Dijk, Sarah van

Abstract Sensing of extracellular metabolites controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin-1 (Panx1). Here, we report that T cell-specific Panx1 is needed for efficient CD8+ T cell responses to viral infection and cancer. Panx1 favors both the expansion of effector CD8+ T cells and the survival of memory CD8+ T cells. Our data suggests that, while memory CD8+ T cells require Panx1 for mitochondrial function, Panx1 promotes the glycolytic pathway in effector CD8+ T cells. Panx1 promotes memory CD8+ T cell survival together with the eATP sensor P2RX7. However, Panx1 induces effector CD8+ T cells independently of eATP. Rather, we found an unexpected link between Panx1, lactate export and the activation of effector CD8+ T cells. Therefore, Panx1 regulates effector and memory CD8+ T cells through export of distinct metabolites and by engaging different intracellular pathways.

Abstract Development of universal influenza (flu) vaccines requires better knowledge of lung-resident immune responses. That includes flu-specific CD4 T cells, which establish long-term lung residency and induce a diversified local protective response to subsequent infection. Tissue-resident helper (Trh) cells are a subset of lung-resident CD4 T cells that share phenotypic and functional features with follicular helper cells (Tfh). Trh cells serve to amplify lung-resident immune responses, and express high levels of the extracellular ATP (eATP) sensor P2RX7. Considering that eATP levels in the lung increase in response to flu infection, we used T cell-specific P2RX7-knockout (KO) mice to test the role of P2RX7 for CD4 T cell responses to flu. During acute phase of infection, we found that P2RX7 promotes the accumulation of flu-specific CD4 T cells inside the lung tissue. Moreover, P2RX7 promoted this establishment via induction of CXCR3 upregulation. Despite no specific defects in the Trh pool at the acute phase, T cell-specific P2RX7-KO led to a progressive decay of Trh cells. Consequently, lungs of T cell-P2RX7-KO mice had reduced lung B cell responses. Our single-cell RNAseq of lung flu-specific CD4 T cells showed reduced expression of cell survival and mitochondrial genes in P2RX7-KO Trh cells. Our findings suggest that lung eATP sensing by CD4 T cells induces lung-resident CD4 T cell accumulation in response to flu, impacting the survival of specific lung CD4 T cell subsets. National Institute of Allergy and Infectious Diseases (NIAID) (H.BdS.: R00 AI139381, R01 AI170649).

Abstract Development of central memory (Tcm) and resident memory (Trm) CD8+ T cells, which respectively promote immunity in the circulation and in barrier tissues, is not completely understood. Tcm and Trm cells may arise from common precursors, however their fate-inducing signals are elusive. We found that virus-specific effector CD8+ T cells display heterogeneous expression of the extracellular ATP sensor P2RX7. P2RX7-high expression is confined, at peak effector phase, to CD62L+ memory precursors which preferentially form Tcm cells. Among early effector CD8+ T cells, asymmetrical P2RX7 distribution correlated with distinct transcriptional signatures, with P2RX7-high cells enriched for memory and tissue-residency sets. P2RX7-high early effectors preferentially form both Tcm and Trm cells. Defective Tcm and Trm formation in P2RX7 deficiency is significantly reverted when the transcriptional repressor Zeb2 is ablated. Mechanistically, P2RX7 negatively regulates Zeb2 expression, at least partially through TGF-β sensing in early effector CD8+ T cells. Our study indicates that unequal P2RX7 upregulation in effector CD8+ T cells is a foundational element of the early Tcm/Trm fate. Supported by grants from NIH (R00 AI139381, NIAID).

Abstract Tissue-derived extracellular ATP (eATP) is a “danger signal” sensed by immune cells via purinergic receptors (e.g. P2RX7). In addition, healthy cells can export eATP through transmembrane channels, such as the hemichannel Pannexin-1 (Panx1). CD8+ T cells express Panx1 constitutively throughout developmental and immune response stages. However, the biological role of Panx1 in CD8+ T cells is undefined. Here, we used genetic and pharmacological tools to address this question. T-cell specific Panx1 deletion did not affect CD8+ T cell thymic development. In contrast, CD8+ T cell responses to viral infection (LCMV) are impaired by Panx1 inhibition or deletion. This is true for effector expansion of CD8+ T cells and for the survival of memory CD8+ T cells. Using adoptive transfer of tamoxifen-conditional Panx1 knockout LCMV-specific (P14) cells into LCMV-infected mice, we showed that memory CD8+ T cell survival requires sustained Panx1 – except for small intestinal-resident cells. Mechanistically, Panx1 promotes memory CD8+ T cell survival via P2RX7, which also favors memory CD8+ T cells - as suggested by our data with Panx1/P2RX7-double knockout P14 cells. The role of Panx1 for effector CD8+ T cell expansion, however, is unlikely to be via P2RX7 since this receptor does not have a role in effector CD8+ T cells. Our transcriptional and metabolic data suggests that, while memory CD8+ T cells require Panx1 for optimal mitochondrial function and expression of pro-memory genes, effector CD8+ T cells lacking Panx1 had decreased activation of the glycolytic pathway. Collectively, these results suggest that, in response to acute virus, Panx1 induces CD8+ T cell effector expansion and memory survival through distinct metabolic pathways. Supported by grants from NIH (R00 AI139381, NIAID).

Abstract Development of CD8+ central memory T (Tcm) and resident memory T (Trm) cells, which promote immunity in the circulation and in barrier tissues, respectively, is not completely understood. Tcm and Trm cells may arise from common precursors; however, their fate-inducing signals are elusive. We found that virus-specific effector CD8+ T cells display heterogeneous expression of the extracellular ATP sensor P2RX7. P2RX7-high expression is confined, at peak effector phase, to CD62L+ memory precursors, which preferentially form Tcm cells. Among early effector CD8+ T cells, asymmetrical P2RX7 distribution correlated with distinct transcriptional signatures, with P2RX7-high cells enriched for memory and tissue residency sets. P2RX7-high early effectors preferentially form both Tcm and Trm cells. Defective Tcm and Trm cell formation in P2RX7 deficiency is significantly reverted when the transcriptional repressor Zeb2 is ablated. Mechanistically, P2RX7 negatively regulates Zeb2 expression, at least partially through TGF-β sensing in early effector CD8+ T cells. Our study indicates that unequal P2RX7 upregulation in effector CD8+ T cells is a foundational element of the early Tcm/Trm fate.