Mutation of the glycine residue preceding the sixth tyrosine of the LAT adaptor severely alters T cell development and activation

Abstract

The LAT transmembrane adaptor is essential to transduce intracellular signals triggered by the TCR. Phosphorylation of its four C-terminal tyrosine residues (136, 175, 195, and 235 in mouse LAT) recruits several proteins resulting in the assembly of the LAT signalosome. Among those tyrosine residues, the one found at position 136 of mouse LAT plays a critical role for T cell development and activation. The kinetics of phosphorylation of this residue is delayed as compared to the three other C-terminal tyrosines due to a conserved glycine residue found at position 135. Mutation of this glycine into an aspartate residue (denoted LAT^G135D) increased TCR signaling and altered antigen recognition in human Jurkat T cells and ex vivo mouse T cells. Here, using a strain of LAT^G135D knockin mice, we showed that the LAT^G135D mutation modifies thymic development, causing an increase in the percentage of CD4+CD8+ double-positive cells, and a reduction in the percentage of CD4+ and CD8+ single-positive cells. Interestingly, the LAT^G135D mutation alters thymic development even in a heterozygous state. In the periphery, the LAT^G135D mutation reduces the percentage of CD8+ T cells and results in a small increment of γδ T cells. Remarkably, the LAT^G135D mutation dramatically increases the percentage of central memory CD8+ T cells. Finally, analysis of the proliferation and activation of T lymphocytes shows increased responses of T cells from mutant mice. Altogether, our results reinforce the view that the residue preceding Tyr136 of LAT constitutes a crucial checkpoint in T cell development and activation.

Keywords: T cell; T cell development; T cell memory; TCR (T cell receptor); anergy; linker for activation of T cell.

Figure 1

Thymic development analysis of LAT^G135D mice. (A) Total thymocytes from wild-type (LAT^+/+), heterozygous (LAT^+/G135D) and homozygous (LAT^G135D/G135D) mutant mice were analyzed for the expression of CD4 and CD8 (upper panels) by flow cytometry. The numbers indicated in each gate represent the percentage of cells. Upper panels show a representative experiment. Lower panels represent bar graphs obtained after the analysis of LAT^+/+ (n = 15), LAT^+/G135D (n = 9) and LAT^G135D/G135D (n = 13) mice, showing the percentages of cells in each of the indicated compartments. The brackets on each bar represent the mean standard error. (B) Dot plots showing CD5 and CD3 expression in DP cells from 6 to 14 weeks old wild-type (LAT^+/+, n = 13), heterozygous (LAT^+/G135D, n = 8) and homozygous (LAT^G135D/G135D, n = 12) mice. Lower panels represent the quantification of percentages of preselection DP cells (DP1), DP cells undergoing selection (DP2), and post selection DP thymocytes (DP3). * indicates p<0.05; ** indicates p<0.01. (C) Analysis of TCR signaling in thymocytes. 5 x 10⁶ fresh thymocytes were obtained from wild-type (LAT^+/+) and homozygous (LAT^G135D/G135D) mutant mice and incubated with the indicated concentrations of biotin-conjugated anti-CD3 for 30 min, and then stimulated with 10 µg/ml of streptavidin for 3 min at 37°C. Cells were then lysed and analyzed by Western blot with the indicated specific antibodies. Membranes were stripped and reanalyzed with antibodies β-actin to show total protein load. Numbers below the images represent the densitometric quantification of each band.

Figure 2

Peripheral lymphocyte populations are altered by the LAT-G135D mutation. (A) CD4 and CD8 expression was analyzed in splenocytes from wild-type (LAT^+/+), heterozygous (LAT^+/G135D), and homozygous (LAT^G135D/G135D) mutant mice (upper panels) by flow cytometry. The numbers indicated in each quadrant represent the percentage of cells. Upper panels show a representative experiment. Lower bar graph represents the average of the indicated populations after the analysis of LAT^+/+ (n = 14), LAT^+/G135D (n = 9), and LAT^G135D/G135D (n = 13) mice. The brackets on each bar represent the mean standard error. (B) Dot plots showing CD3 and TCR-γδ expression in splenocytes cells from the indicated types of mice. The bar graph on the right shows the percentages of γδ-T cells in each mouse type: n = 13 for LAT^+/+, n = 9 for LAT^+/G135D, and n = 11 for LAT^G135D/G135D. (C) Analysis of memory and naive CD4 (upper dot plots) and CD8 (lower dot plots) T cells in spleen from wild-type (LAT^+/+), heterozygous (LAT^+/G135D), and homozygous (LAT^G135D/G135D) mutant mice. The numbers in the depicted regions represent the percentage of cells.

Figure 3

LAT^G135D mutation increases the percentage of anergic T cells. Splenocytes from the indicated types of mice were stained with CD4 and CD25 specific monoclonal antibodies (upper dot plots). The numbers indicated in each gate represent the percentage of CD4⁺CD25^- and CD4⁺CD25⁺ cells. Middle panels: CD4⁺CD25^- cells were analyzed for the expression of CD73 and FR4, and the percentage of anergic T cells is indicated. Lower bar graphs: average of the indicated populations after the analysis of LAT^+/+ (n = 13), LAT^+/G135D (n = 8) and LAT^G135D/G135D (n = 12) mice. The brackets on each bar represent the mean standard error. * indicates p<0.05.

Figure 4

Ex vivo stimulation assays. (A) T cells purified from the spleens of wild-type LAT (LAT^+/+) and homozygous (LAT^G135D/G135D) mutant mice were stimulated with the indicated bead:cell ratios of anti-CD3/CD28 microbeads, and analysis of CD69 surface expression 24H post-stimulation was performed on live cells (Annexin V negative). CD69 expression in CD4+ and CD8+ cell populations are shown separately. The numbers indicated in each histogram represent the percentage of CD69 positive cells (red histograms). Blue histograms show CD69 expression in unstimulated cells. One representative experiment is shown (n=3). (B) Analysis of proliferation performed at 72H post-stimulation. Decrease of CTV staining indicates cell proliferation. Blue histograms correspond to unstimulated cells and red histograms to anti-CD3/CD28 stimulated cells. Proliferative capacity was analyzed separately in CD4+ and CD8+ cell populations. The numbers shown in each histogram represent the percentage of cells that have proliferated. One representative experiment is shown (n=3).