Migration of Th1 Lymphocytes Is Regulated by CD152 (CTLA-4)-Mediated Signaling via PI3 Kinase-Dependent Akt Activation

Efficient adaptive immune responses require the localization of T lymphocytes in secondary lymphoid organs and inflamed tissues. To achieve correct localization of T lymphocytes, the migration of these cells is initiated and directed by adhesion molecules and chemokines. It has recently been shown that the inhibitory surface molecule CD152 (CTLA-4) initiates Th cell migration, but the molecular mechanism underlying this effect remains to be elucidated. Using CD4 T lymphocytes derived from OVA-specific TCR transgenic CD152-deficient and CD152-competent mice, we demonstrate that chemokine-triggered signal transduction is differentially regulated by CD152 via phosphoinositide 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt). In the presence of CD152 signaling, the chemoattractant CCL4 selectively induces the full activation of Akt via phosphorylation at threonine 308 and serine 473 in pro-inflammatory Th lymphocytes expressing the cognate chemokine receptor CCR5. Akt signals lead to cytoskeleton rearrangements, which are indispensable for migration. Therefore, this novel Akt-modulating function of CD152 signals affecting T cell migration demonstrates that boosting CD152 or its down-stream signal transduction could aid therapies aimed at sensitizing T lymphocytes for optimal migration, thus contributing to a precise and effective immune response

Effect of induction with the chemokine CCL4 on signaling molecules.

<p><b>A</b> Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice were analyzed for ERK phosphorylation on day 2 and day 5 of a recall response. Signaling via CCR5 was induced in serum-starved lymphocytes by treatment with 20 nM CCL4 for 5 (heavy black line) or 20 min (heavy grey line). Filled histograms indicate ERK phosphorylation before CCL4 application. <b>B</b> Migration assay in Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice on day 5 of a recall response. A portion of the lymphocytes was incubated with the PKCθ-specific inhibitor Rottlerin for 20 h. <b>C</b> Western blot detection of signaling proteins in lysates of TCR^tgCD152-deficient and TCR^tgCD152-competent Th1 lymphocytes on day 5 of a recall response. The cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031391#pone-0031391-g002" target="_blank">Fig. 2 A</a>. <b>D</b> The band intensity of pGRK2 relative to GRK2 was quantified using Image Gauge 4.0. Representative data from at least two experiments are shown.</p

Chemokine receptor signaling affects signaling molecules involved in cytoskeleton rearrangements.

<p><b>A</b> Activated Rac was detected by G-LISA in Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice on day 5 of a recall response. Rac activation was induced in serum-starved lymphocytes by treatment with 20 nM CCL4 for 5 or 20 min. <b>B</b> Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice on day 5 of a recall response were incubated with or without 20 nM CCL4 for 5 or 20 min. Fixed and permeabilized lymphocytes were analyzed by flow cytometry for Akt activation using antibodies specific for phosphorylated Akt or total Akt. A protion of the lymphocyteswere incubated for 20 h with the PI3K inhibitor Ly294.002 (filled histograms). Histograms show the expression of Akt on T lymphocytes. <b>C</b> Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice were analyzed to determine their migration affinities for the inflammatory chemokine CCL4 in a transwell system on day 5 of a recall response. A portion of the Th1 lymphocytes were incubated for 20 h with a PI3K inhibitor or Akt inhibitor II, as indicated. The dotted line indicates basal migration toward medium (ns, not significant). Representative data from at least two experiments are shown.</p

Chemokine receptor expression in activated T lymphocytes does not reflect the chemotactic capacities of corresponding chemokines.

<p>CD4⁺ T lymphocytes from TCR^tgCD152-deficient (CD152^−/−) and TCR^tgCD152-competent (CD152^+/+) mice were analyzed to detect chemokine receptor expression by flow cytometry (upper panels) and in chemotaxis assays (lower panels) on day 3 after the initiation of recall responses. <b>A</b> CCR7 expression and migration toward medium or CCL19 were detected in T lymphocytes. <b>B</b> Th1-differentiated CD4⁺ T lymphocytes were analyzed for CCR7 expression and migration behavior toward medium or CCL19. <b>C</b> Th1 lymphocytes exhibited similar CCR5 expression levels (M1: TCR^tgCD152-deficient, 60%; TCR^tgCD152-competent, 60%) but different migration rates in transwell systems. Representative data from two experiments are shown.</p

Role of CD28 and CD152 signals in signal transduction via the chemokine receptor CCR5 in Th1 lymphocytes.

<p>Upon binding of its ligand, CCL4, the CCR5 receptor is phosphorylated by CD28-induced GRK2. β-arrestins can now bind to CCR5 and initiate desensitization, which contributes to the degradation or recycling of CCR5. CD152 engagement leads to the inactivation of GRK2, and the phosphorylation of CCR5 is prevented. CD28 and CD152 signal-induced activation of integrins by the Gβγ subunit and via the GTPases Rac1 and Cdc42 or Rap1 ultimately leads to lymphocyte adhesion. Chemokine-induced activation of PI3K and the subsequent phosphorylation and activation of Akt are only initiated in the presence of CD152 signaling, and it is only under these conditions that specific migration occurs along chemokine gradients (orange arrows indicate signal transduction under CD28 signaling, and blue arrows indicate signal transduction under CD152 signaling). The figure shows only those signaling pathways controlled by CD152.</p

The differentiation and plasticity of Tc17 cells are regulated by CTLA-4-mediated effects on STATs

As the blockade of inhibitory surface-molecules such as CTLA-4 on T cells has led to recent advances in antitumor immune therapy, there is great interest in identifying novel mechanisms of action of CD8+ T cells to evoke effective cytotoxic antitumor responses. Using in vitro and in vivo models, we investigated the molecular pathways underlying the CTLA-4-mediated differentiation of IL-17-producing CD8+ T cells (Tc17 cells) that strongly impairs cytotoxicity. Our studies demonstrate that Tc17 cells lacking CTLA-4 signaling have limited production of STAT3-target gene products such as IL-17, IL-21, IL-23R and RORγt. Upon re-stimulation with IL-12, these cells display fast downregulation of Tc17 hallmarks and acquire Tc1 characteristics such as IFNγ and TNF-α co-expression, which is known to correlate with tumor control. Indeed, upon adoptive transfer, these cells were highly efficient in the antigen-specific rejection of established OVA-expressing B16 melanoma in vivo. Mechanistically, in primary and re-stimulated Tc17 cells, STAT3 binding to the IL-17 promoter was strongly augmented by CTLA-4, associated with less binding of STAT5 and reduced relative activation of STAT1 which is known to block STAT3 activity. Inhibiting CTLA-4-induced STAT3 activity reverses enhancement of signature Tc17 gene products, rendering Tc17 cells susceptible to conversion to Tc1-like cells with enhanced cytotoxic potential. Thus, CTLA-4 critically shapes the characteristics of Tc17 cells by regulating relative STAT3 activation, which provides new perspectives to enhance cytotoxicity of antitumor responses

An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells

Abstract During B cell maturation, transitional and mature B cells acquire cell-intrinsic features that determine their ability to exit quiescence and mount effective immune responses. Here we use label-free proteomics to quantify the proteome of B cell subsets from the mouse spleen and map the differential expression of environmental sensing, transcription, and translation initiation factors that define cellular identity and function. Cross-examination of the full-length transcriptome and proteome identifies mRNAs related to B cell activation and antibody secretion that are not accompanied by detection of the encoded proteins. In addition, proteomic data further suggests that the translational repressor PDCD4 restrains B cell responses, in particular those from marginal zone B cells, to a T-cell independent antigen. In summary, our molecular characterization of B cell maturation presents a valuable resource to further explore the mechanisms underpinning the specialized functions of B cell subsets, and suggest the presence of ‘poised’ mRNAs that enable expedited B cell responses

PAG/Cbp suppression reveals a contribution of CTLA-4 to setting the activation threshold in T cells.

PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation

Additional file 1: Figure S1. of Early changes in the metabolic profile of activated CD8+ T cells

Purified CD8+ T cells were treated with OT-I-streptamers for the indicated time periods. Samples were analyzed by Western blotting using the indicated Abs to determine the inhibition of mTOR (A) and AKT (B). Purified CD8+ T cells were treated with OT-I-streptamers for 24 h in presence or absence of aIL-2. Samples were analyzed for pSTAT5 activation to determine the function of aIL-2 antibody (C) Toxicity was assessed for the inhibitors rotenone and oxamate and the aIL-2 antibody by AnnexinV PI staining 24 h after stimulation (D). (TIF 189 kb

Bifidobacteria shape antimicrobial T-helper cell responses during infancy and adulthood

Abstract Microbial infections early in life are challenging for the unexperienced immune system. The SARS-CoV-2 pandemic again has highlighted that neonatal, infant, child, and adult T-helper(Th)-cells respond differently to infections, and requires further understanding. This study investigates anti-bacterial T-cell responses against Staphylococcus aureus aureus, Staphylococcus epidermidis and Bifidobacterium longum infantis in early stages of life and adults and shows age and pathogen-dependent mechanisms. Beside activation-induced clustering, T-cells stimulated with Staphylococci become Th1-type cells; however, this differentiation is mitigated in Bifidobacterium-stimulated T-cells. Strikingly, prestimulation of T-cells with Bifidobacterium suppresses the activation of Staphylococcus-specific T-helper cells in a cell-cell dependent manner by inducing FoxP3+CD4+ T-cells, increasing IL-10 and galectin-1 secretion and showing a CTLA-4-dependent inhibitory capacity. Furthermore Bifidobacterium dampens Th responses of severely ill COVID-19 patients likely contributing to resolution of harmful overreactions of the immune system. Targeted, age-specific interventions may enhance infection defence, and specific immune features may have potential cross-age utilization