Targeting PARP11 to avert immunosuppression and improve CAR T therapy in solid tumors

Abstract

Evasion of antitumor immunity and resistance to therapies in solid tumors are aided by an immunosuppressive tumor microenvironment (TME). We found that TME factors, such as regulatory T cells and adenosine, downregulated type I interferon receptor IFNAR1 on CD8⁺ cytotoxic T lymphocytes (CTLs). These events relied upon poly-ADP ribose polymerase-11 (PARP11), which was induced in intratumoral CTLs and acted as a key regulator of the immunosuppressive TME. Ablation of PARP11 prevented loss of IFNAR1, increased CTL tumoricidal activity and inhibited tumor growth in an IFNAR1-dependent manner. Accordingly, genetic or pharmacologic inactivation of PARP11 augmented the therapeutic benefits of chimeric antigen receptor T cells. Chimeric antigen receptor CTLs engineered to inactivate PARP11 demonstrated a superior efficacy against solid tumors. These findings highlight the role of PARP11 in the immunosuppressive TME and provide a proof of principle for targeting this pathway to optimize immune therapies.

Extended Data Fig. 1. Downregulation of IFNAR1 on the intratumoral CTL undermines their activities.

A. Growth of MC38 tumors (1×10⁶/mouse) after s.c. injection into WT and SA mice. Tumor volumes were measured 3 times per week. Data are shown as mean ± SEM (WT, n = 8 mice; SA, n = 5 mice). Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ***P = 0.0004. B. t-SNE plots showing the expression of Itgam, Cd3d, Cd8a and Cd4. Transcript levels are color-coded. N=9,725 cells. C. Dot plots showing the expression of CD8⁺ T cell function relevant genes that are highly expressed in each cluster. The size of the dot corresponds to the percentage of cells expressing the gene in each group and the color represents the average expression level. WT, n = 2075 cells; SA, n = 2038 cells. D. Flow cytometry gating strategies in analysis of cellular components of tumor tissues. E. Flow cytometry analysis of CD69⁺ and FasL⁺ GzmB⁺ cells gated on CD45⁺CD3⁺CD8⁺ T cells in indicated tumor tissues. Data are shown as mean ± SEM (n = 6 mice for each group). Two-tailed unpaired t-test was performed for the comparisons between groups.

Extended Data Fig. 2. Downregulation of CTL IFNAR1 by Treg and adenosine

A. WT or SA OT-1 cells were first co-cultured with or without iTregs for 24h (Treg:OT-1=1:3) and then combined with MC38OVA-luc cells at indicated E:T ratios. Killing of MC38OVA-luc cells was analyzed using luciferase assay as described in Methods. Data are shown as mean ± SEM (n = 3 co-cultures.) Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. *P = 0.0288. B. Tumor associated Tregs were isolated from MC38 WT tumors and cocultured with OT-1 cells for 24h (Treg: OT-1=1:3). Then analysis of IFNAR1 levels on OT-1 CTL as well as killing of MC38OVA-luc cells (co-cultured at ratio E:T=10:1) was performed as described in Methods. Data are shown as mean ± SEM (WT and SA IFNAR1, n = 3 co-cultures; WT and SA lysis%, n = 6 co-cultures Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. C. Representative flow cytometry analysis of levels of IFNAR1 on the surface of CD3⁺CD8⁺ T cells treated with or without adenosine (Ado, 1mM) prostaglandin E2 (PGE2, 1μg/ml) or tumor growth factor-beta (TGF-β, 5ng/ml) for 2h. D. Lysis of MC38OVA-luc cells co-cultured in vitro with OT-1 cells pretreated or not with adenosine (Ado, 1mM for 24h) at indicated E:T ratios. Data are shown as mean ± SEM (n = 3 co-cultures). Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. E. Schematic of crosses to generate the Ifnar1^ΔCD8 mice. F. Genotyping analysis of Ifnar1^ΔCD8 mice by PCR. G. Validation of Ifnar1 ablation in CD8⁺ cells using the flow cytometry analysis of IFNAR1 levels on the surface of CD4⁺ and CD8⁺ T cells in Ifnar1^+/+ and Ifnar1^ΔCD8 mice. It is representative of n = 3 independent experiments.

Extended Data Fig. 3. PARP11 regulates IFNAR1 downregulation in CD8⁺ T cells

A. qPCR analysis of mRNA of indicated genes in Jurkat cells treated with adenosine (Ado, 1mM) for 30 min. Data are shown as mean ± SEM (n = 3 independently treated cell cultures.). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. B. Flow cytometry analysis of IFNAR1 levels in EL4 cells, in which indicated genes were knocked out by sgRNA mediated CRISPR-Cas9. Data are shown as mean ± SEM. n = 6 independently treated cell cultures. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. C. Immunoblot and qPCR analysis of efficiency of indicated genes knock out (due to the lack of PARP11-specific antibodies, levels of expression of PARP11 is demonstrated by showing the product of qPCR). Levels of β-actin and β-tubulin are shown as loading controls. D. ADP-ribosylation of HA-β-TrCP immunoprecipitated from 293T cells treated or not with adenosine (Ado, 1mM, 30min) as indicated. After treatment, levels of HA-β-TrCP in the whole cell lysate (WCL) was analyzed by immunoblotting. Normalized amounts of lysate containing comparable levels of HA-β-TrCP were taken into immunoprecipitation with HA antibody, which was then analyzed by immunoblotting using anti-ADP-ribose antibody and HA antibody. It is representative of 3 independent repeats with similar results. E. Representative genotyping of Parp11^−/− and Ifnar1^−/− mice. F. Representative flow cytometry data for Fig 3G–H. G. Representative flow cytometry data for Fig 3J. H. Representative flow cytometry data for Fig 3K.

Extended Data Fig. 4. Inactivation of intratumoral CTL and robust tumor growth require IFNAR1-dependent function of PARP11 in the TME

A. Flow cytometry analysis of levels of IFNAR1 on the surface of CD45⁺CD3⁺CD8⁺ T cells in tumor tissues from WT, Parp11^−/−, and Parp11^−/−Ifnar1^−/− mice 13 days after inoculation of s.c. B16F10 tumors (0.5×10⁵/ mouse). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. n = 5 mice. B. Growth of s.c. LLC tumors (1×10<sup>6</sup>/ mouse) inoculated into WT or Parp11<sup>−/−</sup> mice. Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. WT, n = 6 mice; Parp11^−/−, n= = 5 mice. C. Growth of s.c. B16F10 tumor cells (0.5×10⁵/ mouse) inoculated into WT, Parp11^−/−, or Parp11^−/−Ifnar1^−/− mice. Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. *P =0.0387, ****P < 0.0001. n = 5 mice. D. Quantification of B16F10 tumor weights in mice of indicated genotypes at day 13 after inoculation. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. n = 5 mice. E. Flow cytometry analysis of numbers of CD8⁺ T cells in B16F10 tumors growing in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ***P = 0.0007. n = 5 mice. F. Flow cytometry analysis of IFN-γ<sup>+</sup> cells gated on CD45<sup>+</sup>CD3<sup>+</sup>CD8<sup>+</sup> T cells in B16F10 tumors growing in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. n = 5 mice. G. Flow cytometry analysis of TNF-α⁺ cells gated on CD45⁺CD3⁺CD8⁺ T cells in B16F10 tumors growing in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. n = 5 mice. H. Flow cytometry analysis of CD69<sup>+</sup> cells gated on CD45<sup>+</sup>CD3<sup>+</sup>CD8<sup>+</sup> T cells in B16F10 tumors growing in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. n = 5 mice.

Extended Data Fig. 5. PARP11 undermines tumoricidal activities of CAR T cells.

A. Proliferation of CD19-BBz CAR T cells prepared from WT or Parp11^−/− mouse splenic T cells. Data are shown as mean ± SEM (n = 5 samples for each group). Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ns, P = 0.6599. B. Flow cytometry analysis of CAR expression in mouse T cells of indicated genotypes after Meso-BBz transduction. C. Representative flow cytometry analysis of CAR expression in mouse T cells of indicated genotypes after CD19-BBz transduction. D. Weight of hCD19-B16F10 s.c. tumors that grew in NSG mice treated with PBS (Control) or adoptively transferred with CD19-BBz CAR T cells (1×10<sup>6</sup>/ mouse, i.v) of indicated genotypes as in Fig 7D. Data are shown as mean ± SEM (n = 5 mice for each group). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P = 0.0087, ****P < 0.0001, *P = 0.0175. E. Cell surface expression of human CD19 on the surface of hCD19-B16F10 malignant cells is checked by flow cytometry. Data are shown as mean ± SEM (n = 5 mice for each group). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. F. Expression of TIM-3, PD-1, and LAG-3 exhaustion markers by WT or Parp11<sup>−/−</sup> CAR T cells isolated from hCD19-B16 subcutaneous tumors from mice described in Fig S5D. Data are shown as mean ± SEM (n = 5 mice for each group). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P = 0.0270. G. Numbers of WT or Parp11^−/− CAR T cells in the blood, spleen or hCD19-B16 subcutaneous tumors from mice described in Fig S5D. Data are shown as mean ± SEM (n = 5 mice for each group). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P = 0.0038, *P = 0.0212.

Extended Data Fig. 6. Increased efficacy of CAR T cells engineered to inactivate PARP11.

A. qPCR analysis of PARP11 mRNA in shCON-CD19-BBz or shPARP11-CD19-BBz CAR T cells. Data are shown as mean ± SEM (n = 5 independently treated cell cultures.). Two-tailed unpaired t-test was performed for the comparisons between groups. ****P < 0.0001. B. Flow cytometry analysis of CAR expression in human T cells 3 days after shCON-CD19-BBz or shPARP11-CD19-BBz transduction. C. In vitro proliferation of shCON-CD19-BBz and shPARP11-CD19-BBz CAR T cells following stimulation with anti-CD3/CD28 microbeads. Data are shown as mean ± SEM (n = 3 independently treated cell cultures). Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. D. qPCR analysis of PARP11 mRNA in human WT or PARP11 knockout (PARP11 sgRNA) Meso-BBz CAR T cells. Data are shown as mean ± SEM (n = 5 independently treated cell cultures). Two-tailed unpaired t-test was performed for the comparisons between groups. ****P < 0.0001. E. Flow cytometry analysis of IFNAR1 cell surface levels on human WT or PARP11 knockout (PARP11 sgRNA) Meso-BBz CAR T cells. Data are shown as mean ± SEM (n = 5 independently treated cell cultures). Two-tailed unpaired t-test was performed for the comparisons between groups. *P = 0.0128. F. Analysis of killing of EM-Meso-GFP-Luc cells cocultured with human WT or PARP11 knockout (PARP11 sgRNA) Meso-BBz CAR T cells. Data are shown as mean ± SEM (n = 5 independently treated cell cultures). Two-tailed unpaired t-test was performed for the comparisons between groups. ***P = 0.0006.

Extended Data Fig. 7. Inhibitor of p38 kinase increases the efficacy of CAR T cell therapies.

A. Representative flow cytometry analysis of CAR expression in human T cells after Meso-BBz transduction and treatment with vehicle or p38 inhibitor Ralimetinib (LY2228820, LY, 5μM, 72h). B. Flow cytometry analysis of levels of IFNAR1 on the surface of Meso-BBz CAR T cells pretreated with indicated inhibitors (LY2228820, LY, 5μM, 72h) and then treated by adenosine (Ado, 1mM) for 24h. Data are shown as mean ± SEM (n = 4 independently treated cell cultures). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. C. Lysis of EM-Meso-GFP-Luc cells by Meso-BBz CAR T pretreated with inhibitors (LY2228820, LY, 5μM, 72h) at indicated conditions (E: T =10:1). Data are shown as mean ± SEM (Veh treated with or without Ado, n = 5 co-cultures; LY treated with or without Ado, n = 5 co-cultures.). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P < 0.0056. D. Tumor growth of NSG mice that were injected s.c. with 1×10⁶ EM-Meso-GFP-Luc cells. Mice were i.v. treated with 2×10⁶ BBZ-SS1 CAR T cells on day 7. BBZ-SS1 CAR T cells were pretreated with LY (5μM) or Veh for 72h before injected to mice. Data are shown as mean ± SEM (Control, n = 7 mice; BBZ-SS1, n = 9 mice; BBZ-SS1 +LY, n = 10 mice). Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. E. The Kaplan-Meier analysis of survival of animals from experiment described in Fig S7D (n = 5 mice for each group). Statistical analysis was performed using Gehan-Breslow-Wilcoxon test.

Figure 1.. Downregulation of IFNAR1 on the intratumoral CTL undermines their activities.

F. Flow cytometry analysis of IFNAR1 levels on the surface of CD45⁺CD3⁺CD8⁺ T cells in spleen (Sp) and tumor (Tu) tissues from WT or SA mice on day 21 after inoculation of s.c. MC38 tumors (1×10⁶ cells/mouse). Quantification data (on the right) are shown as mean ± SEM (n = 6 mice for each group). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. G. Immune cells were isolated from MC38 tumors growing in WT or SA mice on day 14 post-inoculation. N=9,725 cells were used for the scRNA-seq analyses. t-SNE plot of CD8+ T cells with clusters demarcated by colors demonstrating WT (blue, n = 2075 cells) and SA (red, n = 2038 cells). H. Gene Set Enrichment Analysis (GSEA) of differentially expressed genes comparing CD8<sup>+</sup> T cells in WT and SA groups. Black and red colors denote genesets enriched in WT and SA, respectively. I. Leading-edge plots showing results from Gene Set Enrichment Analysis (GSEA) of CD8<sup>+</sup> T cell function relevant genesets comparing CD8<sup>+</sup> T cells in WT and SA groups. J. Dot plot showing the expression of T cells exhaustion (underlined below) and function related genes. The size of the dot corresponds to the percentage of cells expressing the gene in each group and the color represents the average expression level. WT, n = 2075 cells; SA, n = 2038 cells. K. t-SNE plot of CD8+ T cells with clusters demarcated by colors demonstrating seven clusters based on gene expression (as in Fig S1C). L. t-SNE plot analysis of proportion of CD8<sup>+</sup> T cell clusters (as in Fig 1F) in WT and SA samples. WT, n = 2075 cells; SA, n = 2038 cells. M. Flow cytometry analysis of IFN-γ positive CD8+ T cells in naive spleens and tumor tissues from mice of indicated genotypes. Data are shown as mean ± SEM (WT and SA spleen, n = 5 mice; WT and SA tumor, n = 6 mice). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P = 0.0050.

Figure 2.. Downregulation of CTL IFNAR1 by Treg and adenosine

A. iTregs were cocultured with OT-1 cells (Treg: OT-1=1:3) for 24h. Then, IFNAR1 on the surface of OT-1 cells were analyzed by flow cytometry. Two-tailed unpaired t-test. **P = 0.0085. n = 3 co-cultures. B. Lysis of MC38OVA-Luc cells by WT or SA OT-1 cells cocultured with iTregs at indicated conditions (Treg: OT-1=1:3, E:T ratio=10:1). Two-tailed unpaired t-test. *P = 0.0259. n = 3 co-cultures. C. iTregs were cocultured (Treg: OT-1=1:3) with OT-1 cells (± CPI-444, 10μM 1h). Then IFNAR1 on the surface of OT-1 cells were analyzed by flow. One-way ANOVA. **P = 0.0022, *P = 0.0416. n = 5 co-cultures. D. Lysis of MC38OVA-Luc cells by OT-1 cells (± CPI-444, 10μM 1h) cocultured with iTregs at indicated conditions (Treg: OT-1=1:3, E:T ratio=10:1). One-way ANOVA. ***P = 0.0002, *P = 0.0410. n = 6 co-cultures. E. IFNAR1 on the surface of indicated CD8⁺ T cells (±Ado, 1mM, 2h). One-way ANOVA. **P = 0.0018, ****P < 0.0001. n = 9 mice. F. qPCR analysis of mRNA in the indicated mouse splenocytes pretreated with adenosine (Ado, 1mM) for 2h and then stimulated with IFN-β (1000IU/ml) for 16h. One-way ANOVA.. **P < 0.01, ****P < 0.0001. n = 3 mice. G. IFN-γ levels in indicated CD8⁺ T cells (±Ado, 1mM, 24h). One-way ANOVA. **P = 0.0068. n = 3 mice. H. Lysis of MC38OVA-luc cells by OT-1 cells (±Ado, 1mM 24h). One-way ANOVA. **P = 0.0024. n = 3 co-cultures. I. Growth of MC38 tumors (1×10⁶ cells/mouse, s.c.) inoculated into WT (Ifnar1^+/+) or Ifnar1^ΔCD8 mice and treated daily with CPI-444 (10mg/kg, oral gavage) or vehicle on days 1–12. Ifnar1^+/+, Ifnar1^ΔCD8, n = 13 mice; Ifnar1^+/++CPI444, n = 7 mice; Ifnar1^+/++CPI444, n = 8 mice. Two-way ANOVA. *P = 0.0364, ****P < 0.0001. J. The Kaplan-Meier analysis of survival from experiment described in Fig 2I (Ifnar1^+/+, n = 12 mice; Ifnar1^ΔCD8, n = 11 mice; Ifnar1^+/++CPI444, n = 11 mice; Ifnar1^ΔCD8 +CPI444, n = 8 mice). Log-rank (Mantel-Cox) test. *P = 0.0295, ***P = 0.0008.

Figure 3.. PARP11 regulates IFNAR1 downregulation in CD8⁺ T cells

A. Immunoblot analysis of ubiquitination, phosphorylation, and total levels of IFNAR1 in Jurkat cells treated with adenosine (Ado, 1mM) at indicated time points. B. Immunoblot analysis of phosphorylation and levels of p38α, and levels of IFNAR1 and β-TrCP in Jurkat cells treated with adenosine (Ado, 1mM) at indicated time points. A and B are representative of 3 independent repeats with similar results. C. qPCR analysis of mRNA for Parp11 in T cells treated with serum-free media (SFM) or with tumor condition media (TCM) for 48h. Two-tailed unpaired t-test. ***P = 0.0009. n = 4 mice. D. qPCR analysis of Parp11 mRNA in CTLs isolated from tumors or spleens of MC38 tumor bearing mice. Two-tailed unpaired t-test. **P = 0.004. Sp, n = 9 mice; Tu, n = 9 mice. E. ADP-ribosylation of β-TrCP immunoprecipitated from human T cells treated or not with adenosine (1mM, 30min) as indicated was analyzed by immunoblot. F. In vitro ADP-ribosylation of Myc-β-TrCP immunoprecipitated with Myc or control antibody and incubated on beads with biotinylated NAD⁺ in the presence of soluble Flag-PARP11 purified from 293T cells treated or not with adenosine (1mM, 30min) as indicated. Reactions were probed with streptavidin-HRP conjugate. Input levels of Myc-β-TrCP and Flag-PARP11 are also shown. E and F are representative of 3 independent repeats with similar results. G. Flow cytometry analysis of phospho-CREB in (±Ado, 1mM, 2h). One-way ANNOVA. n = 5 mice. H. Flow cytometry analysis of phospho-PKA in (±Ado, 1mM, 2h). One-way ANNOVA. n = 5 mice. I. Immunoblot analysis of β-TrCP levels in indicated splenocytes treated with or without adenosine (Ado, 1mM, 30 min) as indicated. It is representative of 3 independent repeats with similar results. J. Flow cytometry analysis of levels of IFNAR1 in indicated CTL (± CPI-444, 10 μM, 1h) and then treated with adenosine (Ado, 1mM) or tumor conditioned media (TCM) as indicated for 2h. One-way ANNOVA. ****P < 0.0001. n = 4 mice. K. Flow cytometry analysis of levels of IFNAR1 in Parp11^−/− CTL transduced with empty virus (GFP-con) or viruses for expression of human WT PARP11 or HYm PAPR11 mutant and treated with adenosine (Ado, 1mM, 2h). One-way ANNOVA. ****P < 0.0001. n = 4 mice.

Figure 4.. Inactivation of intratumoral CTL and robust tumor growth require IFNAR1-dependent function of PARP11 in the TME

A. Flow cytometry analysis of levels of IFNAR1 on the surface of CD45⁺CD3⁺CD8⁺ T cells in tumor tissues from WT, Parp11^−/−, and Parp11^−/−Ifnar1^−/− mice on days 20 after inoculation of s.c. MC38 tumors (0.5×10⁶ cells/ mouse). Quantification is shown on the right. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. *P = 0.0111, ***P = 0.0002. n = 5 mice. B. Volume of MC38 s.c. tumors in mice of indicated genotypes. Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ***P = 0.0009. n = 5 mice. C. Tumor weight of MC38 tumors grown in mice of indicated genotypes (Day 20). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. *P = 0.0387, ***P = 0.0007. n = 5 mice. D. Flow cytometry analysis of numbers of CD8<sup>+</sup> T cells in MC38 tumors growing in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. *P < 0.05. n = 5 mice. E. Flow cytometry analysis of IFN-γ⁺ cells gated on CD45⁺CD3⁺CD8⁺ T cells isolated from MC38 tumors grown in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P < 0.01. n = 5 mice. F. Flow cytometry analysis of TNF-α<sup>+</sup> cells gated on CD45<sup>+</sup>CD3<sup>+</sup>CD8<sup>+</sup> T cells isolated from MC38 tumors grown in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P = 0.0081, ***P = 0.0005. n = 5 mice. G. Flow cytometry analysis of CD69⁺cells gated on CD45⁺CD3⁺CD8⁺ T cells isolated from MC38 tumors grown in indicated mice. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. *P = 0.0164, **P = 0.0016. n = 5 mice.

Figure 5.. PARP11 undermines tumoricidal activities of CAR T cells.

N. Relative expression of PARP11 in CD8⁺ T cell subsets isolated from colorectal cancer patients . Data in naïve, effector memory and highly active effector CD103⁺CD39⁺ subpopulations are shown. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. *P = 0.0312, ****P < 0.0001, **P = 0.0052. n = 7 samples. O. Flow cytometry analysis of the IFNAR1 levels on indicated Meso-BBz CAR T cells treated with or without Ado (Ado, 1mM, 24h). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001, **P = 0.0021. n = 6 independently treated cell cultures. P. Lysis of EM-Meso-GFP-Luc cells by Meso-BBz CAR T cells produced from WT and Parp11^−/− mice treated as indicated (E:T ratio=10:1). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P < 0.0027, *P = 0.0144. n = 6 independently treated cell cultures. Q. Flow cytometry analysis of the IFNAR1 levels on CD19-BBz CAR T cells produced from WT, Parp11<sup>−/−</sup>, and Parp11<sup>−/−</sup>Ifnar1<sup>−/−</sup> mice treated as indicated. Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. n = 5 mice. R. Lysis of hCD19-B16F10 cells by CD19-BBz CAR T cells produced from WT, Parp11^−/−, and Parp11^−/−Ifnar1^−/− mice treated as indicated (E:T ratio=10:1). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ***P = 0.0002, ****P < 0.0001. n = 6 independently treated cell cultures. S. Tumor growth (s.c.) of hCD19-B16F10 tumor-bearing C57BL/6 mice adoptively transferred with CD19-BBz CAR T cells (2×10<sup>6</sup>/mouse, i.v. at day 7) as indicated. Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. **P = 0.0019. Control, n = 7 mice, Parp11^−/−, n = 6 mice; WT, n = 5 mice, Parp11^−/− Ifnar1^−/−, n = 5 mice. T. The Kaplan-Meier analysis of survival of animals from experiment described in Fig 5F. Statistical analysis was performed using Gehan-Breslow-Wilcoxon test. ***P = 0.0007. Control, n = 7 mice, Parp11^−/−, n = 6 mice; WT, n = 5 mice, Parp11^−/− Ifnar1^−/−, n = 5 mice.

Figure 6.. Increased efficacy of CAR T cells engineered to inactivate PARP11

A. Schematic representation of shCON and shPARP11-CD19-BBz chimeric receptors. B. Flow cytometry analysis of levels of IFNAR1 on the surface of indicated CAR T cells treated with or without adenosine (Ado, 1mM, 24h). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. **P = 0.0033, ***P = 0.0009. n = 5 samples. C. Lysis of hCD19-B16F10 cells by shCON-CD19-BBz or shPARP11-CD19-BBz CAR T cells treated with or without adenosine (Ado, 1mM, 24h). (E:T ratio=10:1). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. n = 6 independently treated cell cultures. D. Lysis of NALM6 cells that express endogenous CD19 by shCON-CD19-BBz or shPARP11-CD19-BBz CAR T cells treated with or without adenosine (Ado, 1mM, 24h). (E:T ratio=10:1). Statistical analysis was performed using ordinary one-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001, *P = 0.0426. n = 6 independently treated cell cultures. E. hCD19-B16F10 tumor growth in NSG mice inoculated with 5×10<sup>5</sup> hCD19-B16 cells (s.c.) and 7 days later administered with indicated CAR T cells (1×10<sup>6</sup>/ mouse, i.v). Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001, ***P = 0.0005. Con, n = 4 mice; shCon, n = 5 mice; shPARP11, n = 5 mice. F. The Kaplan-Meier analysis of survival of animals from experiment described in Fig 6E (Con, n = 4 mice; shCon, n = 5 mice; shPARP11, n = 5 mice). Statistical analysis was performed using Log-rank (Mantel-Cox) test. *P = 0.0138, **P = 0.0026. G. Growth of EM-Meso-GFP-Luc tumors (1×10⁶/mouse, s.c.) in NSG mice that were administered with control (PBS) or WT or PARP11 knockout Meso-BBz CAR T cells (1×10⁶/ mouse, i.v. on day 7). Statistical analysis was performed using two-way ANOVA with Tukey`s multiple comparisons test. ****P < 0.0001. n=5 mice. H. The Kaplan-Meier analysis of survival of animals from experiment described in Fig. Statistical analysis was performed using Log-rank (Mantel-Cox) test. **P = 0.0019, *P = 0.0174. n = 5 mice.

Figure 7.. Rucaparib increases the efficacy of CAR T cell therapies.

A. Immunoblot analysis of β-TrCP and IFNAR1 levels in Jurkat cells (± Ruc, 1uM, 2h) and then treated with or without adenosine (Ado, 1mM) for 30 min. It is representative of 3 independent repeats with similar results. B. Volume of MC38 tumors (5×10⁵ cells/mouse, s.c.) in Ifnar1^+/+ and Ifnar1^ΔCD8 mice that were administered with Rucaparib (Ruc, 40mg/kg) or vehicle every other day from d7. Two-way ANNOVA. **P = 0.0034, ****P < 0.0001. Ifnar1^+/+ +Veh, Ifnar1^ΔCD8 +Veh, and Ifnar1^ΔCD8 +Ruc, n = 5 mice; Ifnar1^+/+ +Ruc, n = 7 mice. C. The Kaplan-Meier analysis of survival from Fig. 7B. Log-rank (Mantel-Cox) test. **P = 0.0046, *P = 0.0230. Ifnar1^+/+ +Veh, Ifnar1^ΔCD8 +Veh, and Ifnar1^ΔCD8 +Ruc, n = 5 mice; Ifnar1^+/+ +Ruc, n = 7 mice. D. Growth of hCD19-B16F10 tumors (5×10⁵ cells/mouse, s.c.) in NSG mice that were administered with WT or Parp11^−/− CD19-BBz CAR T cells (10⁶ /mouse, i.v. at d7) and Rucaparib (20mg/kg by oral gavage at d10, 12, 14) or vehicle. Two-way ANNOVA. ****P < 0.0001. n = 5 mice. E. IFNAR1 levels on Meso-BBz CAR T (±Ruc, 10μM, 72h) and treated with or without adenosine (Ado, 1mM, 24h). Two-tailed unpaired t-test. ****P < 0.0001. Veh treated with or without Ado, n = 4 independently treated cell cultures; Ruc treated with Veh, n = 6 independently treated cell cultures; Ruc treated with Ado, n = 5 independently treated cell cultures. F. Killing efficacy of human Meso-BBz CAR T (± Ruc, 10μM, 72h), then treated with adenosine (Ado, 1mM, 24h) cocultured with EM-Meso-GFP-Luc cells (E:T =10:1). One-way ANNOVA. ****P < 0.0001. n = 6 independently treated cell cultures. G. Growth of EM-Meso-GFP-Luc tumors (1×10⁶ cells/mouse, s.c.) in NSG mice that were administered with 0.5×10⁶ Meso-BBz CAR T cells (i.v) on Day 7 and Rucaparib (40mg/kg) or vehicle every other day from Day 7 for 6 times. Two-way ANNOVA. ****P < 0.0001. Control, n = 3 mice; Ruc, Meso CAR T +Veh, Meso CAR T+Ruc, n = 5 mice. H. The Kaplan-Meier analysis of survival from Fig. 7G. Log-rank (Mantel-Cox) test. **P < 0.01. Control, n = 3 mice; Ruc, Meso CAR T +Veh, Meso CAR T+Ruc, n = 5 mice.