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Background Wharton's jelly derived stem cells (WJMSCs) are gaining attention as a possible clinical alternative to bone marrow derived mesenchymal stem cells (BMMSCs) owing to better accessibility, higher expansion potential and low immunogenicity. Usage of allogenic mesenchymal stem cells (MSC) could be permissible in vivo only if they retain their immune properties in an inflammatory setting. Thus the focus of this study is to understand and compare the immune properties of BMMSCs and WJMSCs primed with key pro-inflammatory cytokines, Interferon-γ (IFNγ) and Tumor Necrosis Factor-α (TNFα). Methodology/Principal Findings Initially the effect of priming on MSC mediated suppression of alloantigen and mitogen induced lymphoproliferation was evaluated in vitro. Treatment with IFNγ or TNFα, did not ablate the immune-suppression caused by both the MSCs. Extent of immune-suppression was more with WJMSCs than BMMSCs in both the cases. Surprisingly, priming BMMSCs enhanced suppression of mitogen driven lymphoproliferation only; whereas IFNγ primed WJMSCs were better suppressors of MLRs. Further, kinetic analysis of cytokine profiles in co-cultures of primed/unprimed MSCs and Phytohematoagglutinin (PHA) activated lymphocytes was evaluated. Results indicated a decrease in levels of pro-inflammatory cytokines. Interestingly, a change in kinetics and thresholds of Interleukin-2 (IL-2) secretion was observed only with BMMSCs. Analysis of activation markers on PHA-stimulated lymphocytes indicated different expression patterns in co-cultures of primed/unprimed WJMSCs and BMMSCs. Strikingly, co-culture with WJMSCs resulted in an early activation of a negative co-stimulatory molecule, CTLA4, which was not evident with BMMSCs. A screen for immune suppressive factors in primed/unprimed WJMSCs and BMMSCs indicated inherent differences in IFNγ inducible Indoleamine 2, 3-dioxygenase (IDO) activity, Hepatocyte growth factor (HGF) and Prostaglandin E-2 (PGE2) levels which could possibly influence the mechanism of immune-modulation. Conclusion/Significance This study demonstrates that inflammation affects the immune properties of MSCs distinctly. Importantly different tissue derived MSCs could utilize unique mechanisms of immune-modulation.

Clinically reported reparative benefits of mesenchymal stromal cells (MSCs) are majorly attributed to strong immune‐modulatory abilities not exactly shared by fibroblasts. However, MSCs remain heterogeneous populations, with unique tissue‐specific subsets, and lack of clear‐cut assays defining therapeutic stromal subsets adds further ambiguity to the field. In this context, in‐depth evaluation of cellular characteristics of MSCs from proximal oro‐facial tissues: dental pulp (DPSCs) and periodontal ligament (PDLSCs) from identical donors provides an opportunity to evaluate exclusive niche‐specific influences on multipotency and immune‐modulation. Exhaustive cell surface profiling of DPSCs and PDLSCs indicated key differences in expression of mesenchymal (CD105) and pluripotent/multipotent stem cell–associated cell surface antigens: SSEA4, CD117, CD123 and CD29. DPSCs and PDLSCs exhibited strong chondrogenic potential, but only DPSCs exhibited adipogenic and osteogenic propensities. PDLSCs expressed immuno‐stimulatory/immune‐adhesive ligands like HLA‐DR and CD50, upon priming with IFNγ, unlike DPSCs, indicating differential response patterns to pro‐inflammatory cytokines. Both DPSCs and PDLSCs were hypo‐immunogenic and did not elicit robust allogeneic responses despite exposure to IFNγ or TNFα. Interestingly, only DPSCs attenuated mitogen‐induced lympho‐proliferative responses and priming with either IFNγ or TNFα enhanced immuno‐modulation capacity. In contrast, primed or unprimed PDLSCs lacked the ability to suppress polyclonal T cell blast responses. This study indicates that stromal cells from even topographically related tissues do not necessarily share identical MSC properties and emphasizes the need for a thorough functional testing of MSCs from diverse sources with respect to multipotency, immune parameters and response to pro‐inflammatory cytokines before translational usage.

BackgroundEpigenetic reinforcement of T cell exhaustion is a well-established barrier limiting multiple modalities of T cell-based immunotherapies for cancer. Disruption of the epigenetic regulators DNMT3A and TET2 has been previously used to preserve the proliferative capacity of chronically stimulated CD8 T cells in the setting of both chimeric antigen receptor (CAR) T cells and PD-1 blockade-mediated therapy. These epigenetic regulators, in addition to ASXL1, are commonly mutated in clonal hematopoiesis, providing a survival advantage to stem cells and suggesting a universal role of these factors in controlling stemness. This concept was recently supported by clinical data showing that ASXL1 mutations in the T cell compartment of myelodysplastic syndrome (MDS) patients are associated with extended survival after immune checkpoint blockade (ICB). Given the established role of DNMT3A and TET2 in controlling cellular multipotency, we investigated the impact of ASXL1 in the development of functional and exhausted CD8 T cells.MethodsASXL1 and AAVS1 (WT) knock-out P14 T cells were generated using CRISPR-Cas9 technology. Acute lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells were generated by adoptive transfer of ~3000 congenically distinct naive ASXL1 KO and WT P14 CD8 T cells. One day later, mice were infected with chronic LCMV by i.v. injection of 2x106 PFU LCMV per mouse. Mice were treated with PD-L1 on days 31, 34, 37, 40 and 42 post infection. Longitudinal phenotypic and epigenetic analysis were performed. Tumor experiments were performed using LLC1-GP33 and tracking tumor size and survival in mice who received WT versus ASXL1 KO P14 T cells.ResultsDisruption of Asxl1 in murine LCMV-specific CD8 T cells enabled the stem-like subset to persist in chronically infected mice for one year. These cells retained the capacity to give rise to potent effector T cells in adoptive transfer settings. Additionally, the Asxl1 KO LCMV-specific CD8 T cells retained a heightened ability to undergo a proliferative burst in response to ICB and exhibited significant efficacy against historically aggressive tumors. These data document clonal hematopoiesis-associated epigenetic checkpoints control the transition of stem-like progenitor T cells into terminally exhausted T cells.ConclusionsCollectively, these data demonstrate that ASXL1 is a novel epigenetic regulator controlling the durability of the stem-like T cell population that is responsible for sustaining clinical response during T cell-based cancer immunotherapy.AcknowledgementsThis work was supported by the National Institutes of Health (R01CA237311 to BY; K08CA279926 to CZ), the National Comprehensive Cancer Network Young Investigator Award (to CZ), the Alex’s Lemonade Stand Young Investigator Award (to CZ), SU2C (to BY) and the American Lebanese Syrian Associated Charities (ALSAC to CZ and BY).

Mesenchymal stem cells (MSCs) are speculated to act at macrophage-injury interfaces to mediate efficient repair. To explore this facet in-depth this study evaluates the influence of MSCs on human macrophages existing in distinct functional states. MSCs promoted macrophage differentiation, enhanced respiratory burst and potentiated microbicidal responses in naïve macrophages (Mφ). Functional attenuation of inflammatory M1 macrophages was associated with a concomitant shift towards alternatively activated M2 state in MSC-M1 co-cultures. In contrast, alternate macrophage (M2) activation was enhanced in MSC-M2 co-cultures. Elucidation of key macrophage metabolic programs in Mo/MSC, M1/MSC and M2/MSC co-cultures indicated changes in Glucose transporter1 (GLUT1 expression/glucose uptake, IDO1 protein/activity, SIRTUIN1 and alterations in AMPK and mTOR activity, reflecting MSC-instructed metabolic shifts. Inability of Cox2 knockdown MSCs to attenuate M1 macrophages and their inefficiency in instructing metabolic shifts in polarized macrophages establishes a key role for MSC-secreted PGE in manipulating macrophage metabolic status and plasticity. Functional significance of MSC-mediated macrophage activation shifts was further validated on human endothelial cells prone to M1 mediated injury. In conclusion, we propose a novel role for MSC secreted factors induced at the MSC-macrophage interface in re-educating macrophages by manipulating metabolic programs in differentially polarized macrophages.

Mesenchymal stem cells (MSCs) are speculated to act at macrophage-injury interfaces to mediate efficient repair. To explore this facet in-depth this study evaluates the influence of MSCs on human macrophages existing in distinct functional states. MSCs promoted macrophage differentiation, enhanced respiratory burst and potentiated microbicidal responses in naïve macrophages (Mφ). Functional attenuation of inflammatory M1 macrophages was associated with a concomitant shift towards alternatively activated M2 state in MSC-M1 co-cultures. In contrast, alternate macrophage (M2) activation was enhanced in MSC-M2 co-cultures. Elucidation of key macrophage metabolic programs in Mo/MSC, M1/MSC and M2/MSC co-cultures indicated changes in Glucose transporter1 ( GLUT1 expression/glucose uptake, IDO1 protein/activity, SIRTUIN1 and alterations in AMPK and mTOR activity, reflecting MSC-instructed metabolic shifts. Inability of Cox2 knockdown MSCs to attenuate M1 macrophages and their inefficiency in instructing metabolic shifts in polarized macrophages establishes a key role for MSC-secreted PGE 2 in manipulating macrophage metabolic status and plasticity. Functional significance of MSC-mediated macrophage activation shifts was further validated on human endothelial cells prone to M1 mediated injury. In conclusion, we propose a novel role for MSC secreted factors induced at the MSC-macrophage interface in re-educating macrophages by manipulating metabolic programs in differentially polarized macrophages.

Wharton's jelly derived stem cells (WJMSCs) are gaining attention as a possible clinical alternative to bone marrow derived mesenchymal stem cells (BMMSCs) owing to better accessibility, higher expansion potential and low immunogenicity. Usage of allogenic mesenchymal stem cells (MSC) could be permissible in vivo only if they retain their immune properties in an inflammatory setting. Thus the focus of this study is to understand and compare the immune properties of BMMSCs and WJMSCs primed with key pro-inflammatory cytokines, Interferon-gamma (IFNgamma) and Tumor Necrosis Factor-alpha (TNFalpha). Initially the effect of priming on MSC mediated suppression of alloantigen and mitogen induced lymphoproliferation was evaluated in vitro. Treatment with IFNgamma or TNFalpha, did not ablate the immune-suppression caused by both the MSCs. Extent of immune-suppression was more with WJMSCs than BMMSCs in both the cases. Surprisingly, priming BMMSCs enhanced suppression of mitogen driven lymphoproliferation only; whereas IFNgamma primed WJMSCs were better suppressors of MLRs. Further, kinetic analysis of cytokine profiles in co-cultures of primed/unprimed MSCs and Phytohematoagglutinin (PHA) activated lymphocytes was evaluated. Results indicated a decrease in levels of pro-inflammatory cytokines. Interestingly, a change in kinetics and thresholds of Interleukin-2 (IL-2) secretion was observed only with BMMSCs. Analysis of activation markers on PHA-stimulated lymphocytes indicated different expression patterns in co-cultures of primed/unprimed WJMSCs and BMMSCs. Strikingly, co-culture with WJMSCs resulted in an early activation of a negative co-stimulatory molecule, CTLA4, which was not evident with BMMSCs. A screen for immune suppressive factors in primed/unprimed WJMSCs and BMMSCs indicated inherent differences in IFNgamma inducible Indoleamine 2, 3-dioxygenase (IDO) activity, Hepatocyte growth factor (HGF) and Prostaglandin E-2 (PGE2) levels which could possibly influence the mechanism of immune-modulation. This study demonstrates that inflammation affects the immune properties of MSCs distinctly. Importantly different tissue derived MSCs could utilize unique mechanisms of immune-modulation.

Chronological aging correlates with epigenetic modifications at specific loci, calibrated to species lifespan. Such ‘epigenetic clocks’ appear conserved among mammals, but whether they are cell autonomous and restricted by maximal organismal lifespan remains unknown. We used a multilifetime murine model of repeat vaccination and memory T cell transplantation to test whether epigenetic aging tracks with cellular replication and if such clocks continue ‘counting’ beyond species lifespan. Here we found that memory T cell epigenetic clocks tick independently of host age and continue through four lifetimes. Instead of recording chronological time, T cells recorded proliferative experience through modification of cell cycle regulatory genes. Applying this epigenetic profile across a range of human T cell contexts, we found that naive T cells appeared ‘young’ regardless of organism age, while in pediatric patients, T cell acute lymphoblastic leukemia appeared to have epigenetically aged for up to 200 years. Thus, T cell epigenetic clocks measure replicative history and can continue to accumulate well-beyond organismal lifespan.

Wharton's jelly derived stem cells (WJMSCs) are gaining attention as a possible clinical alternative to bone marrow derived mesenchymal stem cells (BMMSCs) owing to better accessibility, higher expansion potential and low immunogenicity. Usage of allogenic mesenchymal stem cells (MSC) could be permissible in vivo only if they retain their immune properties in an inflammatory setting. Thus the focus of this study is to understand and compare the immune properties of BMMSCs and WJMSCs primed with key pro-inflammatory cytokines, Interferon-[gamma] (IFN[gamma]) and Tumor Necrosis Factor-[alpha] (TNF[alpha]). Initially the effect of priming on MSC mediated suppression of alloantigen and mitogen induced lymphoproliferation was evaluated in vitro. Treatment with IFN[gamma] or TNF[alpha], did not ablate the immune-suppression caused by both the MSCs. Extent of immune-suppression was more with WJMSCs than BMMSCs in both the cases. Surprisingly, priming BMMSCs enhanced suppression of mitogen driven lymphoproliferation only; whereas IFN[gamma] primed WJMSCs were better suppressors of MLRs. Further, kinetic analysis of cytokine profiles in co-cultures of primed/unprimed MSCs and Phytohematoagglutinin (PHA) activated lymphocytes was evaluated. Results indicated a decrease in levels of pro-inflammatory cytokines. Interestingly, a change in kinetics and thresholds of Interleukin-2 (IL-2) secretion was observed only with BMMSCs. Analysis of activation markers on PHA-stimulated lymphocytes indicated different expression patterns in co-cultures of primed/unprimed WJMSCs and BMMSCs. Strikingly, co-culture with WJMSCs resulted in an early activation of a negative co-stimulatory molecule, CTLA4, which was not evident with BMMSCs. A screen for immune suppressive factors in primed/unprimed WJMSCs and BMMSCs indicated inherent differences in IFN[gamma] inducible Indoleamine 2, 3-dioxygenase (IDO) activity, Hepatocyte growth factor (HGF) and Prostaglandin E-2 (PGE2) levels which could possibly influence the mechanism of immune-modulation. This study demonstrates that inflammation affects the immune properties of MSCs distinctly. Importantly different tissue derived MSCs could utilize unique mechanisms of immune-modulation.