CD6-mediated inhibition of T cell activation via modulation of Ras

Abstract

Background: CD6 is one of many cell surface receptors known to regulate signal transduction upon T cell activation. However, whether CD6 mediates costimulatory or inhibitory signals is controversial. When T cells engage with antigen presenting cells (APCs), CD6 interacts with its ligand CD166 at the cell-cell interface while the cytosolic tail assembles a complex signalosome composed of adaptors and effector enzymes, that may either trigger activating signaling cascades, or instead modulate the intensity of signaling. Except for a few cytosolic adaptors that connect different components of the CD6 signalosome, very little is known about the mechanistic effects of the cytosolic effectors that bind CD6.

Methods: Jurkat model T cells were transfected to express wild-type (WT) CD6, or a cytoplasmic truncation, signaling-disabled mutant, CD6Δcyt. The two resulting cell lines were directly activated by superantigen (sAg)-loaded Raji cells, used as APCs, to assess the net signaling function of CD6. The Jurkat cell lines were further adapted to express a FRET-based unimolecular HRas biosensor that reported the activity of this crucial GTPase at the immunological synapse.

Results: We show that deletion of the cytosolic tail of CD6 enhances T-cell responses, indicating that CD6 restrains T-cell activation. One component of the CD6-associated inhibitory apparatus was found to be the GTPase activating protein of Ras (RasGAP), that we show to associate with CD6 in a phosphorylation-dependent manner. The FRET HRas biosensor that we developed was demonstrated to be functional and reporting the activation of the T cell lines. This allowed to determine that the presence of the cytosolic tail of CD6 results in the down-regulation of HRas activity at the immunological synapse, implicating this fundamental GTPase as one of the targets inhibited by CD6.

Conclusions: This study provides the first description of a mechanistic sequence of events underlying the CD6-mediated inhibition of T-cell activation, involving the modulation of the MAPK pathway at several steps, starting with the coupling of RasGAP to the CD6 signalosome, the repression of the activity of Ras, and culminating in the reduction of ERK1/2 phosphorylation and of the expression of the T-cell activation markers CD69 and IL-2R α chain. Video abstract.

Keywords: Cell surface molecules; FRET; GTPases; Immunological synapse; Inhibitory receptors; Signaling transduction.

Fig. 1

CD6 inhibits CD69 and CD25 upregulation following T cell activation. a E6.1 cells were stably transduced with vectors encoding CD6WT or CD6∆cyt. Cells were stained with fluorochrome-conjugated CD6 mAbs, and protein expression was assessed by flow cytometry. Histograms show the expression of the corresponding CD6 forms in each cell line. b E6.1-CD6WT and E6.1-CD6Δcyt cells were cultured for 24 h in the presence of unloaded or sAg-loaded Raji cells, and expression of CD69 and CD25 was then assessed by flow cytometry. Shown are representative histograms of CD69 and CD25 expression of one of three independent experiments. c Percentage of CD69-positive (left panels) and CD25-positive (right panels) E6.1-CD6WT or E6.1-CD6ΔDcyt cells cultured with Raji cells without or with sAg. Each dot represents one independent experiment. Bars represent mean and SD for each condition, that were compared using a paired t test. **, p < 0.01

Fig. 2

RasGAP associates with the cytoplasmic tail of CD6 depending on tyrosine phosphorylation. E6.1-CD6WT, E6.1-CD6∆cyt, as well as untransfected E6.1 cells were incubated with pervanadate for 5 min at 37 °C, lysed in Triton X-100 lysis buffer, and CD6 was immunoprecipitated. RasGAP was detected by WB in CD6WT but not in CD6∆cyt immunoprecipitates in activated cells (left panels). WB of cell lysates (right panels) confirms the presence of the indicated molecules at equivalent levels. Images are of one of two independent experiments. MM, molecular mass; IP, immunoprecipitation; WB, western blotting

Fig. 3

The FRET biosensor MRS2 reports on HRas activation. a Schematic representation of the MRS2 fusion protein and its conformation when HRas is active or inactive. b MRS2- and MRS2-S17N-encoding plasmids were stably transduced into E6.1 cells, and the expression of the fusion proteins was assessed by WB, displaying a size of ~ 120 kDa. Protein species of 58 and 60 kDa presumably represent proteolytic cleavage by-products of the full probe. c Distribution of MRS2 and MRS2-S17N in E6.1 cells assessed by fluorescence microscopy. d MRS2- and MRS2-S17N-expressing E6.1 cells were filmed for 5 min at 37 °C (1 min frames), medium was then added, and cells were filmed for a further 30 min (see Additional file 2: Movie S1). FRET/Clover ratio was assessed for each time-point, and the ratio from MRS2-expressing E6.1 cells was normalized to that of MRS2-S17N-expressing cells. e E6.1 cells expressing MRS2 and MRS2-S17N formed conjugates with Raji cells, unloaded or pre-loaded with sAg (see Additional file 3: Movie S2). Raji cells were added at the 5 min mark. FRET/Clover ratios from MRS2-expressing E6.1 cells were divided by those of MRS2-S17N-expressing E6.1 cells for each condition, and normalized for the average of the first three time points to establish the basal activity before the addition of Raji cells. d, e Dots and lines represent mean ± SD of 5 independent experiments. In each experiment, 10 individual cells (in d) or 9–10 cell pairs (in e) were assessed for both MRS2 and MRS2-S17N biosensors. f Area under the curve of graph in (e), with the baseline starting at 1. Each dot represents one of 5 independent experiments, and lines represent paired experiments. 9–10 cells were assessed in all conditions of each individual experiment. *, p < 0.05, paired t test

Fig. 4

The cytosolic tail of CD6 constrains HRas activation in E6.1 cells upon APC-mediated activation. a Representative images of E6.1-CD6WT and E6.1-CD6∆cyt cells, expressing either MRS2 or MRS2-S17N, during the formation of conjugates with sAg-loaded Raji cells (full movies at Additional file 4: Movie S3). FRET/Clover ratio is presented as a thermal range (scale in bottom right). b FRET/Clover ratios from MRS2-expressing cells (see Additional file 1: Fig. S2c) were divided by those of MRS2-S17N-bearing cells and normalized to the average of the first three points before the addition of Raji cells. Dots and lines represent mean ± SD of 3 independent experiments. c Area under the curve of graph in B, where baseline = 1. Bars represent mean and SD, and each dot represents an independent experiment. 9–10 cells were assessed in all conditions of each individual experiment. Comparisons were performed using a paired t test. **, p < 0.01

Fig. 5

CD6 downmodulates ERK1/2 phosphorylation in activated cells. a Parental E6.1 cells, E6.1-CD6WT and E6.1-CD6∆cyt cells were left undisturbed or were activated with CD3 (3 μg/ml) and CD28 (5 μg/ml) mAbs for 5 min at 37 °C, and lysed. Total and phosphorylated ERK1/2 levels were detected in lysates by WB, and quantified using Fiji. b Quantification of ERK1/2 phosphorylation from three independent experiments. Bars represent mean and SD. The probability that the phosphorylation of ERK was similar between E6.1-CD6WT and parental E6.1 or E6.1-CD6∆cyt cells was assessed using a paired t test. *, p < 0.05