Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation

CD25, a component of the IL-2 receptor, is important in T cell proliferation, activation induced cell death, as well as the actions of both regulatory (Treg) and effector (Teff) T cells. Recent genome wide association studies have implicated the CD25 locus as an important region for genetic susceptibility to a number of autoimmune disorders, with serum levels of soluble CD25 receptor (sCD25) serving as a potential phenotypic marker for this association. However, the functional impact of CD25 cleavage, as well as the influence of sCD25 on immunoregulatory activities, remain largely unknown and form the basis of this effort.The generation of sCD25 by Treg (CD4(+)CD25(+)) and Teff (CD4(+)CD25(-)) cells was examined during in vitro suppression assays, efforts that demonstrated constitutive and stable surface CD25 expression on Treg throughout the period of in vitro assessment. In contrast, Teff cells increased CD25 expression during the process of in vitro suppression, with supernatant sCD25 levels correlating to the amount of cellular proliferation. Interestingly, under serum-free conditions, Tregs partially lost their characteristic anergic and suppressive properties. sCD25 supplementation at physiological concentrations to serum free in vitro suppression assays reduced Teff proliferation without specifically influencing suppression. Indeed, sCD25 production within these cultures correlated with cell death.These results support the notion that sCD25 functions as both a surrogate marker of T cell activation as well as an indicator of subsequent cellular death. In addition, the role of CD25 in immunomodulation is likely dependent on the local inflammatory milieu, with molecules capable of modulating surface CD25 expression playing a key role in defining immune responsiveness

Modeling cell-mediated immunity in human type 1 diabetes by engineering autoreactive CD8+ T cells

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing β-cells is thought to be mediated primarily by antigen-specific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human β-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells

Expansion of Human Regulatory T-Cells From Patients With Type 1 Diabetes

OBJECTIVE—Regulatory T-cells (Tregs) have catalyzed the field of immune regulation. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires robust methods for the isolation and expansion of these cells—a need forming the basis for these studies

geneBasis: an iterative approach for unsupervised selection of targeted gene panels from scRNA-seq.

Funder: European Molecular Biology Laboratory (EMBL) (4843)scRNA-seq datasets are increasingly used to identify gene panels that can be probed using alternative technologies, such as spatial transcriptomics, where choosing the best subset of genes is vital. Existing methods are limited by a reliance on pre-existing cell type labels or by difficulties in identifying markers of rare cells. We introduce an iterative approach, geneBasis, for selecting an optimal gene panel, where each newly added gene captures the maximum distance between the true manifold and the manifold constructed using the currently selected gene panel. Our approach outperforms existing strategies and can resolve cell types and subtle cell state differences

Antithymocyte Globulin Plus G-CSF Combination Therapy Leads to Sustained Immunomodulatory and Metabolic Effects in a Subset of Responders With Established Type 1 Diabetes.

Low-dose antithymocyte globulin (ATG) plus pegylated granulocyte colony-stimulating factor (G-CSF) preserves β-cell function for at least 12 months in type 1 diabetes. Herein, we describe metabolic and immunological parameters 24 months following treatment. Patients with established type 1 diabetes (duration 4-24 months) were randomized to ATG and pegylated G-CSF (ATG+G-CSF) (N = 17) or placebo (N = 8). Primary outcomes included C-peptide area under the curve (AUC) following a mixed-meal tolerance test (MMTT) and flow cytometry. "Responders" (12-month C-peptide ≥ baseline), "super responders" (24-month C-peptide ≥ baseline), and "nonresponders" (12-month C-peptide < baseline) were evaluated for biomarkers of outcome. At 24 months, MMTT-stimulated AUC C-peptide was not significantly different in ATG+G-CSF (0.49 nmol/L/min) versus placebo (0.29 nmol/L/min). Subjects treated with ATG+G-CSF demonstrated reduced CD4+ T cells and CD4+/CD8+ T-cell ratio and increased CD16+CD56hi natural killer cells (NK), CD4+ effector memory T cells (Tem), CD4+PD-1+ central memory T cells (Tcm), Tcm PD-1 expression, and neutrophils. FOXP3+Helios+ regulatory T cells (Treg) were elevated in ATG+G-CSF subjects at 6, 12, and 18 but not 24 months. Immunophenotyping identified differential HLA-DR expression on monocytes and NK and altered CXCR3 and PD-1 expression on T-cell subsets. As such, a group of metabolic and immunological responders was identified. A phase II study of ATG+G-CSF in patients with new-onset type 1 diabetes is ongoing and may support ATG+G-CSF as a prevention strategy in high-risk subjects

The autoimmune disease-associated SNP rs917997 of IL18RAP controls IFNγ production by PBMC

AbstractType 1 Diabetes (T1D) is an autoimmune disorder characterized by aberrant T cell responses. Innate immune activation defects may facilitate a T helper 1 (Th1) phenotype. The cytokine IL-18 synergizes with IL-12 to induce IFNγ production and Th1 differentiation. The IL-18R subunit (IL18RAP) SNP rs917997 has been linked to decreased IL18RAP gene expression. Prior reports link rs917997 allele A with protection from T1D, and conversely with susceptibility to Celiac disease. However, few studies have investigated the IL-18 pathway in T1D. In this study, we analyzed responsiveness to IL-18 in T1D, and the effect of rs917997 genotype on IL18RAP gene expression post-activation. Upon IL-12 and IL-18 treatment, peripheral blood mononuclear cells from subjects carrying susceptibility alleles at rs917997 produced higher levels of IFNγ than those with protective genotypes. Additionally, the SNP modified IL18RAP surface protein expression by NK cells and gene expression in activated T cells. Taken together, these data suggest that the disease-associated rs917997 allele G permits hyperresponsiveness to IL-18, providing a novel target for therapeutic intervention in T1D