CXCR4 Physically Associates with the T Cell Receptor to Signal in T Cells

SummarySDF-1α (CXCL12) signaling via its receptor, CXCR4, stimulates T cell chemotaxis and gene expression. The ZAP-70 tyrosine kinase critically mediates SDF-1α-dependent migration and prolonged ERK mitogen-activated protein (MAP) kinase activation in T cells. However, the molecular mechanism by which CXCR4 or other G protein-coupled receptors activate ZAP-70 has not been characterized. Here we show that SDF-1α stimulates the physical association of CXCR4 and the T cell receptor (TCR) and utilizes the ZAP-70 binding ITAM domains of the TCR for signal transduction. This pathway is responsible for several of the effects of SDF-1α on T cells, including prolonged ERK MAP kinase activity, increased intracellular calcium ion concentrations, robust AP-1 transcriptional activity, and SDF-1α costimulation of cytokine secretion. These results suggest new paradigms for understanding the effects of SDF-1α and other chemokines on immunity

CAML Is a p56Lck-Interacting Protein that Is Required for Thymocyte Development

SummaryCalcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein implicated in T cell signaling, although its mechanism and physiologic role in the immune system are unknown. We show here that CAML is essential for peripheral T cell development. Inactivation of CAML in mouse thymocytes lowered the numbers of double-positive and single-positive thymocytes, concomitant with reduced positive and enhanced negative selection. We found that CAML interacts with p56Lck and appears to regulate subcellular localization of the kinase in both resting and T cell receptor (TCR)-stimulated cells. CAML-deficient cells displayed enhanced p56lck and ZAP-70 phosphorylation and increased IL2 production and cell death after TCR stimulation, suggesting that CAML may act as a negative regulator of p56lck. Our data establish a novel role for CAML as an essential mediator of T cell survival during thymopoiesis and indicate that its loss deregulates p56Lck signaling

Relationship of relapse-over-expressed immune genes to the ribosomal gene signature.

<p>(A) Association of increased <i>HLA-DRB1</i> and <i>HLA-DRB5</i> expression with history of relapse. Proportions were compared using Fisher’s exact test. Y-axes are in normalized units. Expression levels >400 normalized units were considered high. (B) Positive correlation of the patient ribosomal gene signature with expression of seven of eight immune genes over-expressed in relapse patients. Spearman r values are shown. Y-axes are in normalized units.</p

Association of RGS, HLA DR gene expression or combination thereof with relapse in ADAMTS13-deficient TTP.

<p>^a Odds ratios (OR) estimated by modified median unbiased estimation (MMUE) due to zero cells;</p><p>^b Confidence intervals (CI) estimated by exact, bootstrapped MMUE;</p><p>^c Fisher’s exact test, 2-tailed.</p><p>Association of RGS, HLA DR gene expression or combination thereof with relapse in ADAMTS13-deficient TTP.</p

Relationship between peripheral blood markers and number of episodes.

<p>Expression of five immune transcripts overexpressed in patients with history of relapse correlates with number of past TTP episodes. Serum ADAMTS13 activity does not correlate with number of past episodes (upper left panel). Y-axis is percent of full activity for ADAMTS13 as measured by FRET assay (upper left panel). Y-axis is gene expression in normalized units for the five immune genes (all other panels). Spearman R values are shown.</p

Direct comparison of gene expression of peripheral blood samples between patients with or without a history of relapse.

<p>(A) Average normalized ribosomal gene signature expression values in patients with or without a history of relapse. Error bars depict standard error of the mean. Expression values were compared using Student’s t-test. (B) Relative expression levels of genes differentially expressed between relapse and non-relapse patients presented following clustering. Colors indicate relative increased (red) or decreased (green) expression.</p

Type I IFN and ribosomal gene expression signature associations.

<p>(A) Association of the type I IFN gene signature with antibodies to RNA binding proteins (RBP) but not relapse history. (B) Association of the ribosomal gene signature (RGS) with relapse history but not antibodies to RBP. Proportions were compared using Fisher’s exact test. Odds ratios (OR) were calculated as described in Materials and Methods.</p

Characteristics of TTP remission cohort^a.

<p>^a Individuals with a history of clinically diagnosed TTP (<10% ADAMTS13 activity during a prior acute episode), in remission for at least 2 months, who fail to meet criteria for any known rheumatic disease;</p><p>^b t-test;</p><p>^c Fisher’s exact test;</p><p>^d Kolmogorov-Smirnov test.</p><p>Characteristics of TTP remission cohort<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117614#t001fn001" target="_blank">^a</a>.</p

Expression of CD52, CD244 and CD161 protein on the cell surface of CD3^-CD56^dim NK and CD3⁺ T cells isolated from relapsed ADAMTS13-deficient TTP patients in remission.

<p>Mean fluorescence intensity (MFI) of CD52, CD244 and CD161 on CD3^-CD56^dim and CD3^-CD56^bright NK cells (A) and on CD3⁺ T cells (B) derived from relapsed (R) or non-relapsed (NR) TTP patients. Representative histograms of fluorescence intensity of markers exhibiting significant differences between relapse (thick, dashed lines) and non-relapsed (thin, solid lines) patients are shown in C. (relapse n = 9, non-relapse n = 13, <i>Mann-Whitney test</i>; **p<0.01).</p