Journal: Journal for Immunotherapy of Cancer

Article Title: PD-L1 blockade restores CAR T cell activity through IFN-γ-regulation of CD163+ M2 macrophages

doi: 10.1136/jitc-2021-004400

Figure Lengend Snippet: CAR T cells induce PD-L1 expression in M2 macrophages. (A–C) PD-L1 expression in macrophages and DU145-PSCA tumor cells in the immune-suppression assay. Data represent three independent experiments using three different donors, in duplicate. (D) Immunostaining of CD163, CD68, PD-L1 and CD3 in a humanized MISTRG mouse model following CAR T cell therapy against intratibial LAPC9 prostate xenografts. (E, F) PD-L1 induction at the protein (E) and messenger RNA (F) levels following inhibition of IFN-γ signaling. Anti-IFN-γR1 antibody was used to block IFN-γ signaling in the presence of recombinant IFN-γ or PSCA-CAR T cell-derived CM collected from the DU145-PSCA tumor cell killing assay. (G) PD-L1 induction following inhibition of various signaling pathways. PSCA-CAR T cell-derived CM was applied to M2 macrophages in the presence of various small molecule inhibitors: fludarabine (STAT1 i), C188-9 (STAT3 i), itacitinib (JAK1 i), AG490 (JAK2 i), AZD1480 (JAK1/2 i), Bay11-7082 (NFκB i), Akti VIII (AKT i), CZC24832 (PI3K i), rapamycin (mTOR i). PD-L1 induction was evaluated by flow cytometry 48 hours after CM stimulation. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; CM, conditioned media; IFN, interferon; PD-L1, programmed death ligand-1; PSCA, prostate stem cell antigen; UTD, untransduced.

Article Snippet: Tissue was incubated with rabbit anti-human CD68 (1:200, Cell Signaling Technology, 76 437T) and goat anti-human PD-L1 (1:50, Leinco Technologies, B560) at 4°C overnight and washed in PBS containing 0.1% Tween 20 for 5 min three times.

Techniques: Expressing, Suppression Assay, Immunostaining, Inhibition, Blocking Assay, Recombinant, Derivative Assay, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-L1 blockade restores CAR T cell activity through IFN-γ-regulation of CD163+ M2 macrophages

doi: 10.1136/jitc-2021-004400

Figure Lengend Snippet: PD-L1 blockade restores CAR T cell function in the presence of suppressive M2 macrophages. CAR T cell function was evaluated in the prostate cancer immune-suppression assay in the presence of PD-L1 blockade. (A) Flow cytometry plots indicating the number of viable DU145-PSCA tumor cells in each condition in the presence or absence of anti-PD-L1 antibody, atezolizumab (Atezo). (B–D) Quantification of PSCA-CAR T cell-mediated killing of DU145-PSCA tumor cells (B), T cell activation (C), and IFN-γ secretion (D). (E) DU145-PSCA tumor cell killing of CAR T cells in the presence or absence of two clinically approved anti-PD-L1 antibodies, Atezo and avelumab (Ave), and Ave that lacks CH 2 domain (Ave (ΔCH 2 )). Tumor killing and T cell activation were evaluated by flow cytometry, and IFN-γ secretion was evaluated by ELISA. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; IFN, interferon; PD-L1, programmed death ligand-1; PSCA, prostate stem cell antigen; UTD, untransduced.

Article Snippet: Tissue was incubated with rabbit anti-human CD68 (1:200, Cell Signaling Technology, 76 437T) and goat anti-human PD-L1 (1:50, Leinco Technologies, B560) at 4°C overnight and washed in PBS containing 0.1% Tween 20 for 5 min three times.

Techniques: Cell Function Assay, Suppression Assay, Flow Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-L1 blockade restores CAR T cell activity through IFN-γ-regulation of CD163+ M2 macrophages

doi: 10.1136/jitc-2021-004400

Figure Lengend Snippet: Combination of PD-L1 blockade and CAR T cell therapy depletes M2 macrophages via IFN-γ signaling. (A, B) Analysis of M2 macrophages in the prostate cancer immune-suppression assay in the presence or absence of PD-L1 blockade. (C, D) Analysis of M2 macrophages stimulated with PSCA-CAR T cell-derived CM in the presence or absence of PD-1 or PD-L1 blockade. (E–G) Representative brightfield images and analysis of M2 macrophage stimulated with PSCA-CAR T cell-derived CM in the presence or absence of PD-L1 and/or IFN-γR1 blockade. The number of total viable M2 macrophages (A, C, F) and the frequency and number of CD163 + M2 macrophages (B, D, G) were evaluated by flow cytometry. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; CM, conditioned media; IFN, interferon; PD-L1, programmed death ligand-1; PD-1, programmed cell death protein-1; PSCA, prostate stem cell antigen; UTD, untransduced.

Article Snippet: Tissue was incubated with rabbit anti-human CD68 (1:200, Cell Signaling Technology, 76 437T) and goat anti-human PD-L1 (1:50, Leinco Technologies, B560) at 4°C overnight and washed in PBS containing 0.1% Tween 20 for 5 min three times.

Techniques: Suppression Assay, Derivative Assay, Flow Cytometry

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: a, Schedule of mouse treatments. b–g, Tumor growth profiles (b,e), mouse survival (c,f) and SK plots (d,g) after various treatments in TC-1 (b–d) and B16 (e–g) tumor models. Tumor growth and survival data are the average of two independent experiments with the indicated numbers of mice per group. The error bars indicate the s.e.m. For tumor growth, statistical analysis was performed by unpaired, one-tailed Student’s t-test, with the asterisks colored to indicate the comparison: purple, comparison to untreated; blue, comparison to anti-PD-1; brown, comparison to Vax; green, comparison to Vax + anti-PD-1 (pre). Individual P values (for the same order of comparisons) are as follows: ***P = 0.001, ****P ≤ 0.0001 (b). **P = 0.0078, **P = 0.0078, **P = 0.0022 and **P = 0.0016 (day (D) 34) (d). *P = 0.0244, **P = 0.0037, *P = 0.0313 and *P = 0.0451 (day 18); ***P = 0.0001, ****P ≤ 0.0001, *P = 0.0262 and **P = 0.0086 (day 21) (e). Survival in various groups was compared using log-rank (Mantel–Cox) tests. Individual P values (for the same order of comparisons except where indicated) are as follows: ****P ≤ 0.0001 (all comparisons) (c); ****P ≤ 0.0001, ****P ≤ 0.0001, ****P ≤ 0.0001, ***P = 0.0003 (day 34) (d); ****P ≤ 0.0001, ****P ≤ 0.0001, ***P = 0.0002, **P = 0.0027 (f); ****P ≤ 0.0001, ****P ≤ 0.0001, ***P = 0.0002, **P = 0.0027 (day 31) (g). h,i, Profiles of total CD8+ (h) and antigen-specific CD8+ T cells (i) in the TME of TC-1-bearing mice after various treatments, 3 d after the second vaccination (D20). Flow cytometry data are the average from two independent experiments. Each dot corresponds to one mouse with the indicated number of mice per group given in parentheses. The error bars indicate the s.e.m. Statistical analysis was performed by unpaired, one-tailed Student’s t-test. Individual P values are as follows: *P = 0.0255 (left), *P = 0.0322 (middle), *P = 0.0146 (right), *P = 0.0365 (upper), **P = 0.0018 (lower), **P = 0.01 (upper), ***P = 0.0002 (h). *P = 0.0059 (lower), *P = 0.0273 (upper), **P = 0.0035 (lower), **P = 0.0039 (upper), ***P = 0.0008 (left), ***P = 0.0001 (right), ****P ≤ 0.0001 (i). NS, not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For in vitro activation, magnetically enriched (Miltenyi Biotec) CD8 + T cells (>95% purity) from Pmel-1 mice were cultured in T cell medium containing RPMI 1640 (Lonza) supplemented with 10% FBS, penicillin (100 U ml −1 ), streptomycin (100 mg ml −1 ), 0.1% β-mercaptoethanol and either interleukin-2 (IL-2, 100 U ml −1 ) (PeproTech) or anti-PD-1 + IL-2 (clone- RMP1–14, 25 μg ml −1 ; MedImmune) at 37 °C, 5% CO 2 .

Techniques: One-tailed Test, Flow Cytometry

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: a–d, MFI and frequency of PD-1+CD38hi T cells in total (a,c) and antigen-specific (b,d) CD8+ T cells in TC-1 (a,b) and B16 (c,d) tumor-bearing mice at day 13 post-tumor implantation. Data are the average of two independent experiments. Each dot corresponds to one mouse with the indicated number of mice per group given in parentheses. The error bars indicate the s.e.m. For statistical comparison, an unpaired, one-tailed Student’s t-test was used. NS; versus untreated: *P = 0.0339 (lower) and *P = 0.0164 (upper); versus Vax: *P = 0.0233 (lower) and *P = 0.0269 (upper), ***P = 0.0005 (left panel); *P = 0.0201 (lower), *P = 0.0416 (upper), **P = 0.006 (right panel) (a); versus untreated: *P = 0.0416 (lower) and *P = 0.0316 (upper); versus Vax: *P = 0.0342 (lower) and *P = 0.0261 (upper), ***P = 0.0005 (left panel); *P = 0.028 (lower), *P = 0.0221 (upper), **P = 0.0015, ****P ≤ 0.0001 (right panel) (b); *P = 0.032 (lower), *P = 0.0137 (middle), *P = 0.0482 (upper), **P = 0.0037, ****P ≤ 0.0001 (left panel); *P = 0.0498 (lower), *P = 0.0241 (upper), **P = 0.01 (right panel) (c); versus untreated: *P = 0.0478 (lower) and *P = 0.0273 (upper); versus Vax: *P = 0.0168 (lower) and *P = 0.0464 (upper), **P = 0.0014 (left panel); versus untreated: *P = 0.0213 (lower) and *P = 0.0202 (upper); versus Vax: *P = 0.0272 (lower) and * P = 0.035 (upper), ***P = 0.0003 (right panel) (d). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For in vitro activation, magnetically enriched (Miltenyi Biotec) CD8 + T cells (>95% purity) from Pmel-1 mice were cultured in T cell medium containing RPMI 1640 (Lonza) supplemented with 10% FBS, penicillin (100 U ml −1 ), streptomycin (100 mg ml −1 ), 0.1% β-mercaptoethanol and either interleukin-2 (IL-2, 100 U ml −1 ) (PeproTech) or anti-PD-1 + IL-2 (clone- RMP1–14, 25 μg ml −1 ; MedImmune) at 37 °C, 5% CO 2 .

Techniques: Tumor Implantation, One-tailed Test

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: a,b, Frequency of CD40L+ or IFN-γ+ PD-1+CD38hi cells in total CD8+ T cells (a) and in the PD-1+CD38hi CD8+ T cell population (b). c,d, Frequencies of annexin V+ PD-1+CD38hi cells in total (c) and antigen-specific (d) CD8+ T cells. e,f, Frequencies of CD62L+CD44+ (e) and CD62L−CD44+ (f) CD8+ T cells after various treatments as shown. Data at day 13 (a–d) and day 20 (e,f) post-TC-1 tumor implantation. Data are representative of two independent experiments. Each dot corresponds to one mouse with the indicated number of mice per group given in parentheses. The error bars indicate the s.e.m. For comparison purposes, an unpaired, one-tailed Student’s t-test was used. *P = 0.0462 (lower), *P = 0.0258 (upper), **P = 0.0052, ***P = 0.0004 (left panel); *P = 0.0148 (lower), *P = 0.0309 (middle), *P = 0.0382 (upper), **P = 0.002, ***P = 0.001 (right panel) (a); *P = 0.0372 (lower), *P = 0.0449 (middle), *P = 0.0225 (upper), ***P = 0.0006 (left panel); *P = 0.0104 (lower), *P = 0.0421 (upper), **P = 0.004 (right panel) (b); ***P = 0.001, ****P ≤ 0.0001 (c); *P = 0.05 (lower), *P = 0.0146 (upper) (d); *P = 0.0299 (lower), *P = 0.012 (upper) (e); *P = 0.0318 (left), *P = 0.0271 (f) (right). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For in vitro activation, magnetically enriched (Miltenyi Biotec) CD8 + T cells (>95% purity) from Pmel-1 mice were cultured in T cell medium containing RPMI 1640 (Lonza) supplemented with 10% FBS, penicillin (100 U ml −1 ), streptomycin (100 mg ml −1 ), 0.1% β-mercaptoethanol and either interleukin-2 (IL-2, 100 U ml −1 ) (PeproTech) or anti-PD-1 + IL-2 (clone- RMP1–14, 25 μg ml −1 ; MedImmune) at 37 °C, 5% CO 2 .

Techniques: Tumor Implantation, One-tailed Test

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: a, Tumor growth and survival of variously treated B16-bearing Rag1−/− mice following transfer of either total or PD-1+CD38+-depleted, in vitro-activated CD8+ T cells (with the indicated number of mice per group given in parentheses); data are the average of two independent experiments. The error bars indicate the s.e.m. Left panel: for comparison purposes, an unpaired, one-tailed Student’s t-test was used; *P = 0.017 (day 14), *P = 0.0127 (day 16), **P = 0.0074 (day 20). Right panel: survival in various groups was compared using the log-rank (Mantel–Cox) test; **P = 0.0054. b, Experimental outline for Pmel-1 CD8+ T cell treatment. c, MFI and protein expression of CD38 in PD-1+CD8+ T cells (shown in the small red box on the left). The protein expression of CD38 in flow-sorted PD-1+CD38+ T cells transfected with CD38 siRNA or scrambled RNA (scRNA) was determined by immunoblot. The expression of β-actin was used as a loading control (the uncropped full scan of the blot is shown in Supplementary Fig. 5b). d, Frequency of Ki-67+, CD40L+ and IFN-γ+ in the PD-1+CD38+ CD8+ T cell population. Data are representative of two independent experiments with at least 3–5 technical replicates per group. The error bars indicate the s.e.m. For comparison purposes, an unpaired, one-tailed Student’s t-test was used. ***P = 0.0003 (c); ***P = 0.0007 (left panel); ****P ≤ 0.0001 (middle panel); **P = 0.0012 (right panel) (d). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For in vitro activation, magnetically enriched (Miltenyi Biotec) CD8 + T cells (>95% purity) from Pmel-1 mice were cultured in T cell medium containing RPMI 1640 (Lonza) supplemented with 10% FBS, penicillin (100 U ml −1 ), streptomycin (100 mg ml −1 ), 0.1% β-mercaptoethanol and either interleukin-2 (IL-2, 100 U ml −1 ) (PeproTech) or anti-PD-1 + IL-2 (clone- RMP1–14, 25 μg ml −1 ; MedImmune) at 37 °C, 5% CO 2 .

Techniques: In Vitro, One-tailed Test, Expressing, Transfection, Western Blot

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: a, Schedule of mouse treatment. b–e, Estimation of CD38 MFI and frequency of PD-1+CD38hi CD8+ T cells (b,d) and frequency of total CD8+, Annexin V+ CD8+ and Annexin V+ PD-1+CD38hi CD8+ T cells (c,e) in variously treated TC-1 (b,c) and B16 (d,e) tumor-bearing mice. Day 10 data after tumor implantation; data are the average of two independent experiments. Each dot corresponds to one mouse with the indicated number of mice per group given in parentheses. The error bars indicate the s.e.m. For comparison purposes, an unpaired, one-tailed Student’s t-test was used. *P = 0.0201 (left panel), *P = 0.0435 (right panel) (b); NS (left panel); ****P ≤ 0.0001 (middle panel); **P = 0.0096 (right panel) (c); **P = 0.0083 (left panel), *P = 0.0129 (right panel) (d); NS (left panel), *P = 0.0291 (middle panel), *P = 0.0392 (right panel) (e). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For in vitro activation, magnetically enriched (Miltenyi Biotec) CD8 + T cells (>95% purity) from Pmel-1 mice were cultured in T cell medium containing RPMI 1640 (Lonza) supplemented with 10% FBS, penicillin (100 U ml −1 ), streptomycin (100 mg ml −1 ), 0.1% β-mercaptoethanol and either interleukin-2 (IL-2, 100 U ml −1 ) (PeproTech) or anti-PD-1 + IL-2 (clone- RMP1–14, 25 μg ml −1 ; MedImmune) at 37 °C, 5% CO 2 .

Techniques: Tumor Implantation, One-tailed Test

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: a, Treatment schedule for in vitro activation of OT I CD8+ T cells with OVA or OVA-V as indicated. b,c, Frequency of CD40L+ (b) and IFN-γ+ (c) CD8+ T cells after various treatments. d, MFI of CD38 in PD-1+ CD8+ T cells. e, Frequency of PD-1+CD38hi cells in CD8+ T cells after various treatments. f, Frequency of annexin V+ cells in PD-1+CD38hi CD8+ T cells after various treatments. In vitro data are representative of two independent experiments with at least four technical replicates. g, Schedule of mouse treatment. h,i, Frequency of PD-1+CD38hi (h) and IFN-γ+ (i) CD8+ T cells after various treatments in TC-1 tumor-bearing mice. Day 10 data after tumor implantation; data are representative of one of two independent experiments. Each dot corresponds to one mouse with the indicated number of mice per group given in parentheses. The error bars indicate the s.e.m. For comparison purposes, an unpaired, one-tailed Student’s t-test was used. ****P ≤ 0.0001 (b); ****P ≤ 0.0001 (c); ****P ≤ 0.0001 (d); NS, OVA versus OVA-V: **P = 0.0047, **P = 0.0067 (lower), **P = 0.009 (middle), **P = 0.0073 (upper) (e); *P = 0.0211 (lower), *P = 0.0436 (middle), *P = 0.0328 (upper) (f); *P = 0.044, **P = 0.0015 (left), **P = 0.0061 (right), ****P ≤ 0.0001 (h); *P = 0.0312 (lower), *P = 0.0112 (middle), *P = 0.0172 (upper) (i). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For in vitro activation, magnetically enriched (Miltenyi Biotec) CD8 + T cells (>95% purity) from Pmel-1 mice were cultured in T cell medium containing RPMI 1640 (Lonza) supplemented with 10% FBS, penicillin (100 U ml −1 ), streptomycin (100 mg ml −1 ), 0.1% β-mercaptoethanol and either interleukin-2 (IL-2, 100 U ml −1 ) (PeproTech) or anti-PD-1 + IL-2 (clone- RMP1–14, 25 μg ml −1 ; MedImmune) at 37 °C, 5% CO 2 .

Techniques: In Vitro, Activation Assay, Tumor Implantation, One-tailed Test

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: a, The posttreatment average frequency of PD-1+CD38+ CD8+ T cells in 21 non-responding and 8 responding tumor lesions, as determined by single-cell RNA sequencing analysis, is shown on the left and the individual frequencies are shown on the right. The red line depicts the cutoff limit where at least 4% or more CD8+ T cells were PD-1+CD38+ in the tumors. b, The pretreatment average frequency of PD-1+CD38+ CD8+ T cells in 10 non-responding and 9 responding tumors lesions is shown on left and the individual frequencies are shown on the right. The red line depicts the cutoff limit where at least 10% or more CD8+ T cells were PD-1+CD38+ in the tumors. The error bars indicate the s.e.m. Left panels: an unpaired, one-tailed Student’s t-test was used. **P = 0.0045 (a); **P = 0.0048 (b). The post- and pretreatment cutoffs that best predicted responders from non-responders were determined a priori and were further confirmed using ROC analysis (right panels). c, Absolute numbers of CD8+ (top) and PD-1+CD38+ CD8+ (bottom) T cells in total viable cells in the TME in the pretreatment (non-responders: n = 10; responders: n = 9), posttreatment (non-responders: n = 21; responders: n = 8) or total (non-responders: n = 31; responders: n = 17) number of responding and non-responding tumor lesions. Each dot corresponds to one tumor lesion. The error bars indicate the s.e.m. For comparison purposes, a one-tailed Student’s t-test was used. Top panels: NS; bottom panels: *P = 0.019 (pretreatment); *P = 0.034 (posttreatment); **P = 0.0034 (total). d, Flow cytometry measurements of CD38+ cells in PD-1+CD8+ T cells in PBMCs from advanced melanoma patients at 3 and 9 weeks after anti-PD-1 treatment. For two non-responding patients, data are shown at 6 weeks since samples were not available at week 9.

Article Snippet: For in vitro activation, magnetically enriched (Miltenyi Biotec) CD8 + T cells (>95% purity) from Pmel-1 mice were cultured in T cell medium containing RPMI 1640 (Lonza) supplemented with 10% FBS, penicillin (100 U ml −1 ), streptomycin (100 mg ml −1 ), 0.1% β-mercaptoethanol and either interleukin-2 (IL-2, 100 U ml −1 ) (PeproTech) or anti-PD-1 + IL-2 (clone- RMP1–14, 25 μg ml −1 ; MedImmune) at 37 °C, 5% CO 2 .

Techniques: RNA Sequencing Assay, One-tailed Test, Flow Cytometry