Naive CD4 T-cell Research Articles

Predictive and pharmacodynamic biomarkers for combination therapy in stage III-IV melanoma: A Simon phase II trial (NCT03999749).

222 Background: The evaluation of biomarkers that predict response and immune-related adverse events (irAEs) is crucial for optimizing melanoma treatment with Immune checkpoint inhibitors (ICIs). We report predictive and pharmacodynamic biomarkers for response and irAEs using high-dimensional flow cytometry in a phase II trial combining nivolumab (3 mg/kg), ipilimumab (1 mg/ml), and the IL-6 receptor inhibitor tocilizumab (4 mg/kg) in unresectable/advanced melanoma patients. Methods: We analyzed serum and blood from melanoma patients at baseline, weeks 7 and 37, categorized them by irAE severity and clinical response. Patients were categorized based on the severity of irAEs (grades 3-5) (TOX), the absence of toxicity or grades 1-2 irAE (NT), clinical benefit (CB), and non-clinical benefit (NCB). Immune monitoring regimen included ELISA for IL-6, gp130, IL-23, osteopontin, and Luminex oncological panel (LXSAHM-22). We employed multi-parametric flow cytometry panels to examine myeloid cells, cytotoxic T cells, and checkpoint protein expression on immune cells. We processed ~1 million PBMCs per sample using various antibodies, with data acquisition performed via Sony’s ID7000 cytometer. The data underwent quality control, normalization PBMCs per patient, concatenation, dimensionality reduction, and clustering in FlowJo. Results: Among the 70 patients administered treatment, we observed a best overall response rate of 57%. Grade 3-5 irAEs occurred in 22% of patients by week 24. Patients manifesting irAEs exhibited notably higher baseline Th17 cell counts (NT=181±18 vs. Tox=311±49, p<0.01) and CD26+ Naive CD4 T-cell populations (NT=5838±801 vs. Tox=9632±1343, p<0.04), suggesting their potential role as predictive markers for toxicity. Additionally, in vitro investigations led us to identify CD26 co-stimulation as a preliminary step in TH17 cell differentiation. Key pharmacodynamic markers included a significant elevation in Treg cells (NCB=1286±71 vs. CB=1492±95, p=0.02) and reduction in serum gp130 levels (NCB=104±5 vs. CB=83±5 ng/ml, p<0.01) at week 7 in addition to IL-6 (NCB=7.5±1.5 vs. CB=4.6±1.1 pg/ml, p<0.01) and osteopontin (NCB=95±15 vs. CB=25±3 ng/ml, p<0.01) by week 37 when compared to baseline. Conclusions: Our findings suggest that Th17 cells can serve as a toxicity biomarker for combination therapy with ipilimumab, nivolumab and tocilizumab. Circulating Treg cells and serum osteopontin have emerged as promising pharmacodynamic biomarkers. Clinical trial information: NCT03999749 .

ACC1 Inhibition Enhances Treg Gvhd Treatment Efficacy through Regulation of Mitochondrial Fusion and Elongation

Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not uniformly eliminate grades II-IV graft-vs-host disease (GVHD). Treg more dominantly rely on fatty acid oxidation (FAO) as a fuel source, in contrast to glycolytic conventional T-cells. We hypothesized that increasing FAO would augment Treg efficacy by increasing potency and persistence, thereby ultimately decreasing the Treg cell number required to enhance Treg-mediated GVHD reduction. The FAO and fatty acid synthesis (FAS) pathways compete for acetyl-CoA use. Acetyl-CoA carboxylase-1 (ACC1) skews acetyl-CoA toward FAS and results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 deletion or inhibition skews naive CD4 T-cell toward FAO, favoring Treg over Th17 differentiation. To precisely determine the role of ACC1 on in vivo Treg-mediated suppression of GVHD, we tested the effects of a 2-hour treatment of murineTreg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, or Treg-specific ACC1 knockout (KO). ACC1 inhibition or KO increased in vitro Treg suppressive function, and when infused into mice with established bronchiolitis obliterans (BOS) and chronic GVHD (cGVHD), ACC1KO Treg were more potent than wild-type Treg in reversing BOS pulmonary resistance (FigA), a finding consistent in each of 3 replicate transplants. Mechanistically, both ND630 treated Treg and ACC1KO Treg demonstrated an increased oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. This was associated with an increase in uptake of BoDipyC16, a synthetic fatty acid, suggesting exogenous fatty acids may drive the increase in OXPHOS through FAO. IACS, an OXPHOS inhibitor, abrogated OCR augmentation and precluded increased suppressive function after ND630 treatment, indicating a critical role for OXPHOS in driving Treg suppressor function. To directly evaluate effects on Treg mitochondria, where OXPHOS occurs, Treg were stained with MitoTracker dyes that measure mitochondrial mass and membrane potential, two key surrogate measurements for mitochondrial function. Compared to controls, ND630 treated and ACC1KO Treg each showed increased mitochondrial mass and membrane potential, as well as enhanced mitochondrial polarization (membrane potential to mass ratio), which is required for full Treg functionality. Expansion and scanning electron microscopic analysis of Treg mitochondria revealed enhanced mitochondrial fusion and elongation after ND630 treatment ( FigB). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition/KO on mitochondrial fitness. In light of these mitochondrial morphology changes, we hypothesized that directly modifying the mitochondria itself, without inhibiting ACC1, might result in augmented Treg function. Consistent with this hypothesis, a 2-hour treatment with the combination of M1 (mitochondrial fusion promoter) and mDIVI1 (mitochondrial fission inhibitor), significantly enhanced Treg function in vitro, and enhanced Treg OCR to a similar degree as ND630. Electron microscopy confirmed that M1/mDIVI1 treatment resulted in a similar degree of mitochondrial fusion as ND630 treatment. Similar to ACC1KO Treg, M1/mDIVI1 treated Treg were more potent than control Treg in reversing established cGVHD. To further explore mitochondrial fusion effects on Treg function, we used siRNA to knockdown the critical mitochondrial fusion protein mitofusin 1 (MFN1). A 50% decrease in MFN1 protein significantly decreased Treg in vitro functionality and OCR, and treatment of MFN1 knockdown Treg with ND630 had no functional or metabolic effect (FigC), pointing to mitochondrial fusion as a critical component of the mechanism for ACC1 inhibition/KO. In summary, ACC1 inhibition or deletion amplifies murine Treg potency resulting in superior metabolic fitness, and greater efficacy in treating cGVHD mice with established BOS, in part through enhancing mitochondrial fusion. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing Tregs with inhibited or deleted ACC1 to improve GVHD therapy.

AIM2 promotes TH17 cells differentiation by regulating RORγt transcription activity

AIM2 is an interferon-inducible HIN-200 protein family member and is well-documented for its roles in innate immune responses as a DNA sensor. Recent studies have highlighted AIM2's function on regulatory Tcells (Treg) and follicular Tcells (Tfh). However, its involvement in Th17 cell differentiation remains unclear. This study reveals that AIM2 promotes Th17 cell differentiation. AIM2 deficiency decreases IL-17A production and downregulates key Th17 associated proteins (RORγt, IL-1R1, IL-23R). AIM2 is located in the nucleus of Th17 cells, where it interacts with RORγt, enhancing its binding to the Il17a promoter. The absence of AIM2 hinders naive CD4 Tcells from differentiating into functional Th17 cells and from inducing colitis in Rag1-/- mice. This study uncovers AIM2's role as a regulator of Th17 cell transcriptional programming, highlighting its potential as a therapeutic target for Th17 cell-mediated inflammatory diseases.

Role of mouse dendritic cell subsets in priming naive CD4 T cells.

Conventional dendritic cells (cDCs) are potent antigen-presenting cells that consist of developmentally, phenotypically, and functionally distinct subsets. Following immunization, each subset of cDCs acquires the antigen and presents it to CD4T (CD4+ T (cells)) cells with distinct spatiotemporal kinetics in the secondary lymphoid organs, often causing multiple waves of antigen presentation to CD4T cells. Here, we review the current understanding of the kinetics of antigen presentation by each cDC subset and its functional consequences in priming naive CD4T cells, and discuss its implications in the differentiation of CD4T cells.

ACC1 Inhibition Enhances Treg Gvhd Treatment Efficacy through Reciprocal Fatty-Acid Synthesis and Oxidative Phosphorylation Regulation

Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not eliminate grades II-IV graft-vs-host disease (GVHD). We hypothesized that increasing fatty acid oxidation (FAO) would augment Treg potency, and ultimately enhance Treg-mediated GVHD reduction. Acetyl-CoA carboxylase-1 (ACC1) negatively regulates FAO by augmenting fatty acid synthesis (FAS), which results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 genetic deletion or inhibition increases T-cell FAO and skews naive CD4 T-cell differentiation from Th17 to Treg. However, the role of ACC1 in thymic Treg function and metabolic fitness is unknown. Here we show that a 2-hour treatment of murine Treg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, enhances Treg suppressive function. As compared to control Tregs, ND630 treatment also increased expression of suppressive molecules Lag3, TIGIT, and IL-10. Treatment of Tregs with either of two other ACC1 inhibitors (TOFA, sorafin A) resulted in similar augmentations in Treg function, disfavoring the likelihood of off-target effects. Additionally, ND630 treatment increased Treg oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. Similar to murine Treg, ND630 treatment of in vitro expanded human Treg enhanced their suppressive function and OCR/OXPHOS. IACS, an OXPHOS inhibitor, abrogated the positive ND630 effect on in vitro Treg suppressive function and OXPHOS. Given the augmentation in OXPHOS with ND630 treatment, we further evaluated ND630 effects on Treg mitochondria. Compared to controls, ND630 treatment increased mitochondrial mass and membrane potential, essential for energy storage during OXPHOS, as well as mitochondrial polarization, which is required for full Treg functionality. Microscopy analysis revealed enhanced mitochondrial fusion and elongation after ND630 or TOFA treatment (FigA). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition on mitochondrial fitness. Since ACC1-driven FAS ultimately inhibits FAO, we next evaluated contributors to FAO. Expression of CPT1a, a critical enzyme for mitochondrial FAO, fatty acid binding protein 5, a key shuttle of fatty acids (FA), and exogenous FA uptake were all enhanced after ND630 treatment. Using an unbiased metabolomic screen, a metabolic signature was observed that was consistent with enhanced FAO. Together, these data suggest that ND630 increases mitochondrial fitness, and augments both FA uptake and FAO. To identify additional ND603-induced intracellular effects in Treg, activated murine Treg were subjected to RNAseq. Differential gene expression analysis indicated that 82 genes differed between DMSO and ND630 treated Treg. These included increased expression of genes associated with OXPHOS, as well as mTOR, consistent with high OXPHOS and suppressor cell function. Flow cytometry analysis of phosphorylated proteins showed reduced pFOXO3a and pAKT, both mTORC2 targets, but increased mTORC1 downstream phospho-proteins. An imbalance of mTORC1/2 is consistent to our previous studies reflective of higher Treg function and metabolic fitness (McDonald-Hyman C, JCI 2018). To exclude potential bystander effects of ND630 on Treg function, we utilized murine Treg with genetic deletion of ACC1 (ACC1KO). Supporting our studies with chemical ACC1 inhibitors, ACC1KO Treg were functionally and metabolically superior to wild-type Treg. Furthermore, as our goal is to use ACC1 inhibited or deleted Treg in treatment of GVHD, we tested ACC1KO Treg in treating established bronchiolitis obliterans (BOS) in a murine model of chronic GVHD. ACC1KO Treg were more potent than wild-type Treg in reversing BOS pathology (FigB), a finding consistent in each of 3 replicate transplants. In summary, ACC1 inhibition or deletion amplifies murine (and human) Treg potency resulting in superior metabolic fitness, and greater efficacy in treating established BOS in murine chronic GVHD. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing ACC1 inhibited Treg infusion to improve GVHD therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

David John Williams

<h3>Objective:</h3> Our objective was to determine whether altered naive CD4 T-cell biology contributes to development of disease progression in secondary progressive multiple sclerosis (SPMS). <h3>Methods:</h3> We compared the naive CD4...

Atherosclerosis Impairs Naive CD4 T-Cell Responses via Disruption of Glycolysis.

CD4 T cells are important regulators of atherosclerotic progression. The metabolic profile of CD4 T cells controls their signaling and function, but how atherosclerosis affects T-cell metabolism is unknown. Here, we sought to determine the impact of atherosclerosis on CD4 T-cell metabolism and the contribution of such metabolic alterations to atheroprogression. Using PCR arrays, we profiled the expression of metabolism genes in CD4 T cells from atherosclerotic apolipoprotein-E knockout mice fed a Western diet. These cells exhibited dysregulated expression of genes critically involved in glycolysis and fatty acid degradation, compared with those from animals fed a standard laboratory diet. We examined how T-cell metabolism was changed in either Western diet–fed apolipoprotein-E knockout mice or samples from patients with cardiovascular disease by measuring glucose uptake, activation, and proliferation in CD4 T cells. We found that naive CD4 T cells from Western diet–fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet–fed animals. Similarly, we observed that naive CD4 T-cell frequencies were reduced in the circulation of human subjects with high cardiovascular disease compared with low cardiovascular disease. Naive T cells from high cardiovascular disease subjects also showed reduced proliferative capacity. These results highlight the dysfunction that occurs in CD4 T-cell metabolism and immune responses during atherosclerosis. Targeting metabolic pathways within naive CD4 T cells could thus yield novel therapeutic approaches for improving CD4 T-cell responses against atheroprogression.

Microbiome And&amp;nbsp;Metabolome Driven Differentiation Of&amp;nbsp;TGF-β&amp;nbsp;Producing Tregs Leads to Senescence and HIV Latency

Therapeutic interventions to eradicate latent HIV in ART-treated infection have yet to show efficacy. We used a systems biology approach to identify a subset of ART-treated individuals with immune-dysfunction that had the highest frequencies of cells with inducible HIV. Contrary to the prevailing notion that immune activation drives HIV persistence, blood from these individuals was enriched in senescence-inducing genes (high FOXO3, SMAD2 and IRF3), Treg frequencies and TGF-β signaling expression. In these Senescent-INRs, high Firmicute phylum plasma nucleotides and butyrate/bile acids (like a-ketobutyrate) correlated with Treg frequencies and inducible HIV levels. Stimulation of naive CD4 T-cells with a-ketobutyrate led to TGF-β producing Treg differentiation, and PD-1 up-regulation on less differentiated cells. A dose dependent increase in latent HIV-infected memory CD4 T-cells was observed after TGF-β stimulation. Senescence cascades identified here can be targeted by PD-1/TGF-β specific interventions that have shown safety/efficacy in cancer, and can be crucial for HIV eradication.

CLDN10 associated with immune infiltration is a novel prognostic biomarker for clear cell renal cell carcinoma.

Aims: To identify the clinical roles ofCLDN10in clear cell renal cell carcinoma (ccRCC). Materials & methods: Using data from TCGA-KIRC, GEO DataSets and laboratory experiments to determine the prognostic and clinicopathological characteristics of CLDN10. Results: CLDN10 expression was remarkably reduced in ccRCC. Downregulated CLDN10 was related to metastasis and poor prognosis. Multivariate Cox analysis determined that elevated CLDN10 expression was independently correlated with longer OS and DFS. Moreover, CLDN10 expression was negatively associated with the methylation levels of cg10305311 and cg16275739. CLDN10 expression was alsoassociated with naive CD4 andmemory T-cell and dendritic cell infiltration. Conclusions: Immune-related CLDN10 is an independent prognostic biomarker of ccRCC. DNA hypermethylation plays an important role in decreased CLDN10 expression.

Drug efflux transporters and metabolic enzymes in human circulating and testicular T-cell subsets: relevance to HIV pharmacotherapy.

ATP-binding cassette (ABC) drug efflux transporters and drug metabolic enzymes could reduce antiretroviral concentrations in HIV target cells. The testis has been demonstrated to be a sanctuary site, displaying suboptimal antiretroviral concentrations and persistent HIV infection. Therefore, we compared the expression and function of ABC transporters and metabolic enzymes in CD4 and CD8 T cells isolated from human testis and peripheral blood mononuclear cells (PBMCs), and assessed their expression in circulating naive and memory CD4 T-cell phenotypes. Testicular tissue and blood were collected from 15 uninfected donors undergoing gender affirmation surgery. Testicular interstitial cells were isolated by enzymatic digestion, whereas PBMCs were isolated from blood by density gradient centrifugation. The expression and/or function of ABC transporters and metabolic enzymes were examined in blood and testicular T-cell subsets by flow cytometry. ABC transporters (P-gp, BCRP, MRP1) and metabolic enzymes (CYP3A4, UGT1A1) were expressed in testicular and circulating CD4 and CD8 T cells, as well as in circulating naive, central, transitional, and effector memory T-cell phenotypes. MRP1 demonstrated lower frequencies in T cells from testis compared with PBMCs, as well as in circulating naive T cells compared with the memory T-cell phenotypes. Functional activity of P-gp and BCRP was detected in T-cell subsets from testis and PBMCs. Our findings demonstrate for the first time that antiretroviral drug efflux transporters and metabolic enzymes are functionally expressed in T-cell subsets infiltrating the human testis. These transporters and enzymes can reduce antiretroviral intracellular concentrations, potentially contributing to residual HIV replication in the testis, and negatively impact HIV cure strategies.

A role for cell-autocrine interleukin-2 in regulatory T-cell homeostasis.

Activated T-cells make both interleukin-2 (IL2) and its high-affinity receptor component CD25. Regulatory CD4 T-cells (Treg cells) do not make IL2, and the IL2-CD25 circuit is considered a paracrine circuit crucial in their generation and maintenance. Yet, all T-cells are capable of making IL2 at some stage during differentiation, making a cell-intrinsic autocrine circuit additionally possible. When we re-visited experiments with mixed bone marrow chimeras using a wide range of ratios of wild-type (WT) and IL2-/- genotype progenitors, we found that, as expected, thymic Treg cells were almost equivalent between WT and IL2-/- genotypes at ratios with WT prominence. However, at WT-limiting ratios, the IL2-/- genotype showed lower thymic Treg frequencies, indicating a role for cell-intrinsic autocrine IL2 in thymic Treg generation under IL2-limiting conditions. Further, peripheral IL2-/- naive CD4 T-cells showed poor conversion to inducible Tregs (pTregs) both in vivo and in vitro, again indicating a significant role for cell-intrinsic autocrine IL2 in their generation. Peripherally, the IL2-/- genotype was less prominent at all WT:IL2-/- ratios among both thymic Tregs (tTregs) and pTregs, adoptively transferred IL2-/- Tregs showed poorer survival than WT Tregs did, and RNA-seq analysis of WT and IL2-/- Tregs showed interesting differences in the T-cell receptor and transforming growth factor-beta-bone morphogenetic protein-JNK pathways between them, suggesting a non-titrating role for cell-intrinsic autocrine IL2 in Treg programming. These data indicate that cell-intrinsic autocrine IL2 plays significant roles in Treg generation and maintenance.

Immunophenotyping of B-Cell AND T-CELL ABNORMALITIES IN PID USING EUROFLOW-BASED PANELS

Background: Predominantly antibody deficiency (PAD) is characterised by severe and recurrent bacterial infections, and up to 68% of patients develop non-infectious complications (NIC) including autoimmunity. Because the aetiology of NIC is unknown, we aimed to identify immune cell markers that associate with NIC in PAD patients. Methods: We developed an 11-colour flowcytometry panel and performed in-depth analysis of B- and T-cells in 53 adult PAD patients and 59 age-matched controls. Results: The patients were not genetically defined, and 30 (57%) had one or more NIC, 24 patients had reduced B-cell and 17 reduced T-cell numbers. Both PAD-NIC and PAD+NIC groups had significantly reduced Ig class-switched memory B cells, and naive CD4 and CD8 T-cell numbers. Naive and IgM memory B cells, Treg, Th17, and Tfh17 cells were specifically reduced in the PAD+NIC group. CD21lo B-cell and Tfh cells were increased in frequencies, but not in absolute numbers in PAD+NIC. Conclusion: The increased CD21lo B-cell and Tfh-cell frequencies are the indirect result of reduced naive B-cell and T-cell numbers. Hence, absolute cell counts are critical for correct interpretation of immunophenotyping of immunodeficiencies. The defects in naive B- and T-cell numbers suggest a mild combined immunodeficiency in PAD patients with NIC.

Predominantly Antibody-Deficient Patients With Non-infectious Complications Have Reduced Naive B, Treg, Th17, and Tfh17 Cells.

Background: Patients with predominantly antibody deficiency (PAD) suffer from severe and recurrent infections that require lifelong immunoglobulin replacement and prophylactic antibiotic treatment. Disease incidence is estimated to be 1:25,000 worldwide, and up to 68% of patients develop non-infectious complications (NIC) including autoimmunity, which are difficult to treat, causing high morbidity, and early mortality. Currently, the etiology of NIC is unknown, and there are no diagnostic and prognostic markers to identify patients at risk.Objectives: To identify immune cell markers that associate with NIC in PAD patients.Methods: We developed a standardized 11-color flow cytometry panel that was utilized for in-depth analysis of B and T cells in 62 adult PAD patients and 59 age-matched controls.Results: Nine males had mutations in Bruton's tyrosine kinase (BTK) and were defined as having X-linked agammaglobulinemia. The remaining 53 patients were not genetically defined and were clinically diagnosed with agammaglobulinemia (n = 1), common variable immunodeficiency (CVID) (n = 32), hypogammaglobulinemia (n = 13), IgG subclass deficiency (n = 1), and specific polysaccharide antibody deficiency (n = 6). Of the 53, 30 (57%) had one or more NICs, 24 patients had reduced B-cell numbers, and 17 had reduced T-cell numbers. Both PAD–NIC and PAD+NIC groups had significantly reduced Ig class-switched memory B cells and naive CD4 and CD8 T-cell numbers. Naive and IgM memory B cells, Treg, Th17, and Tfh17 cells were specifically reduced in the PAD+NIC group. CD21lo B cells and Tfh cells were increased in frequencies, but not in absolute numbers in PAD+NIC.Conclusion: The previously reported increased frequencies of CD21lo B cells and Tfh cells are the indirect result of reduced naive B-cell and T-cell numbers. Hence, correct interpretation of immunophenotyping of immunodeficiencies is critically dependent on absolute cell counts. Finally, the defects in naive B- and T-cell numbers suggest a mild combined immunodeficiency in PAD patients with NIC. Together with the reductions in Th17, Treg, and Tfh17 numbers, these key differences could be utilized as biomarkers to support definitive diagnosis and to predict for disease progression.

Abstract 2400: K01401-020 is a novel anti-VISTA antibody for cancer treatment: Pharmacodynamic impact on non-human primates and human immune system

Abstract VISTA (V-domain Ig suppressor of T cell activation), also known as PD-1 homolog (PD-1H), is a transmembrane protein which is highly expressed in myeloid cells such as neutrophils, monocytes, macrophages, and dendritic cells, and at low levels on naive CD4, CD8 and regulatory T-cells. VISTA is also expressed in cancer cells in some patient subsets (Villarroel-Espindola, 2017; Böger, 2017; Liao, 2018). VISTA is functionally described as a negative checkpoint regulator of immune response. Hence, VISTA blockade increases proliferation when T cells are stimulated with VISTA expressing myeloid antigen presenting cells (Wang et al, 2011). Immune suppression by VISTA-positive cells has been hypothesized in the primary or acquired resistance to cancer immunotherapy (Kakhavand, 2017); and even a negative impact of VISTA on immune response has been functionally demonstrated in prostate cancer samples from patients having received prior immunotherapy (Gao, 2017). Here we show that VISTA RNA and protein are expressed in lung cancer samples. We also show by using bioinformatics, that VISTA expression is linked to the higher myeloid infiltrated tumors and can also be found in tumor cells. K01401-020 is novel monoclonal, humanized IgG antibody that binds to cynomolgus monkey and human-VISTA (hVISTA) with a high affinity (Kd ~0.1 nM). The antibody binds to VISTA extracellular domain and blocks its associated function. The pharmacology of K01401-020 was studied in a series of in vitro (ex vivo) experiments with human blood cells, and in vivo studies in non-human primates. Peripheral Blood Mononuclear cells (PBMCs) from several couples of human donors were mixed together in a Mixed Lymphocyte Reaction (MLR) setting, which showed that K01401-020 stimulated IFNg release after 5 days of culture. K01401-020 stimulates NK cells to proliferate and induces activating cytokines by NK cells and monocytes, contributing to T cell activation. PDL1 expression is also clearly induced on myeloid cells by K01401-020, suggesting that combination therapy with anti-PD1/L1 may be required to optimize the anti-tumor efficacy. In non-human primates, preliminary results show that both K01401-020 monotherapy, given weekly at 3 dose levels for 4 weeks and anti-VISTA antibody combination with anti-PD1 antibody (pembrolizumab) were well tolerated. Pharmacodynamic exploration using peripheric blood sampling demonstrated full VISTA occupation by the antibody, and notably, dendritic cell activation. K01401-020 antibody drug is thus expected to increase global and specific anti-tumor immune response in situations where VISTA is expressed in cancer patients and may be synergistic with anti-PD1/L1 antibodies. Citation Format: Noureddine Loukili, Isabelle Vandenberghe, Olivier Delfour, Françoise Fraboul, Eric Chetaille, Francisco Cruzalegui, Nathalie Corvaïa, Pierre J. Ferré. K01401-020 is a novel anti-VISTA antibody for cancer treatment: Pharmacodynamic impact on non-human primates and human immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2400.

Model-Based Assessment of the Role of Uneven Partitioning of Molecular Content on Heterogeneity and Regulation of Differentiation in CD8 T-Cell Immune Responses.

Activation of naive CD8 T-cells can lead to the generation of multiple effector and memory subsets. Multiple parameters associated with activation conditions are involved in generating this diversity that is associated with heterogeneous molecular contents of activated cells. Although naive cell polarisation upon antigenic stimulation and the resulting asymmetric division are known to be a major source of heterogeneity and cell fate regulation, the consequences of stochastic uneven partitioning of molecular content upon subsequent divisions remain unclear yet. Here we aim at studying the impact of uneven partitioning on molecular-content heterogeneity and then on the immune response dynamics at the cellular level. To do so, we introduce a multiscale mathematical model of the CD8 T-cell immune response in the lymph node. In the model, cells are described as agents evolving and interacting in a 2D environment while a set of differential equations, embedded in each cell, models the regulation of intra and extracellular proteins involved in cell differentiation. Based on the analysis of in silico data at the single cell level, we show that immune response dynamics can be explained by the molecular-content heterogeneity generated by uneven partitioning at cell division. In particular, uneven partitioning acts as a regulator of cell differentiation and induces the emergence of two coexisting sub-populations of cells exhibiting antagonistic fates. We show that the degree of unevenness of molecular partitioning, along all cell divisions, affects the outcome of the immune response and can promote the generation of memory cells.

Peeking under the Hood of Naive T Cells

Signaling and transcriptional regulation of metabolic reprogramming upon Tcell activation has been studied intensively. In this issue of Cell Metabolism, Ricciardi etal. (2018) show that translational regulation of key metabolic enzymes GLUT1 and ACC1 plays a novel role in human naive CD4 Tcell activation and subset differentiation.

Age-Related Decline in Primary CD8+ T Cell Responses Is Associated with the Development of Senescence in Virtual Memory CD8+ T Cells

Age-associated decreases in primary CD8+ Tcell responses occur, in part, due to direct effects on naive CD8+ Tcells to reduce intrinsic functionality, but the precise nature of this defect remains undefined. Aging also causes accumulation of antigen-naive but semi-differentiated "virtual memory" (TVM) cells, but their contribution to age-related functional decline is unclear. Here, we show that TVM cells are poorly proliferative in aged mice and humans, despite being highly proliferative in young individuals, while conventional naive Tcells (TN cells) retain proliferative capacity in both aged mice and humans. Adoptive transfer experiments in mice illustrated that naive CD8 Tcells can acquire a proliferative defect imposed by the aged environment but age-related proliferative dysfunction could not be rescued by a young environment. Molecular analyses demonstrate that aged TVM cells exhibit a profile consistent with senescence, marking an observation of senescence in an antigenically naive Tcell population.

Kmt2d regulates naive CD8 T-cell development and activation-related survival

Abstract Lysine specific methyl-transferase 2D (Kmt2d) catalyzes the mono-methylation of histone 3 lysine 4 (H3K4me1) and play a critical role in enhancer-mediated gene expression regulation. However, its role in CD8 T cell development and differentiation has not been addressed. Here, we analyzed a T-cell specific KMT2D knockout mouse (KMT2Dfl/flCD4cre+) and found that the number of CD8 T cells in spleen and blood was greatly decreased compared to wild type (KMT2Dfl/flCD4cre-, WT) mice. The decrease in CD8 T cells is caused by defective production of T cells in the thymus based on the following observations. First, numbers of recent thymic emigrant (RTE) CD8 cells in blood and spleen of KMT2Dfl/flCD4cre+ mice were significantly less in KMT2Dfl/flCD4cre+RAG-GFP mice. Second, thymic development of T cells was compromised (CD4+/CD8+ population was less and immature CD8SP cells are increased in KMT2Dfl/flCD4cre+ mice). Third, naïve CD8 T cells (CD62Lhi/CD44lo) were more significantly reduced (WT:KO=7:1) than memory (CD62Llo/CD44hi, WT:KO=2:1) CD8 T cells. Furthermore, naïve CD8 T cells isolated from KMT2Dfl/flCD4cre+ mice with anti-CD3 and anti-CD28 in vitro showed increased apoptosis compared to that from WT mice. RNA-seq analysis of naïve CD8 T cells revealed increased expressions of genes related to apoptosis prior stimulation, suggesting Kmt2d regulates activation-induced apoptosis. Together, our findings reveal a role of Kmt2d in naïve CD8 T cell production and survival after activation. This research was supported entirely by the Intramural Research Programs of the NIH, National Institute on Aging and National Institute of Diabetes, Digestive and Kidney Diseases.

292 - Regulation of Foxp3 expression in TGF-β induced regulatory T cells derived from human umbilical cord blood vs adult blood CD4 T cells

Natural T regulatory cells (nTreg) have shown the potential to safely and effectively induce immune tolerance in several in vivo applications. However, the limited proliferative capacity of nTreg remains a challenge to broader clinical applications. Inducible Treg (iTreg) can restore immune tolerance in settings where nTregs are decreased or defective. However, the instability of expression of the transcription factor Fork head box P3 (Foxp3) poses a significant barrier to successful immune therapy. Further understanding of Foxp3 binding partners and their function is emerging. The transcription factor BACH2 has been shown to repress effector programs to stabilize Treg-mediated immune homeostasis in murine KO studies. However, the potential of BACH2 to stabilize Foxp3 expression in human CD4 T-cells has not been explored. In the current studies, we compared the influence of BACH2 on Foxp3 expression in UCB vs. AB naive CD4 T-cells. UCB naïve CD4 T-cells highly express BACH2, 21-fold higher compared to AB naive CD4 T-cells, at the protein and mRNA levels (p < 0.01). Expression of Foxp3 is 2.5-fold higher in iTreg derived from UCB vs. AB naïve CD4 T-cells (p < 0.001). Also, the absolute number of Foxp3+ iTreg generated from UCB vs. AB naïve CD4 T-cells is 4-fold higher. Of particular interest, the suppressive function of UCB Foxp3+ iTreg is more potent in mixed lymphocyte cultures compared to AB Foxp3+ iTreg (p < 0.001). UCB iTreg also exhibit higher surface CD25 and CTLA-4 expression as measured by flow cytometry (p < 0.001). Transient BACH2 shRNA knockdown in UCB CD4 T-cells results in ~ 42% loss of Foxp3 expression (Empty shRNA: 78.8% vs. Bach2 shRNA: 46.2%, p < 0.001) and reduced expression of molecules down-stream of Foxp3, including CTLA-4, as measured by qPCR (p < 0.01) . Chromatin immunoprecipitation (ChIP) analysis revealed that BACH2 binds to the Foxp3 promoter in UCB iTreg but not AB iTreg (p < 0.01). Furthermore, Foxp3 luciferase reporter assays demonstrated that BACH2 is transcriptionally active at the Foxp3 promoter. Overall, our results suggest that BACH2 is an important transcription factor partner that stabilizes Foxp3 expression in UCB naïve CD4 T-cells.

Insufficiency of DNA repair enzyme ATM promotes naive CD4 T-cell loss in chronic hepatitis C virus infection

T cells have a crucial role in viral clearance and vaccine response; however, the mechanisms regulating their responses to viral infections or vaccinations remain elusive. In this study, we investigated T-cell homeostasis, apoptosis, DNA damage, and repair machineries in a large cohort of subjects with hepatitis C virus (HCV) infection. We found that naive CD4 T cells in chronically HCV-infected individuals (HCV T cells) were significantly reduced compared with age-matched healthy subjects. In addition, HCV T cells were prone to apoptosis and DNA damage, as evidenced by increased 8-oxoguanine expression and γH2AX/53BP1-formed DNA damage foci—hallmarks of DNA damage responses. Mechanistically, the activation of DNA repair enzyme ataxia telangiectasia mutated (ATM) was dampened in HCV T cells. ATM activation was also diminished in healthy T cells exposed to ATM inhibitor or to HCV (core protein) that inhibits the phosphoinositide 3 kinase pathway, mimicking the biological effects in HCV T cells. Importantly, ectopic expression of ATM was sufficient to repair the DNA damage, survival deficit, and cell dysfunctions in HCV T cells. Our results demonstrate that insufficient DNA repair enzyme ATM leads to increased DNA damage and renders HCV T cells prone to apoptotic death, which contribute to the loss of naive T cells in HCV infection. Our study reveals a novel mechanism for T-cell dysregulation and viral persistence, providing a new strategy to improve immunotherapy and vaccine responses against human viral diseases.