Silva, Henrique Borges da

Abstract The tumor microenvironment (TME) is characterized by high levels of extracellular ATP (eATP) and adenosine, which are sensed by purinergic receptors. CD8+ T cells express two important receptors that bind these ligands: the extracellular receptor P2RX7 binds eATP and is generally immunostimulatory, while the adenosine receptor A2AR promotes immunosuppression. However, the role of the TME itself in the T cell expression of purinergic receptors is not clear. We used in vitro co-cultures of tumor cells and CD8+ T cells, and specifically found that A2AR is significantly upregulated after activation, in a tumor cell number-correlated manner. These tumor-induced changes in A2AR expression are independent of TCR:MHC contact, which was evidenced by co-cultures using transwells to physically separate tumor cells and T cells, as well as distinct combination of transgenic T cells and tumors. In vivo murine solid tumor models confirmed that tumor-infiltrating CD8+ T cells upregulate A2AR. As previously found by our group, P2RX7 expression is limited to a small percentage of tumor-infiltrating CD8+ T cells. Interestingly, we identified that these few P2RX7-expressing tumor-infiltrating CD8+ T cells are enriched for high A2AR expression – suggesting that co-expression of these two receptors is favored in T cells exposed to tumors. Overall, our findings suggest that TME exposure – likely through TCR-independent factors – directly promote the expression of A2AR in CD8+ T cells. We are currently investigating the nature of the tumor-derived factor(s) promoting this upregulation, as well as the possible role of A2AR/P2RX7 co-expressing tumor-infiltrating CD8+ T cells as effector antitumor cells with longer intratumoral lifespan. 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 Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

AbstractIdentifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.

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.

Abstract Memory CD8+ T cells promote protective immunity against viruses or cancer. Our field has done a terrific job identifying how CD8+ T cell memory forms in response to Ag. However, many studies focused on systems in which inflammation recedes over time. These situations, while relevant, do not cover all situations in which CD8+ T cell memory is relevant. It is increasingly clear that CD8+ T cells with a memory phenotype form in response to infections with extensive or prolonged tissue inflammation, for example, influenza, herpes, and more recently, COVID-19. In these circumstances, inflammatory mediators expectedly affect forming memory CD8+ T cells, especially in tissues in which pathogens establish. Notwithstanding recent important discoveries, many outstanding questions on how inflammation shapes CD8+ T cell memory remain unanswered. We will discuss, in this review, what is already known and the next steps to understand how inflammation influences CD8+ T cell memory.