Sequential JAK inhibition boosts antitumor T cell responses to combined anti-PD-1 and anti-CTLA4

While immune Checkpoint Inhibition (CPI) has reshaped cancer treatment, the majority of cancer patients do not benefit from this treatment approach, and this treatment can lead to immune related adverse events. While induction of IFNγ responses are thought be necessary for anti-tumor immunity, growing evidence also implicates IFNγ as a tumor-intrinsic mediator of CPI resistance. CPI-induced IFNγ also mediates activation induced cell death in T cells as an immune-intrinsic mechanism of resistance. In this study, we show that transient block of IFNγ signaling through administration of the JAK1 inhibitor ABT-317 enhances anti-tumor T cell responses with CPI in pre-clinical models. Importantly sequential but not concomitant ABT-317 treatment, led to significantly reduced toxicity and improved tumor efficacy. Sequential treatment reduced activation-induced T cell death and enhanced expansion of tumor-reactive T cell subsets with increased effector function in vivo and ex vivo. Only CPI in combination with ABT-317 was able to protect mice from tumor rechallenge. These results demonstrate that the JAK inhibition within a window following CPI can used to address an immune-intrinsic mechanism of therapeutic resistance. 8- to 10-week-old male WT C57BL/6J mice were implanted s.c. with 1 million TRAMP-C2 or MC38 cells and treated i.p. with Isotype or CPI (anti-CTLA4+antiPD-1) on days 3, 6 and 9, followed by vehicle (DPBS) or 20 mg/kg ABT-317 on day 12. On day 15 after implantation, tumor, and TDLNs from mice were harvested and processed to single cell suspension as described. To enrich for CD45+ cells in tumors, total tumor lysates were stained for Live/Dead and CD45. Live CD45- or CD45+ (tumors) were sorted with a FACS Aria into separate tubes and barcoded by with mouse TotalSeq-C03 hashtag antibodies 1-10 (155861, 155863, 155865, 155867, 155869, 155871, 155873, 155875, 155877, 155879, Biolegend) at a 1:200 dilution for 40 minutes at 4C to identify individual samples and treatment groups. Then, individual reactions of 6 x 104 cells each with 1:1 CD45+:CD45- (tumors) ratios (unsorted total cells for TDLNs) and equal hashtag representation for batch correction purposes were prepared at the Chromium Controller (10X Genomics) using the Chromium Next GEM Single Cell 5' Kit v2 (1000263, 10X Genomics) and the Chromium Single Cell Mouse TCR Amplification kit (1000254, 10X Genomics) and sequencing was performed on an Illumina NovaSeq 6000 with paired-end 100 base pair read lengths.