30 research outputs found
N-WASP is required for B-cell–mediated autoimmunity in Wiskott-Aldrich syndrome
No full textMutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott-Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling by modulating B-cell receptor (BCR) clustering and internalization. We have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-cell-dependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS
ARHGEF1 deficiency reveals Gα13-associated GPCRs are critical regulators of human lymphocyte function
No full textAbstract A202: Lysophosphatidic acid impedes the effector function of CD8+ T-cells through LPA5R
No full textAnti-CD8 monoclonal antibody-mediated depletion alters the phenotype and behavior of surviving CD8+ T cells
No full textIt is common practice for researchers to use antibodies to remove a specific cell type to infer its function. However, it is difficult to completely eliminate a cell type and there is often limited or no information as to how the cells which survive depletion are affected. This is particularly important for CD8+ T cells for two reasons. First, they are more resistant to mAb-mediated depletion than other lymphocytes. Second, targeting either the CD8α or CD8β chain could induce differential effects. We show here that two commonly used mAbs, against either the CD8α or CD8β subunit, can differentially affect cellular metabolism. Further, in vivo treatment leaves behind a population of CD8+ T cells with different phenotypic and functional attributes relative to each other or control CD8+ T cells. The impact of anti-CD8 antibodies on CD8+ T cell phenotype and function indicates the need to carefully consider the use of these, and possibly other “depleting” antibodies, as they could significantly complicate the interpretation of results or change the outcome of an experiment. These observations could impact how immunotherapy and modulation of CD8+ T cell activation is pursued.</div
LPA5 Is an Inhibitory Receptor That Suppresses CD8 T-Cell Cytotoxic Function via Disruption of Early TCR Signaling
No full textPersistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity
CD8+ T cells surviving depletion differentially localize dependent on mAb used for depletion and type of challenge.
No full text(A-B) 106 CD45.1+ OT1 T cells were transferred i.v. into CD45.2+ mice, treated with anti-CD8α or –β the next day, and immunized or VV-ova infected the day after. Spleens were harvested on day 7 post-infection or immunization and either used for flow cytometry or fixed for immunofluorescence imaging. (A) Post-depletion OT1 T cells were stained for levels of CXCR3 and CD62L. (B) CD45.1+ OT1 T cells were quantified as either white pulp (WP) or red pulp (RP) localized and plotted as a ratio. Points represent the average value per mouse determined from at least 3 sections, pooled from 2 experiments. (C) Representative images of spleen sections 7 days after immunization. Top: blue IgM staining is used to highlight the architecture and delineate RP and WP and false color spots were superimposed to show OT1 T cells scored as WP localized (green) and RP localized (red). White bar represents 100μm. Bottom: images magnified to show individual WP regions; blue MOMA-1 (CD169) staining shows metallophilic marginal macrophages and outlines WP regions and red CD45.1 staining depicts CD45.1+ OT1 T cells.</p
Anti-CD8α boosts the metabolism of CD8+ T cells during activation.
No full text(A-D) CD8+ T cells were isolated from unmanipulated mice and anti-CD3ε /anti-CD28 stimulated for 2–3 days in the presence of either anti-CD8α or –β prior to metabolic flux profiling. Dashed line represents control value. (A) Resting ECAR and OCR of blasting CD8+ T cells. (B) Maximum glycolysis was obtained by injection of oligomycin and glycolytic reserve was then calculated by subtracting basal ECAR from the maximum. (C) Maximum respiration was obtained by injection of FCCP and spare capacity was then calculated by subtracting basal OCR from the maximum. (D) Plot of ECAR and (E) OCR measurements overtime with drug injections indicated. Data are representative of 3 experiments performed with at least 3 technical replicates per treatment group.</p
CD8+ T cells are resistant to mAb-mediated depletion and more difficult to identify post-depletion.
No full text(A-C) Naïve mice were treated i.p. with a total of 10μg of: anti-CD8α only, anti-CD8β only, or 5μg of both. Splenocytes were isolated and stained the next day. CD8+ T cells were considered CD3+ CD4- B220-. (A) CD8+ T cells as a percentage of lymphocytes and total numbers were calculated. (B) Remaining CD8+ T cell surface bound depleting mAb was determined by staining with Goat anti-Rat IgG. (C) Levels of unbound CD8α and CD8β on surviving CD8+ T cells was determined by staining with the same clones used to deplete. (D) Unmanipulated splenocytes were kept on ice and stained with either anti-CD8α or –β and then stained with the other. Data representative of at least 3 independent experiments. Each point represents the average value of a mouse with 3–5 mice per group.</p
IDENTIFICADOS TRES TIPOS DE RESPUESTAS INMUNOLÓGICAS EN PACIENTES DE COVID-19
No full textMuchos pacientes con enfermedad por COVID-19, causada por una infección por coronavirus 2 (SARS-CoV-2) del síndrome respiratorio agudo severo, presentan una enfermedad respiratoria grave que requiere hospitalización y ventilación mecánica. Aunque la mayoría de los pacientes se recuperan, la enfermedad es compleja y la letalidad puede llegar al 10%. Actualmente, no se comprende bien cómo las respuestas inmunitarias humanas controlan o exacerban el COVID-19, y definir la naturaleza de las respuestas inmunitarias durante el COVID-19 agudo podría ayudar a identificar terapias y vacunas eficaces. Con este objetivo, se publicó un estudio en Science prominentes y distintos de pacientes COVID-19 hospitalizados, los cuales están asociados con trayectorias clínicas deficientes frente a la mejora de la salud. Estos inmunotipos pueden tener implicaciones para el diseño de terapias y vacunas para COVID-19
Anti-CD8 mAbs have direct effects on target recognition and indirect effects on cytotoxic function.
No full text(A-B) IncuCyte cytotoxicity assays presented as the ratio of target cell death/total target cells over time. Activated OT1 T cells were added to previously plated B16.OVA.RFP tumor target cells. (A) in vitro SIINFEKL activated OT1 T cells were plated at an effector to target ratio of 10:1. Anti-CD8 mAbs were then added at multiple concentrations with at least 4 technical replicates per condition. Data is representative of 3 independent experiments. (B) OT1 T cells were adoptively transferred into naïve mice that were anti-CD8 treated the next day. The following day the mice were immunized and the OT1 T cells FACS isolated from pooled lymph nodes and spleens 7 days later. OT1 T cells were then plated at an effector to target ratio of 1:1 with at least 3 technical replicates per treatment group. Data is representative of 2 independent experiments.</p
