT + anti-CTLA4 (Res 499 and Res 177). Resistance was confirmed in vivo and was not due to intrinsic RT resistance (Extended Data Fig. 2a–c). Random forest (RF) machine learning analysis8,9 of tumor infiltrating lymphocytes (TILs) demonstrated that the top predictor of resistance, as measured by variable importance scores and selection, was the CD8+CD44+ to Treg (CD8/Treg) ratio (Fig. 1e, Extended Data Fig. 2d). In resistant tumors, the CD8/Treg ratio failed to increase after RT + anti-CTLA4 as it did in sensitive tumors because CD8+CD44+ T cells did not significantly expand despite reduction in Tregs (Fig. 1f). Other immune variables associated with resistance were also related to the failure to accumulate CD8 TILs.

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