CD38-RyR2 axis mediated signaling impedes CD8+  T cell response to anti-PD1 therapy in  cancer
PD-1 blockade therapy, harnessing the cytotoxic potential of CD8+  T cells, has yielded clinical success in treating malignancies. However, its efficacy is often limited due to the progressive differentiation of intratumoral CD8+ T cells into a hypofunctional state known as terminal exhaustion. Despite identifying CD8+ T cell subsets associated with immunotherapy resistance, the molecular pathway triggering the resistance remains elusive. Given the clear association of CD38 with CD8+ T cell subsets resistant to anti-PD1 therapy, we investigated its role in inducing resistance. Phenotypic and functional characterization, along with single-cell RNA sequencing analysis of both in vitro chronically stimulated and intratumoral CD8+ T cells, revealed that CD38-expressing CD8+ T cells are terminally exhausted. Exploring the molecular mechanism, we discovered that CD38 expression was crucial in promoting terminal differentiation of CD8+ T cells by suppressing TCF1 expression, thereby rendering them unresponsive to anti-PD1 therapy. Genetic ablation of CD38 in tumor reactive CD8+ T cells restored TCF1 levels and improved the responsiveness to anti-PD1 therapy in mice. Mechanistically, CD38 expression on exhausted CD8+ T cells elevated intracellular Ca2+ levels through RyR2 calcium channel activation. This, in turn, promoted chronic AKT activation, leading to TCF1 loss. Knockdown of RyR2 or inhibition of AKT in CD8+ T cells maintained TCF1 levels, induced a sustained anti-tumor response, and enhanced responsiveness to anti-PD1 therapy. Thus, targeting CD38 represents a potential strategy to improve the efficacy of anti-PD1 treatment in cancer. Overall design: Peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-hypaque gradient centrifugation from de-identified buffy coats obtained from two healthy human subjects (Donor 1 and Donor2). Naïve CD3 T cells were purified using the Dynabeads Untouched Human CD3+ T-cell isolation kit (Invitrogen). Purified CD3+ T cells were activated with plate-bound anti-CD3 (5 µg/ml) and anti-CD28 (2 µg/ml), along with recombinant human IL-2 (100 units/ml), in complete RPMI-1640 supplemented with 10% FBS (Invitrogen) for 72 hours. Following 72 h of activation, T cells were passaged at every 48h at 1×10^6 cells/ml in RPMI-1640 medium containing 10% FBS, with (chronic) or without (referred to as control in the text) plate-bound anti-CD3 (2 µg/ml) until 11 days. The chronic group was maintained in recombinant human IL-2 (25 units/ml), while the control group was maintained in recombinant human IL-2 (100 units/ml). On day 11, live cells were sorted using FACS. Cell viability was determined using Trypan Blue on the ThermoFisher Scientific, Countess® II FL Automated Cell Counter. Approximately, 0.1X10^6 T cells were labelled using BDTM Single-Cell Multiplexing Kit-Human with unique tags. The control samples i.e. Control1 (obtained from Donor1) and Control2 (obtained from Donor2) were labelled with SampleTag 1 and Sample Tag3 respectively while the chronically stimulated samples i.e. Chr. St. 1 (obtained from Donor1) and Chr. St. 2 (obtained from Donor2) were labelled with SampleTag 2 and Sample Tag 4 respectively. Thereafter, these samples were taken for further steps of single cell sequencing. Finally, all sequencing libraries were prepared using BD RhapsodyTM WTA Amplification kit as per the manufacturer's guideline. The unique nucleotide sequences of individual sampleTag are provided as text file. The raw data fastq file is a pooled sample file, while the processed files are matrix tables containing raw gene counts for individual genes and single cells. Each processed file represents one sample and is generated from processing the aforementioned FASTQ file.