The Extracellular ATP Receptor P2RX7 Imprints a Promemory Transcriptional Signature in Effector CD8+ T Cells.

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-ß 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.

The ATP-exporting channel Pannexin 1 promotes CD8+ T cell effector and memory responses.

Sensing of extracellular ATP (eATP) controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin 1 (Panx1). Whether Panx1 controls CD8+ T cell immune responses in vivo, however, has not been previously addressed. Here, we report that T-cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 promotes both effector and memory CD8+ T cell responses. Panx1 favors initial effector CD8+ T cell activation through extracellular ATP (eATP) export and subsequent P2RX4 activation, which helps promote full effector differentiation through extracellular lactate accumulation and its subsequent recycling. In contrast, Panx1 promotes memory CD8+ T cell survival primarily through ATP export and subsequent P2RX7 engagement, leading to improved mitochondrial metabolism. In summary, Panx1-mediated eATP export regulates effector and memory CD8+ T cells through distinct purinergic receptors and different metabolic and signaling pathways.

Pannexin-1 channels promote CD8+ T cell effector and memory responses through distinct intracellular pathways.

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). Whether Panx1 controls CD8+ T cell immune responses to antigen, however, has not been previously addressed. Here, we report that T cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 favors memory CD8+ T cell survival primarily through ATP export and induction of mitochondrial metabolism. CD8-specific Panx1 is also crucial for the effector expansion of CD8+ T cells, however this regulation occurs independently of eATP. Instead, our results suggest a connection between Panx1-induced extracellular lactate accumulation and the complete activation of effector CD8+ T cells. In summary, Panx1 regulates effector and memory CD8+ T cells through export of distinct metabolites and by engaging different metabolic and signaling pathways.

T cell-specific P2RX7 favors lung parenchymal CD4+ T cell accumulation in response to severe lung infections.

CD4+ T cells are key components of the immune response during lung infections and can mediate protection against tuberculosis (TB) or influenza. However, CD4+ T cells can also promote lung pathology during these infections, making it unclear how these cells control such discrepant effects. Using mouse models of hypervirulent TB and influenza, we observe that exaggerated accumulation of parenchymal CD4+ T cells promotes lung damage. Low numbers of lung CD4+ T cells, in contrast, are sufficient to protect against hypervirulent TB. In both situations, lung CD4+ T cell accumulation is mediated by CD4+ T cell-specific expression of the extracellular ATP (eATP) receptor P2RX7. P2RX7 upregulation in lung CD4+ T cells promotes expression of the chemokine receptor CXCR3, favoring parenchymal CD4+ T cell accumulation. Our findings suggest that direct sensing of lung eATP by CD4+ T cells is critical to induce tissue CD4+ T cell accumulation and pathology during lung infections.

Extracellular vesicle proteomic analysis leads to the discovery of HDGF as a new factor in multiple myeloma biology.

Identifying 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.

Metabolic regulation of tissue-resident memory CD8+ T cells.

Intracellular metabolic adaptations help define the function and homeostasis of memory CD8+ T cells. These cells, which promote protection against infections or cancer, undergo consecutive metabolic shifts, ultimately relying on mitochondrial-related pathways. Past CD8+ T cell metabolism studies focused on circulating memory cells, which are exclusive to secondary lymphoid organs or recirculate between lymphoid and non-lymphoid organs. Yet, now there is unequivocal evidence that memory CD8+ T cells reside in many non-lymphoid organs and mediate protective immunity in barrier tissues. The metabolic adaptations occurring in forming and established tissue-resident memory CD8+ T cells are currently subject of intense research. In this review, we discuss the latest breakthroughs on the transcriptional and protein control of tissue-resident memory CD8+ T cell metabolism.

Multipeptide stimulated PBMCs generate TEM/TCM for adoptive cell therapy in multiple myeloma.

Multiple Myeloma (MM) patients suffer disease relapse due to the development of therapeutic resistance. Increasing evidence suggests that immunotherapeutic strategies can provide durable responses. Here we evaluate the possibility of adoptive cell transfer (ACT) by generating ex vivo T cells from peripheral blood mononuclear cells (PBMCs) isolated from MM patients by employing our previously devised protocols. We designed peptides from antigens (Ags) including cancer testis antigens (CTAs) that are over expressed in MM. We exposed PBMCs from different healthy donors (HDs) to single peptides. We observed reproducible Ag-specific cluster of differentiation 4+ (CD4+) and CD8+ T cell responses on exposure of PBMCs to different single peptide sequences. These peptide sequences were used to compile four different peptide cocktails. Naïve T cells from PBMCs from MM patients or HDs recognized the cognate Ag in all four peptide cocktails, leading to generation of multiclonal Ag-specific CD4+ and CD8+ effector and central memory T (TEM and TCM, respectively) cells which produced interferon-gamma (IFN-γ), granzyme B and perforin on secondary restimulation. Furthermore, this study demonstrated that immune cells from MM patients are capable of switching metabolic programs to induce effector and memory responses. Multiple peptides and cocktails were identified that induce IFN-γ+, T1-type, metabolically active T cells, thereby paving the way for feasibility testing of ACT in phase I clinical trials.

Metformin Reduces Desmoplasia in Pancreatic Cancer by Reprogramming Stellate Cells and Tumor-Associated Macrophages.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with a dismal prognosis for most patients. Fibrosis and inflammation are hallmarks of tumor desmoplasia. We have previously demonstrated that preventing the activation of pancreatic stellate cells (PSCs) and alleviating desmoplasia are beneficial strategies in treating PDAC. Metformin is a widely used glucose-lowering drug. It is also frequently prescribed to diabetic pancreatic cancer patients and has been shown to associate with a better outcome. However, the underlying mechanisms of this benefit remain unclear. Metformin has been found to modulate the activity of stellate cells in other disease settings. In this study, we examine the effect of metformin on PSC activity, fibrosis and inflammation in PDACs. METHODS/RESULTS: In overweight, diabetic PDAC patients and pre-clinical mouse models, treatment with metformin reduced levels of tumor extracellular matrix (ECM) components, in particular hyaluronan (HA). In vitro, we found that metformin reduced TGF-ß signaling and the production of HA and collagen-I in cultured PSCs. Furthermore, we found that metformin alleviates tumor inflammation by reducing the expression of inflammatory cytokines including IL-1ß as well as infiltration and M2 polarization of tumor-associated macrophages (TAMs) in vitro and in vivo. These effects on macrophages in vitro appear to be associated with a modulation of the AMPK/STAT3 pathway by metformin. Finally, we found in our preclinical models that the alleviation of desmoplasia by metformin was associated with a reduction in ECM remodeling, epithelial-to-mesenchymal transition (EMT) and ultimately systemic metastasis. CONCLUSION: Metformin alleviates the fibro-inflammatory microenvironment in obese/diabetic individuals with pancreatic cancer by reprogramming PSCs and TAMs, which correlates with reduced disease progression. Metformin should be tested/explored as part of the treatment strategy in overweight diabetic PDAC patients.

Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer.

PURPOSE: While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model. EXPERIMENTAL DESIGN: Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma. RESULTS: Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity. CONCLUSION: Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.