CD38-RyR2 axis-mediated signaling impedes CD8+ T cell response to anti-PD1 therapy in cancer.

PD1 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 found 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.

Nature and Dimensions of Systemic Hyperinflammation and its Attenuation by Convalescent Plasma in Severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), has led to significant morbidity and mortality. While most suffer from mild symptoms, some patients progress to severe disease with acute respiratory distress syndrome (ARDS) and associated systemic hyperinflammation. METHODS: First, to characterize key cytokines and their dynamics in this hyperinflammatory condition, we assessed abundance and correlative expression of a panel of 48 cytokines in patients progressing to ARDS as compared to patients with mild disease. Then, in an ongoing randomized controlled trial of convalescent plasma therapy (CPT), we analyzed rapid effects of CPT on the systemic cytokine dynamics as a correlate for the level of hypoxia experienced by the patients. RESULTS: We identified an anti-inflammatory role of CPT independent of its neutralizing antibody content. CONCLUSIONS: Neutralizing antibodies, as well as reductions in circulating interleukin-6 and interferon-γ-inducible protein 10, contributed to marked rapid reductions in hypoxia in response to CPT. CLINICAL TRIAL REGISTRY OF INDIA: CTRI/2020/05/025209. http://www.ctri.nic.in/.

Thioredoxin-1 improves the immunometabolic phenotype of antitumor T cells.

Adoptive transfer of tumor epitope-reactive T cells has emerged as a promising strategy to control tumor growth. However, chronically-stimulated T cells expanded for adoptive cell transfer are susceptible to cell death in an oxidative tumor microenvironment. Because oxidation of cell-surface thiols also alters protein functionality, we hypothesized that increasing the levels of thioredoxin (Trx), an antioxidant molecule facilitating reduction of proteins through cysteine thiol-disulfide exchange, in T cells will promote their sustained antitumor function. Using pre-melanosome protein (Pmel)-Trx1 transgenic mouse-derived splenic T cells, flow cytometry, and gene expression analysis, we observed here that higher Trx expression inversely correlated with reactive oxygen species and susceptibility to T-cell receptor restimulation or oxidation-mediated cell death. These Trx1-overexpressing T cells exhibited a cluster of differentiation 62Lhi (CD62Lhi) central memory-like phenotype with reduced glucose uptake (2-NBDGlo) and decreased effector function (interferon γlo). Furthermore, culturing tumor-reactive T cells in the presence of recombinant Trx increased the dependence of T cells on mitochondrial metabolism and improved tumor control. We conclude that strategies for increasing the antioxidant capacity of antitumor T cells modulate their immunometabolic phenotype leading to improved immunotherapeutic control of established tumors.

Selective induction of apoptosis in various cancer cells irrespective of drug sensitivity through a copper chelate, copper N-(2 hydroxy acetophenone) glycinate: crucial involvement of glutathione.

Drug induced toxicity and drug resistance are the major impediments to successful application of cancer chemotherapy. Therefore, selective targeting of the key biochemical events of the malignant cells may have a great therapeutic potential in specifically kill the cancer cells. We have evaluated in vitro the cytotoxic efficacy of a previously reported copper complex viz. copper N-(2-hydroxy acetophenone) glycinate (CuNG) on different drug sensitive and resistant cancer cell lines by MTT, annexin V positivity and caspase 3 activation assays. We have also investigated the underlying signalling events in CuNG mediated apoptosis of cancer cells by Western blotting technique. We have found that CuNG preferentially induces apoptosis to malignant cells irrespective of drug sensitivity and spares the normal cells. Our studies disclose that CuNG causes cellular redox imbalance in cancer cells through depletion of intracellular GSH level. CuNG mediated depletion of intracellular GSH level induces mitochondrial superoxide generation, which detaches cyto C from mitochondrial membrane through lipid peroxidation. The detached cyto C then release into the extra mitochondrial milieu in Bax mediated pathway where CuNG facilitates the binding of Bax through dissociation of hexokinase II from mitochondrial membrane. The present study opens the possibility of developing effective chemotherapeutic drugs by synthesizing numerous chemical compounds capable of targeting cellular redox environment and thus specifically kills cancer cells of broad spectrum.

Intratumoral PD1+CD38+Tim3+ CD8+ T Cells in Pre-BCG Tumor Tissues Are Associated with Poor Responsiveness to BCG Immunotherapy in Patients with Non-Muscle Invasive Bladder Cancer.

Intravesical immunotherapy with Bacillus Calmette-Guerin (BCG) is a standard of care therapy for non-muscle invasive bladder cancer (NMIBC), which accounts for about 75% of newly diagnosed urothelial cancer. However, given the frequent recurrence and progression, identification of a pre-treatment biomarker capable of predicting responsiveness to BCG in NMIBC is of utmost importance. Herein, using multiparametric flow cytometry, we characterized CD8+ T cells from peripheral blood and tumor tissues collected from 27 pre-BCG patients bearing NMIBC to obtain immune correlates of bladder cancer prognosis and responsiveness to BCG therapy. We observed that intratumoral CD8+ T cell subsets were highly heterogenous in terms of their differentiation state and exist at different proportions in tumor tissues. Remarkably, among the different CD8+ T cell subsets present in the tumor tissues, the frequency of the terminally exhausted-like CD8+ T cell subset, marked as PD1+CD38+Tim3+ CD8+ T cells, was inversely correlated with a favorable outcome for patients and a responsiveness to BCG therapy. Moreover, we also noted that the intratumoral abundance of the progenitor exhausted-like PD1+CD8+ T cell subset in pre-BCG NMIBC tumor tissues was indicative of better recurrence-free survival after BCG. Collectively, our study led to the identification of biomarkers that can predict the therapeutic responsiveness of BCG in NMIBC.

Circulating Interleukin-8 Dynamics Parallels Disease Course and Is Linked to Clinical Outcomes in Severe COVID-19.

Severe COVID-19 frequently features a systemic deluge of cytokines. Circulating cytokines that can stratify risks are useful for more effective triage and management. Here, we ran a machine-learning algorithm on a dataset of 36 plasma cytokines in a cohort of severe COVID-19 to identify cytokine/s useful for describing the dynamic clinical state in multiple regression analysis. We performed RNA-sequencing of circulating blood cells collected at different time-points. From a Bayesian Information Criterion analysis, a combination of interleukin-8 (IL-8), Eotaxin, and Interferon-γ (IFNγ) was found to be significantly linked to blood oxygenation over seven days. Individually testing the cytokines in receiver operator characteristics analyses identified IL-8 as a strong stratifier for clinical outcomes. Circulating IL-8 dynamics paralleled disease course. We also revealed key transitions in immune transcriptome in patients stratified for circulating IL-8 at three time-points. The study identifies plasma IL-8 as a key pathogenic cytokine linking systemic hyper-inflammation to the clinical outcomes in COVID-19.

Association of gut microbial dysbiosis with disease severity, response to therapy and disease outcomes in Indian patients with COVID-19.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is associated with systemic hyper-inflammation. An adaptive interaction between gut microbiota and host immune systems is important for intestinal homeostasis and systemic immune regulation. The association of gut microbial composition and functions with COVID-19 disease severity is sparse, especially in India. We analysed faecal microbial diversity and abundances in a cohort of Indian COVID-19 patients to identify key signatures in the gut microbial ecology in patients with severe COVID-19 disease as well as in response to different therapies. The composition of the gut microbiome was characterized using 16Sr RNA gene sequences of genomic DNA extracted from faecal samples of 52 COVID-19 patients. Metabolic pathways across the groups were predicted using PICRUSt2. All statistical analyses were done using Vegan in the R environment. Plasma cytokine abundance at recruitment was measured in a multiplex assay. RESULTS: The gut microbiome composition of mild and severe patients was found to be significantly different. Immunomodulatory commensals, viz. Lachnospiraceae family members and Bifidobacteria producing butyrate and short-chain fatty acids (SCFAs), were under represented in patients with severe COVID-19, with an increased abundance of opportunistic pathogens like Eggerthella. The higher abundance of Lachnoclostridium in severe disease was reduced in response to convalescent plasma therapy. Specific microbial genera showed distinctive trends in enriched metabolic pathways, strong correlations with blood plasma cytokine levels, and associative link to disease outcomes. CONCLUSION: Our study indicates that, along with SARS-CoV-2, a dysbiotic gut microbial community may also play an important role in COVID-19 severity through modulation of host immune responses.

Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo.

Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.

Intracellular Acetyl CoA Potentiates the Therapeutic Efficacy of Antitumor CD8+ T Cells.

Effector CD8+ T cells rely primarily on glucose metabolism to meet their biosynthetic and functional needs. However, nutritional limitations in the tumor microenvironment can cause T-cell hyporesponsiveness. Therefore, T cells must acquire metabolic traits enabling sustained effector function at the tumor site to elicit a robust antitumor immune response. Here, we report that IL12-stimulated CD8+ T cells have elevated intracellular acetyl CoA levels and can maintain IFNγ levels in nutrient-deprived, tumor-conditioned media (TCM). Pharmacological and metabolic analyses demonstrated an active glucose-citrate-acetyl CoA circuit in IL12-stimulated CD8+ T cells supporting an intracellular pool of acetyl CoA in an ATP-citrate lyase (ACLY)-dependent manner. Intracellular acetyl CoA levels enhanced histone acetylation, lipid synthesis, and IFNγ production, improving the metabolic and functional fitness of CD8+ T cells in tumors. Pharmacological inhibition or genetic knockdown of ACLY severely impaired IFNγ production and viability of CD8+ T cells in nutrient-restricted conditions. Furthermore, CD8+ T cells cultured in high pyruvate-containing media in vitro acquired critical metabolic features of IL12-stimulated CD8+ T cells and displayed improved antitumor potential upon adoptive transfer in murine lymphoma and melanoma models. Overall, this study delineates the metabolic configuration of CD8+ T cells required for stable effector function in tumors and presents an affordable approach to promote the efficacy of CD8+ T cells for adoptive T-cell therapy. SIGNIFICANCE: IL12-mediated metabolic reprogramming increases intracellular acetyl CoA to promote the effector function of CD8+ T cells in nutrient-depleted tumor microenvironments, revealing strategies to potentiate the antitumor efficacy of T cells.

Quantitative increase in T regulatory cells enhances bone remodeling in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is characterized by repeated bone fractures. Recent studies have shown that T lymphocytes and regulatory T cells (Tregs) regulate the functions of osteoclasts and osteoblasts, thus playing a role in bone turnover. We demonstrate an activated effector phenotype and higher secretion of pro-inflammatory cytokines, IFN-γ, and TNF-α in OI peripheral T cells as compared with wild-type (WT). Suppressive Tregs (spleen and thymus) were qualitatively similar, whereas there was a quantitative decrease in OI versus WT. Restoring Treg numbers by systemic transplantation in OI mice resulted in reduced T cell activation and effector cytokine secretion that correlated with significant improvements in tibial trabecular and cortical bone parameters and stiffness of femur, along with increased osteoblast mineralization and decreased osteoclast numbers. Therefore, Tregs can dampen the pro-inflammatory environment and enhance bone remodeling in OI mice. Thus, this study will be helpful in developing future autologous immunotherapy-based treatment modalities for OI.

Clinical Trial Subgroup Analyses to Investigate Clinical and Immunological Outcomes of Convalescent Plasma Therapy in Severe COVID-19.

Objective: To assess the clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT). Materials and Methods: A series of subclass analyses were performed on the previously published outcome data and accompanying clinical metadata from a completed randomized controlled trial (RCT) (Clinical Trial Registry of India, number CTRI/2020/05/025209). The subclass analyses were performed on the outcome data and accompanying clinical metadata from a completed RCT (patient recruitment between May 15, 2020 and October 31, 2020). Data on the plasma abundance of a large panel of cytokines from the same cohort of patients were also used to characterize the heterogeneity of the putative anti-inflammatory function of convalescent plasma (CP) in addition to passively providing neutralizing antibodies. Results: Although the primary clinical outcomes were not significantly different in the RCT across all age groups, significant immediate mitigation of hypoxia, reduction in hospital stay, and significant survival benefit were registered in younger (<67 years in our cohort) patients with severe coronavirus disease 2019 and acute respiratory distress syndrome on receiving CPT. In addition to neutralizing the antibody content of CP, its anti-inflammatory proteome, by attenuation of the systemic cytokine deluge, significantly contributed to the clinical benefits of CPT. Conclusion: Subgroup analyses revealed that clinical benefits of CPT in severe coronavirus disease 2019 are linked to the anti-inflammatory protein content of CP apart from the anti-severe acute respiratory syndrome coronavirus 2 neutralizing antibody content.

Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

A phase 2 single center open label randomised control trial for convalescent plasma therapy in patients with severe COVID-19.

A single center open label phase 2 randomised control trial (Clinical Trial Registry of India No. CTRI/2020/05/025209) was done to assess clinical and immunological benefits of passive immunization using convalescent plasma therapy. At the Infectious Diseases and Beleghata General Hospital in Kolkata, India, 80 patients hospitalized with severe COVID-19 disease and fulfilling the inclusion criteria (aged more than 18 years, with either mild ARDS having PaO2/FiO2 200-300 or moderate ARDS having PaO2/FiO2 100-200, not on mechanical ventilation) were recruited and randomized into either standard of care (SOC) arm (N = 40) or the convalescent plasma therapy (CPT) arm (N = 40). Primary outcomes were all-cause mortality by day 30 of enrolment and immunological correlates of response to therapy if any, for which plasma abundance of a large panel of cytokines was quantitated before and after intervention to assess the effect of CPT on the systemic hyper-inflammation encountered in these patients. The secondary outcomes were recovery from ARDS and time taken to negative viral RNA PCR as well as to report any adverse reaction to plasma therapy. Transfused convalescent plasma was characterized in terms of its neutralizing antibody content as well as proteome. The trial was completed and it was found that primary outcome of all-cause mortality was not significantly different among severe COVID-19 patients with ARDS randomized to two treatment arms (Mantel-Haenszel Hazard Ratio 0.6731, 95% confidence interval 0.3010-1.505, with a P value of 0.3424 on Mantel-Cox Log-rank test). No adverse effect was reported with CPT. In severe COVID-19 patients with mild or moderate ARDS no significant clinical benefit was registered in this clinical trial with convalescent plasma therapy in terms of prespecified outcomes.

A prospective study on COVID-19 convalescent plasma donor (CCP) recruitment strategies in a resource constrained blood centre.

Background and Objectives: The COVID-19 pandemic has spread across 87 million people with more than 1·8 million deaths in the world. As there is no definite treatment modality, the use of convalescent plasma has become increasingly popular worldwide. This study aimed to identify an appropriate strategy of donor recruitment and to evaluate the appropriateness of pre-set plasma donation guidelines. Material and Methods: In this prospective study conducted from May to September 2020, the donors were recruited under the following two circumstances: Group I, patients in the post-COVID-19 follow-up in the clinic, and Group II, patients recovered from COVID-19 recruited through mass and electronic media. A pre-set donor selection criteria and laboratory investigation was designed according to national and international guidelines. Approximately 500 ml of COVID-19 convalescent plasma (CCP) was collected from recovered individuals in each group by two different cell separators. The overall donor's attendance rate, deferral rate, adverse events and donor compliance was analysed and compared between the two groups. Results: There was a significant difference in attendance in relation to registration between the groups (P < 0·0001). Donor deferral was significantly higher in group II compared with group I. The single most frequent cause of donor deferral was low antibody index (P = 0·0001). The total donor adverse event rate in CCP donation was significantly lower compared with routine plateletpheresis procedures. The donor's compliance to blood centre's protocol was satisfactory in both the groups. Conclusion: Recruitment of patients in the post-COVID-19 follow-up in the clinic was more effective than the general recruitment through mass and electronic media for convalescence plasma donation in a resource-constrained blood centre.

Plasma Gradient of Soluble Urokinase-Type Plasminogen Activator Receptor Is Linked to Pathogenic Plasma Proteome and Immune Transcriptome and Stratifies Outcomes in Severe COVID-19.

Disease caused by SARS-CoV-2 coronavirus (COVID-19) led to significant morbidity and mortality worldwide. A systemic hyper-inflammation characterizes severe COVID-19 disease, often associated with acute respiratory distress syndrome (ARDS). Blood biomarkers capable of risk stratification are of great importance in effective triage and critical care of severe COVID-19 patients. Flow cytometry and next-generation sequencing were done on peripheral blood cells and urokinase-type plasminogen activator receptor (suPAR), and cytokines were measured from and mass spectrometry-based proteomics was done on plasma samples from an Indian cohort of COVID-19 patients. Publicly available single-cell RNA sequencing data were analyzed for validation of primary data. Statistical analyses were performed to validate risk stratification. We report here higher plasma abundance of suPAR, expressed by an abnormally expanded myeloid cell population, in severe COVID-19 patients with ARDS. The plasma suPAR level was found to be linked to a characteristic plasma proteome, associated with coagulation disorders and complement activation. Receiver operator characteristic curve analysis to predict mortality identified a cutoff value of suPAR at 1,996.809 pg/ml (odds ratio: 2.9286, 95% confidence interval 1.0427-8.2257). Lower-than-cutoff suPAR levels were associated with a differential expression of the immune transcriptome as well as favorable clinical outcomes, in terms of both survival benefit (hazard ratio: 0.3615, 95% confidence interval 0.1433-0.912) and faster disease remission in our patient cohort. Thus, we identified suPAR as a key pathogenic circulating molecule linking systemic hyperinflammation to the hypercoagulable state and stratifying clinical outcomes in severe COVID-19 patients with ARDS.

CD38: T Cell Immuno-Metabolic Modulator.

Activation and subsequent differentiation of T cells following antigenic stimulation are triggered by highly coordinated signaling events that lead to instilling cells with a discrete metabolic and transcriptional feature. Compelling studies indicate that intracellular nicotinamide adenine dinucleotide (NAD+) levels have profound influence on diverse signaling and metabolic pathways of T cells, and hence dictate their functional fate. CD38, a major mammalian NAD+ glycohydrolase (NADase), expresses on T cells following activation and appears to be an essential modulator of intracellular NAD+ levels. The enzymatic activity of CD38 in the process of generating the second messenger cADPR utilizes intracellular NAD+, and thus limits its availability to different NAD+ consuming enzymes (PARP, ART, and sirtuins) inside the cells. The present review discusses how the CD38-NAD+ axis affects T cell activation and differentiation through interfering with their signaling and metabolic processes. We also describe the pivotal role of the CD38-NAD+ axis in influencing the chromatin remodeling and rewiring T cell response. Overall, this review emphasizes the crucial contribution of the CD38-NAD+ axis in altering T cell response in various pathophysiological conditions.

Targeting PIM Kinase with PD1 Inhibition Improves Immunotherapeutic Antitumor T-cell Response.

PURPOSE: Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of ex vivo-engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor. EXPERIMENTAL DESIGN: The role of PIM kinases in T cells was studied either by genetic ablation (PIM1-/-PIM2-/-PIM3-/-) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutaneously and treated by transferring tumor epitope gp100-reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both. RESULTS: With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination. CONCLUSIONS: This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

Pro-Survival Lipid Sphingosine-1-Phosphate Metabolically Programs T Cells to Limit Anti-tumor Activity.

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.

PIM-2 protein kinase negatively regulates T cell responses in transplantation and tumor immunity.

PIM kinase family members play a crucial role in promoting cell survival and proliferation via phosphorylation of their target substrates. In this study, we investigated the role of the PIM kinases with respect to T cell responses in transplantation and tumor immunity. We found that the PIM-2 isoform negatively regulated T cell responses to alloantigen, in contrast to the PIM-1 and PIM-3 isoforms, which acted as positive regulators. T cells deficient in PIM-2 demonstrated increased T cell differentiation toward Th1 subset, proliferation, and migration to target organs after allogeneic bone marrow transplantation, resulting in dramatically accelerated graft-versus-host disease (GVHD) severity. Restoration of PIM-2 expression markedly attenuated the pathogenicity of PIM-2-deficient T cells to induce GVHD. On the other hand, mice deficient in PIM-2 readily rejected syngeneic tumor, which was primarily dependent on CD8+ T cells. Furthermore, silencing PIM-2 in polyclonal or antigen-specific CD8+ T cells substantially enhanced their antitumor response in adoptive T cell immunotherapy. We conclude that PIM-2 kinase plays a prominent role in suppressing T cell responses, and provide a strong rationale to target PIM-2 for cancer immunotherapy.

Hematopoietic Stem Cells as a Novel Source of Dental Tissue Cells.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein+ (GFP+). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP+ cells, which were also CD45+ (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP+ hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP+ cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.