Arid1a promotes thymocyte development through ß-selection-dependent and ß-selection-independent mechanisms.

Early T-cell development from CD4-  CD8- double-negative (DN) stage to CD4+  CD8+ double-positive (DP) stage in the thymus is regulated through multiple steps involving a batch of sequentially expressed factors. Our preliminary data and a recent report showed that AT-rich interaction domain 1A (Arid1a) is required for the transition from DN to DP stages, but the mechanism is not fully understood. In this study, we consolidated that conditional deletion of Arid1a in T-cell lineage intrinsically caused developmental blocks from DN3 to DN4 stages, as well as from DN4 to DP stages using both in vivo adoptive T-cell transfer model and in vitro culture system. The expression of intracellular TCRß is significantly decreased in Arid1a-deficient DN4 cells compared with WT cells. OT1 transgenic TCR can rescue the defect in the transition from DN3 to DN4 stages, but not from DN to DP stages. Furthermore, we observed a comparable or stronger proliferation capacity accompanied by a significant increase in cell death in Arid1a-/- DP cells compared with that in WT controls. RNA-Seq analysis shows a significant enrichment of apoptotic pathway within differentially expressed genes between Arid1a-/- and WT DP cells, including the upregulation of Bim, Casp3 and Trp53 and the downregulation of Rorc, Bcl-XL and Mcl1. Therefore, our study reveals a novel mechanism that Arid1a controls early T-cell development by maintaining intracellular TCRß expression-mediated ß-selection and activating parallel cell survival pathways.

Med1 controls CD8 T cell maintenance through IL-7R-mediated cell survival signalling.

Under steady-state conditions, the pool size of peripheral CD8+ T cells is maintained through turnover and survival. Beyond TCR and IL-7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8+ T cell proportion in spleens but not in thymi of mice with T cell-specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild-type (WT) and Med1-deficient CD8+ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1-deficient CD8+ T cells compared with WT counterparts. Finally, Med1-deficient CD8+ T cells exhibited a decreased expression of interleukin-7 receptor α (IL-7Rα), down-regulation of phosphorylated-STAT5 (pSTAT5) and Bim up-regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8+ T cells through IL-7Rα/STAT5 pathway-mediated cell survival.

NK cell predicts the severity of acute graft-versus-host disease in patients after allogeneic stem cell transplantation using antithymocyte globulin (ATG) in pretreatment scheme.

BACKGROUND: Graft-versus-host disease (GVHD) is one of the most complex complications after allogeneic stem cell transplantation. Current standard of grading system is based on clinical symptoms in skin, liver and intestinal. However, it's difficult to differ GVHD and its extent just by clinical manifestation. Here we retrospectively analyzed cell immune function in patients implemented allogeneic stem cell transplantation in Ningbo first Hospital from Jan 2013 to Jan 2018. RESULTS: the data are collected from 51 patients (mean age was 42; 45.1% women). The average NK cell percentage was 39.31% in severe GVHD (Grade III-IV), was 16.98% in mild GVHD (GradeI-II), while was 21.15% in No GVHD group. The statistical analysis showed difference among each grade. Further analysis was performed in Antithymocyte globulin (ATG) treated group and control group. We showed NK Cell percentage was sharply different in ATG treated group: 47.34% in severe GVHD, 11.98% in mild GVHD group, while 18.3% in no GVHD group. However, in control group, the average percentage of NK cells was 23.27% in severe GVHD, was 23.22%in mild GVHD group, while was 21.13% in no GVHD group. CONCLUSION: The data supports that ATG can prevent GVHD by increasing NK cell percentage. The percentage of NK cell seemed to be a useful probe to evaluate the severity of GVHD in allogeneic stem cell transplantation patients using ATG in pretreatment.

Targeting Glycolysis in Alloreactive T Cells to Prevent Acute Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Effect.

Alloreactive donor T cells undergo extensive metabolic reprogramming to become activated and induce graft-versus-host disease (GVHD) upon alloantigen encounter. It is generally thought that glycolysis, which promotes T cell growth and clonal expansion, is employed in this process. However, conflicting data have been reported regarding the requirement of glycolysis to induce T cell-mediated GVHD due to the lack of T cell-specific treatments using glycolysis inhibitors. Importantly, previous studies have not evaluated whether graft-versus-leukemia (GVL) activity is preserved in donor T cells deficient for glycolysis. As a critical component affecting the clinical outcome, it is necessary to assess the anti-tumor activity following treatment with metabolic modulators in preclinical models. In the present study, we utilized T cells selectively deficient for glucose transporter 1 (Glut1T-KO), to examine the role of glycolysis exclusively in alloreactive T cells without off-targeting effects from antigen presenting cells and other cell types that are dependent on glycolysis. We demonstrated that transfer of Glut1T-KO T cells significantly improved acute GVHD outcomes through increased apoptotic rates, impaired expansion, and decreased proinflammatory cytokine production. In addition to impaired GVHD development, donor Glut1T-KO T cells mediated sufficient GVL activity to protect recipients from tumor development. A clinically relevant approach using donor T cells treated with a small molecule inhibitor of glycolysis, 2-Deoxy-D-glucose ex vivo, further demonstrated protection from tumor development. These findings indicate that treatment with glycolysis inhibitors prior to transplantation selectively eliminates alloreactive T cells, but spares non-alloreactive T cells including those that protect against tumor growth. The present study has established a definitive role for glycolysis in acute GVHD and demonstrated that acute GVHD can be selectively prevented through targeting glycolysis.

Single-cell sequencing reveals antitumor characteristics of intratumoral immune cells in old mice.

BACKGROUND: Aging has long been thought to be a major risk factor for various types of cancers. However, accumulating evidence indicates increased resistance of old animals to tumor growth. An in-depth understanding of how old individuals defend against tumor invasion requires further investigations. METHODS: We revealed age-associated alterations in tumor-infiltrating immune cells between young and old mice using single-cell RNA and coupled T cell receptor (TCR) sequencing analysis. Multiple bioinformatics methods were adopted to analyze the characteristics of the transcriptome between two groups. To explore the impacts of young and old CD8+ T cells on tumor growth, mice were treated with anti-CD8 antibody every 3 days starting 7 days after tumor inoculation. Flow cytometry was used to validate the differences indicated by sequencing analysis between young and old mice. RESULTS: We found a higher proportion of cytotoxic CD8+ T cells, naturally occurring Tregs, conventional dendritic cell (DC), and M1-like macrophages in tumors of old mice compared with a higher percentage of exhausted CD8+ T cells, induced Tregs, plasmacytoid DC, and M2-like macrophages in young mice. Importantly, TCR diversity analysis showed that top 10 TCR clones consisted primarily of exhausted CD8+ T cells in young mice whereas top clones were predominantly cytotoxic CD8+ T cells in old mice. Old mice had more CD8+ T cells with a 'progenitor' and less 'terminally' exhausted phenotypes than young mice. Consistently, trajectory inference demonstrated that CD8+ T cells preferentially differentiated into cytotoxic cells in old mice in contrast to exhausted cells in young mice. Importantly, elimination of CD8+ T cells in old mice during tumor growth significantly accelerated tumor development. Moreover, senescent features were demonstrated in exhausted but not cytotoxic CD8+ T cells regardless of young and old mice. CONCLUSIONS: Our data revealed that a significantly higher proportion of effector immune cells in old mice defends against tumor progression, providing insights into understanding the altered kinetics of cancer development and the differential response to immunotherapeutic modulation in elderly patients.

Calcium/Calmodulin Dependent Protein Kinase Kinase 2 Regulates the Expansion of Tumor-Induced Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are a hetero geneous group of cells, which can suppress the immune response, promote tumor progression and impair the efficacy of immunotherapies. Consequently, the pharmacological targeting of MDSC is emerging as a new immunotherapeutic strategy to stimulate the natural anti-tumor immune response and potentiate the efficacy of immunotherapies. Herein, we leveraged genetically modified models and a small molecule inhibitor to validate Calcium-Calmodulin Kinase Kinase 2 (CaMKK2) as a druggable target to control MDSC accumulation in tumor-bearing mice. The results indicated that deletion of CaMKK2 in the host attenuated the growth of engrafted tumor cells, and this phenomenon was associated with increased antitumor T cell response and decreased accumulation of MDSC. The adoptive transfer of MDSC was sufficient to restore the ability of the tumor to grow in Camkk2-/- mice, confirming the key role of MDSC in the mechanism of tumor rejection. In vitro studies indicated that blocking of CaMKK2 is sufficient to impair the yield of MDSC. Surprisingly, MDSC generated from Camkk2-/- bone marrow cells also showed a higher ability to terminally differentiate toward more immunogenic cell types (e.g inflammatory macrophages and dendritic cells) compared to wild type (WT). Higher intracellular levels of reactive oxygen species (ROS) accumulated in Camkk2-/- MDSC, increasing their susceptibility to apoptosis and promoting their terminal differentiation toward more mature myeloid cells. Mechanistic studies indicated that AMP-activated protein kinase (AMPK), which is a known CaMKK2 proximal target controlling the oxidative stress response, fine-tunes ROS accumulation in MDSC. Accordingly, failure to activate the CaMKK2-AMPK axis can account for the elevated ROS levels in Camkk2-/- MDSC. These results highlight CaMKK2 as an important regulator of the MDSC lifecycle, identifying this kinase as a new druggable target to restrain MDSC expansion and enhance the efficacy of anti-tumor immunotherapy.

A Genetic Model Reveals Biological Features of Neonatal CD4 Helper Cells Undergone Homeostasis in Mice.

CD4+ T cells are essential for regulating effective immune response to pathogens and immune balance. Recent studies have demonstrated the unique features of T cells in neonate mice, such as more sensitive to antigen response and preference toward T helper 2 (Th2) response and regulatory T cells (Tregs) differentiation. However, the biological characteristics of neonatal age-derived CD4+ T cells following homeostasis remain unclear. Here we utilized a lineage tracing model of TCRδ CreER R26 ZsGreen to mark neonatal- and adult-derived CD4+ T cells followed by a combination analysis of activation, proliferation, survival, and differentiation. Our results showed that neonatal CD4+ T cells had higher capacity of activation, proliferation, apoptosis, and differentiation toward Th2 and T helper 17 (Th17) lineages, accompanied by a reduced potential for T helper 1 (Th1), T helper 9 (Th9), and Treg lineages. In contrast, tracked neonatal CD4+ T cells exhibited similar characters of above-mentioned of tracked adult cells in adult mice. Therefore, our data support a natural requirement for CD4+ T cells to acquire fully-equipped functional potentials of adult cells.

T cell metabolism in graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major source of morbidity and mortality following allogeneic hematopoietic stem cell transplant (allo-HSCT), one of the most effective approaches to treat hematopoietic malignancies.1 However, current prophylaxis regimens and treatments that reduce the detrimental effect of acute GVHD can be offset by increased incidence in opportunistic infections and relapse of the primary malignancy.2 In addition, the majority of the approaches that inhibit T cell responses are non-specific, resulting in the inhibition of both alloreactive T cells and protective T cells from the donor. Therefore, there is an increase in the demand to develop novel approaches that selectively target alloreactive T cells. One potential means to address this issue is to take advantage of the unique metabolic profile of activated T cells.