Fig. 3: Loss of components of the IFNγR/JAK/STAT pathway sensitizes tumors to CAR-T therapy in vivo.

a Schematic showing the IFNγR signaling pathway. b, c In vivo competitive assays demonstrate specific depletion of Cas9⁺ RH62 B-ALL cells lacking components of the IFNγR/JAK/STAT pathway and enrichment of B-ALL cells lacking mCD19 after treatment with anti-mCD19 CAR-T cells in b the bone marrow and c spleen. For b, c, n = 9 for the groups Ifngr1 sgRNA#1, Jak2 sgRNA#1 and 2, treated with anti-hEGFRvIII CAR-T cells; n = 8 for the groups Cd19 sgRNA#1 treated with anti-hEGFRvIII or anti-mCD19 CAR-T cells; n = 6 for the group Jak2 sgRNA#1 treated with anti-mCD19; n = 4 for the groups Ifngr1 sgRNA#1 and 2, Stat1 sgRNA#2, and lacZ sgRNA#1, treated with anti-mCD19 CAR-T cells; n = 5 for all other groups. d Kaplan-Meier curves showing survival in immunocompetent mice transplanted with B-ALL cells deficient in the indicated IFNγR/JAK/STAT pathway member. e In vivo competitive assays demonstrate specific depletion of Cas9⁺ RH62 B-ALL cells lacking Ptpn2 or Fitm2 after treatment with anti-mCD19 CAR-T cells in the spleen. Data shown is from flow cytometry analyses examining the proportion of live B-ALL cells that are APC⁺ and therefore, also guide-bearing. In these experiments, n = 5 mice in groups treated with anti-hEGFRvIII CAR-T cells and n = 3 mice in groups treated with anti-mCD19 CAR-T cells. f A Kaplan–Meier curve showing overall survival in leukemia-bearing mice treated with 2.5 × 10⁶ control or anti-mCD19 CAR-T cells in the presence or absence of blocking IFNγ antibody. g A Kaplan-Meier curve of overall survival in leukemia-bearing mice treated with control or 2.5 × 10⁶ anti-mCD19 CAR-T cells in the presence or absence of Ruxolitinib, a JAK1/2 inhibitor. In vivo competition assays were repeated three times. Survival experiments were completed twice. Pharmacologic studies using anti-IFNγ blocking antibodies or JAK inhibitor were completed twice. The significance of survival experiments was determined using log-rank tests. For all other experiments, significance was determined using unpaired two-sided student’s t-tests with Bonferroni correction for multiple comparisons. Data are mean ± s.e.m. *P < 0.05; **P  <  0.01; ***P  <  0.001; ****P  <  0.0001. Exact P values for each comparison shown in (b-d) and (f-g) can be found in Supplementary Data 3.