Tissue-resident memory T (TRM) cells provide infectious, cancer and vaccine-trained immunity across barrier sites. TRM cells are implicated in autoimmunity, successful response to immune checkpoint blockade in the tumor microenvironment and toxicities that occur after immune checkpoint blockade in peripheral tissues. Here, we identified that signaling through the immune checkpoint programmed death receptor 1 (PD-1) strongly impacts the early specification of CD8+ TRM cells in the skin. PD-1 is expressed broadly across mouse and human skin TRM cells, in the absence of persistent infection, and is retained on skin TRM cells in aged mice. PD-1 supports early TRM cell colonization, skin-specific programming and silencing of other differentiation programs and promotes TGFβ responsivity and skin engraftment. Thus, PD-1 signaling mediates skin TRM cell specification during immune initiation. These findings may inform therapeutic PD-1 agonist and antagonist use to modulate successful peripheral memory. Anandasabapathy and colleagues show that the inhibitory receptor PD-1 impacts the specification of tissue-resident memory T cells in the skin.

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 CD8specific 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, Panx1mediated eATP export regulates effector and memory CD8 + T cells through distinct purinergic receptors and different metabolic and signaling pathways.

Supplementary Figure 3 - Expression of VEGFR-1 and IL-1ß in TAMs in PAN02 tumors.

Effects of VEGFR-1 signaling ablation on glucose/insulin tolerance and insulin production by the pancreas during obesity.

Supplementary Figure 5 - Effects of VEGFR-1 signaling ablation on PAN02 tumor progression in body-weight matched mice.

Supplementary Table 1: Protein quantification of cytokines in tumors and plasma of WT and Flt1TK-/- obese using ELISA; Supplementary Table 2: Quantification of the expression of IL-1ÃŽÃ,² and VEGFR-1 in TAMs from PAN02 tumors by mmunofluorescence.

Supplementary Figure 1 - A) Vessel density in PAN02 tumors from lean and obese mice, and effect of VEGFR-1-TK-deletion on tumor vessel density and hypoxia markers in obese mice. Supplementary Figure 2 - Gene expression of M1 / M2 markers in TAMs isolated from PAN02 tumors in lean and obese mice.

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).

Supplementary Figure 7 - Effects of VEGFR-1 signaling ablation on IGF-1R/IR signaling pathways in PAN02 and E0771 tumors implanted in obese mice; Supplementary Figure 8 - Additional effects of VEGFR-1 signaling ablation and metformin on tumor metabolism and vessel density in obese mice.

Supplementary Figure 10 - Additional measurements of PlGF and VEGF-B; Supplementary Figure 11 - Additional effects of PlGF deletion on obesity-induced tumor progression and systemic metabolism.

Effects of VEGFR-1 signaling ablation on E0771 tumor growth, vascular and immune environment in lean and obese mice.

Effects of VEGFR-1 signaling ablation on body weight gain, immune cell infiltration and vasculature in adipose tissues during obesity.

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.

Influenza remains an infection of high medical relevance. Development of universal anti-influenza vaccines will require a better knowledge of lung immune responses. That includes influenza-specific CD4+ T cells, which establish long-term lung residency and induce a diversified local protective response to subsequent infection. Lung-resident memory CD4+ T cells are heterogeneous, including Th1-like cells, and a subset that shares phenotypic and functional features with follicular helper (Tfh) cells – called tissue-resident helper (Trh). Notably, both Tfh and lung Trh cells express the extracellular ATP (eATP) sensor P2RX7 at high levels. Given its known deleterious role in Tfh cells, we investigated if P2RX7 could also limit the Trh pool in influenza-infected lungs. Contrary to our hypothesis, T cell-specific P2RX7 ablation led to a significant decrease in the Trh cell subset. Consequently, lungs of T cell-P2RX7 knockout mice harbor reduced B cell and CD8+ T cell responses. Initial Trh cell establishment is not affected by P2RX7 knockout; rather, P2RX7-deficient Trh cells gradually decay over time. This is associated with increased apoptosis and decreased in situ proliferation of P2RX7-knockout Trh cells. Our bulk RNA-seq analysis of lung CD4+ T cells revealed many cell cycle-associated genes poorly expressed in P2RX7-knockout cells. Some (but not all) Trh-fate genes are also decreased in P2RX7 deficiency. We are currently performing single-cell RNA-seq to define whether P2RX7 promotes the Trh fate in a particular subset of lung precursor cells. Our data shows that, in response to influenza, P2RX7 promotes lung-resident Trh cells, suggesting this eATP sensor has opposite roles in Tfh-like CD4+ T cells in a tissue-specific manner. Supported by grant from NIH (R00 AI139381)

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.

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).

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.

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.

Memory CD8+ T cells protect against secondary viral infections. They develop and maintain exclusively in circulation (e.g. central memory - Tcm) or are excluded from re-circulation (resident memory - Trm). Circulating memory CD8+ T cells arises from memory precursors (MPs) which express memory, pro-survival, and pluripotency molecules. Trm generation, in contrast, is still not fully understood. Previous reports suggest that Trm and Tcm have a common precursor present at the early effector phase; yet, the nature of this precursor is elusive. We have found that the extracellular ATP receptor P2RX7 promotes both Tcm and Trm generation. Moreover, P2RX7 is quickly upregulated in some (but not all) early effectors and early P2RX7 ablation hinders both Tcm and Trm generation. Thus, we postulated that P2RX7 in early effectors may mark a Tcm/Trm common precursor. Comparison between high (P2RX7hi) and low (P2RX7lo) P2RX7-expressing CD8+ T cells shows a correlation between memory markers and P2RX7, with CD62L+ Tcm precursors exclusively found in P2RX7hi cells. RNA-seq of P2RX7hi and P2RX7lo early effectors show survival, memory and pluripotency genes and pathways are upregulated in P2RX7hi cells. Conversely, many terminal effector and apoptosis genes are upregulated in P2RX7lo cells. P2RX7lo early effectors and MPs have increased apoptosis. Upon transfer into infection-matched mice, P2RX7hi MPs preferentially survive and form Tcm compared to P2RX7lo MPs. This is even more apparent when P2RX7hi and P2RX7lo early effectors are compared: approximately 100x more P2RX7hi cells form memory when compared to P2RX7lo cells, both Tcm and Trm. Our data suggest that P2RX7 in early effector CD8+ T cells promotes the generation of a common Tcm/Trm precursor.