A Flow Cytometry-Based Protocol to Measure Lymphocyte Viability Upon Metabolic Stress.

Distinct lymphocyte subpopulations display discrete metabolic profiles and are differently affected by metabolic resource variations, making the analysis of lymphocyte survival in a complex tissue in response to metabolic stress highly challenging. Here we describe a flow cytometry-based method allowing simultaneous cell identification and viable cell counting in mixed lymphocyte populations without extensive cell subset purification procedures. The example provided herein illustrates the role of AMPK in T lymphocyte survival in response to the mitochondrial poison oligomycin.info:eu-repo/semantics/publishe

Naive T cells are resistant to anergy induction by anti-CD3 antibodies.

Anti-CD3 mAbs are potent immunosuppressive agents used in clinical transplantation. It has been generally assumed that one of the anti-CD3 mAb-mediated tolerance mechanisms is through the induction of naive T cell unresponsiveness, often referred to as anergy. We demonstrate in this study that naive T cells stimulated by anti-CD3 mAbs both in vivo and in vitro do not respond to the superantigen staphylococcal enterotoxin B nor to soluble forms of anti-CD3 mAbs and APC, but express increased reactivity to plastic-coated forms of the same anti-CD3 mAbs and to their nominal Ag/class II MHC, a finding that is difficult to rationalize with the concept of anergy. Phenotypic and detailed kinetic studies further suggest that a strong signal 1 delivered by anti-CD3 mAbs in the absence of costimulatory molecules does not lead to anergy, but rather induces naive T cells to change their mitogen responsiveness and acquire features of memory T cells. In marked contrast, Ag-experienced T cells are sensitive to anergy induction under the same experimental settings. Collectively, these studies demonstrate that exposure of naive T cells in vivo and in vitro to a strong TCR stimulus does not induce Ag unresponsiveness, indicating that sensitivity to negative signaling through TCR/CD3 triggering is developmentally regulated in CD4(+) T cells.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

The transcription factor c-Maf promotes the differentiation of follicular helper T cells

Follicular helper T cells (Tfh) have been identified as the primary cell subpopulation regulating B cell responses in germinal centers, thus supporting high-affinity antibody production. Among the transcription factors orchestrating Tfh cell differentiation and function, the role played by the proto-oncogene c-Maf remains poorly characterized. We report herein that selective loss of c-Maf expression in the T cell compartment results in defective development of Tfh cells in response to both antigen/adjuvant vaccinations and commensal intestinal bacteria. Accordingly,c-Maf expression in T cells was essential for the development and high-affinity antibody secretion in vaccinated animals. c-Maf was expressed early, concomitantly to BCL6, in Tfh cell precursors and found to regulate Tfh fate in a cell-autonomous fashion. Altogether, our findings reveal a novel, non-redundant, function for c-Maf in the differentiation of Tfh cells and the regulation of humoral immune responses to T-cell-dependent antigens.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Antigen-presenting cell-derived IL-6 restricts the expression of GATA3 and IL-4 by follicular helper T cells.

Follicular helper T cells (Tfh) support high-affinity Ab production by germinal center B cells through both membrane interactions and secretion of IL-4 and -21, two major cytokines implicated in B-cell survival and Ab class switch. Tfh-2 cells recently emerged in humans as a strong IL-4 producer Tfh cell subset implicated in both autoimmune and allergic diseases. Although the molecular mechanisms governing Tfh cell differentiation from naive T cells have been widely described, much less is known about the regulation of cytokine secretion by mouse Tfh-2 cells. The purpose of our study was to evaluate the role of dendritic cell-derived IL-6 in fine-tuning cytokine secretion by Tfh cells. Our results demonstrate that priming of Th cells by IL-6-deficient antigen-presenting dendritic cells preferentially leads to accumulation of a subset of Tfh cells characterized by high expression of GATA3 and IL-4, associated with reduced production of IL-21. STAT3-deficient Tfh cells also overexpress GATA3, suggesting that early IL-6/STAT3 signaling during Tfh cell development inhibits the expression of a set of genes associated with the Th2 differentiation program. Overall, our data indicate that IL-6/STAT3 signaling restrains the expression of Th2-like genes in Tfh cells, thus contributing to the control of IgE secretion in vivo.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Long-term T cell fitness and proliferation is driven by AMPK-dependent regulation of reactive oxygen species

The AMP-activated kinase (AMPK) is a major energy sensor metabolic enzyme that is activated early during T cell immune responses but its role in the generation of effector T cells is still controversial. Using both in vitro and in vivo models of T cell proliferation, we show herein that AMPK is dispensable for early TCR signaling and short-term proliferation but required for sustained long-term T cell proliferation and effector/memory T cell survival. In particular, AMPK promoted accumulation of effector/memory T cells in competitive homeostatic proliferation settings. Transplantation of AMPK-deficient hematopoïetic cells into allogeneic host recipients led to a reduced graft-versus-host disease, further bolstering a role for AMPK in the expansion and pathogenicity of effector T cells. Mechanistically, AMPK expression enhances the mitochondrial membrane potential of T cells, limits reactive oxygen species (ROS) production, and resolves ROS-mediated toxicity. Moreover, dampening ROS production alleviates the proliferative defect of AMPK-deficient T cells, therefore indicating a role for an AMPK-mediated ROS control of T cell fitness.info:eu-repo/semantics/publishe

Interleukin-6/STAT3 signaling regulates the ability of naive T cells to acquire B-cell help capacities

The conditions leading to the activation/differentiation of T-helper (Th) cells dedicated for B-cell antibody production are still poorly characterized. We now demonstrate that interleukin-6 (IL-6) promotes the differentiation of naive T lymphocytes into helper cells able to promote B-cell activation and antibody secretion. IL-6–driven acquisition of B-cell help capacity requires expression of the signal transducer and activator of transcription 3 (STAT3), but not STAT4 or STAT6 transcription factors, suggesting that the ability to provide help to B cells is not restricted to a well-defined Th1 or Th2 effector population. T cell–specific STAT3-deficient mice displayed reduced humoral responses in vivo that could not be related to an altered expansion of CXCR5-expressing helper T cells. IL-6 was shown to promote IL-21 secretion, a cytokine that was similarly found to promote the differentiation of naive T cells into potent B-cell helper cells. Collectively, these data indicate that the ability to provide B-cell help is regulated by IL-6/IL-21 through STAT3 activation, independently of Th1, Th2, Th17, or follicular helper T cell (TFH) differentiation

Tfh-like cells express MyoR mRNA.

<p>Naive CD62L⁺CD4⁺ T cells purified from the spleen of C57BL/6 mice were stimulated with plastic-coated anti-CD3 and anti-CD28 mAbs under neutral conditions (medium) or in the presence of IL-6 (Tfh-like condition). Expression level of the indicated genes was assessed by quantitative RT-PCR and expressed as relative expression to RPL32 mRNA. (A) Kinetic expression of MyoR under Th0 and Tfh culture conditions; (B) Compilation of individual experiments showing increased MyoR expression in 72 h cultured-Tfh-like cells; (C) Expression of a set of Tfh-associated genes in 72 h-cultured cells in the presence of IL-6; (D) MyoR expression in resting versus TcR activated, IL-6-treated T lymphocytes; (E) Expression of MyoR in 48 h-Tfh-like activated wild type and STAT3-deficient T cells; (F) MyoR, T-bet, GATA-3 and RORγT mRNA expression in 72 h-polarized Th0, Th1, Th2, Th17 and Tfh-like cells. The 72 h activated-Th0 condition (48 h in panel E) was set to 1. Histograms represent the mean ± SD of duplicates and are representative of three independent experiments (A, C-E and F-panels T-bet, GATA-3, RORγT) or the mean ± SD of three independent experiments (F-panel MyoR). Dots in panel B represent individual paired Th0 and Tfh cultures data sets. Differences between groups in B were analyzed with the Mann-Whitney test for 2-tailed data. * p<0.05; n.d. =  not detectable</p