Redox Regulation of LAT Enhances T Cell-Mediated Inflammation

Abstract

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LAT^SS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LAT^SS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1^m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs).

Keywords: NCF1; T cell receptor signaling; linker for activation of T cells; reactive oxygen species; rheumatoid arthritis.

Figure A1

Membrane LAT expression decreases under oxidative pressure. Cells treated with different H₂O₂ concentrations in vitro. LAT quantification in the membrane and cytosol.

Figure A2

Validation of the nucleotide sequence of the LAT^SS mutations. (A) Comparison of the nucleotide sequence of the Lat gene encompassing the sequence coding for the cysteine residue found at position 120 (identified in black C120 top and S120 bottom) from BQ wild-type control mice and homozygous mice for the LAT^SS mutation. The LAT^C120S mutation is associated with a silent SalI restriction site identified with the red star at the bottom and a black star in the wild-type sequence at the top. (B) Comparison of the nucleotide sequence of the Lat gene encompassing the sequence coding for the cysteine residue found at position 172 (identified in black C172 top and S172 bottom) from BQ wild-type control mice and homozygous mice for the LAT^SS mutation. The LAT^C172S mutation is associated with a silent BspEI restriction site identified with the red star at the bottom sequence and a black star in the wild-type sequence. All the other colors correspond to different nucleotides.

Figure A3

LAT^SS does not affect the proliferation or activation of peripheral T cells. (A) Calcium flux analysis of CD4 T cells stimulated with anti-CD3 via FuraRed/Fluo4 staining. (B) Proliferation analysis of CD4 T cells stimulated with anti-CD3/anti-CD28 for 72 h assessed by CFSE dilution. (C) Expression of CD69 on T cells after ex vivo stimulation with either anti-CD3, anti-CD3/anti-CD28, or unstimulated measured by flow cytometry. (D) Representative immunoblot showing phosphorylation of PKC, ZAP70, Src, and ERK in sorted CD4 T cells upon stimulation with anti-CD3/anti-CD28 for 0, 2.5, and 6 min; quantification to the right shows phosphorylated protein expression normalized to total protein expression and loading control BQ (n = 3); LAT^SS (n = 3). Error bars represent mean ± SEM.

Figure A4

Cell phenotyping 12 days after 1 × 10⁶ CD4 T cells were transferred. (A) CD3, CD4, and CD8 cell populations with representative flow cytometry gating on the right. (B) IFN-γ OD values determined by ELISA using ear lymph nodes. * p < 0.05.

Figure 1

Cysteines 120 and 172 mediate LAT activation through redox regulation. (A) Confocal differential interference contrast (DIC) and fluorescence images of LAT-YPet cells with and without treatment with 20 μM H₂O₂. (B) Hypothetical model for LAT displacement in oxidative conditions: (left) the sulfhydryl group of C120/C172 forms a disulfide bond with the sulfhydryl group on C26/C29 causing sterical hindrance and thereby displacement from the membrane; (right) cysteine-to-serine mutations render LAT insensitive to redox regulation. (C) Confocal microscopy image showing LAT distribution (in red) in WT and LAT^SS lymph node cells either untreated or treated with the glutathione inhibitor BSO. (D) LAT Y191 phosphorylation in unstimulated lymph node cells, and 2 and 5 min upon stimulation with anti-CD3/anti-CD28. Each lane represents an individual mouse. On the right, quantification shows phosphorylated protein expression normalized to vinculin loading control. Error bars represent mean ± SEM. * p < 0.05, and ** p < 0.01.

Figure 2

LAT^SS affects thymic selection and peripheral T cell populations. (A–C) Flow cytometry analysis of T cell populations in the thymus of littermate BQ (wild-type) and LAT^SS mice with representative flow cytometry plots shown; DN1 (CD44+ CD25−), DN2 (CD44+ CD25+), DN3 (CD25+ CD44−), DN4 (CD25− CD44−). (D) Percentages of CD4+, CD4+ CD69+, and CD4+ CD25+ Foxp3+ T cells in naive lymph nodes. (E) CD44 and CD62L expression on CD4 T cells in naive lymph nodes. Representative gating is shown. (F) Vβ chain usage on naive CD4/CD8 T cells in the blood measured by flow cytometry. Error bars represent mean ± SEM. * p < 0.05, and ** p < 0.01.

Figure 3

LAT^SS enhances inflammation in the DTH model. (A–C) Mice were immunized according to the DTH protocol with COL2. (A) Mean Δ ear pinna thickness in mm (calculated as COL2-injected ear swelling minus PBS-injected ear swelling) 24, 48, and 72 h after COL2 challenge; BQ (n = 4), LAT^SS (n = 5). Data are representative of 3 experiments. Two-way ANOVA. (B) Percentage of CD45+ cells in wild-type BQ and LAT^SS COL2-injected ear compared to PBS-injected ear measured by flow cytometry and representative gating on the right. (C) Intracellular flow cytometry staining of IFN-γ in inguinal lymph nodes after stimulation with PMA and representative gating on the right. (D) Mean Δ ear pinna thickness in mm in TCRβ knock-out mice post-transfer of purified CD4+ T cells from BQ and LAT^SS mice 10 days after DTH induction. Error bars represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

LAT^SS regulates CIA susceptibility in an NCF1-dependent manner. (A–E) Mice were immunized according to the CIA protocol with COL2. (A,B) Clinical score and incidence of littermate mice immunized with COL2. Data are representative of 3 independent experiments. (C) IL–2 and IFN-γ production by T cell recall assay. (D) Percentage of CD4+ and CD4+ CD40L+ T cells in draining lymph nodes measured by flow cytometry. Representative gating for CD40L is shown. Error bars represent mean ± SEM. (E) Levels of serum antibodies against COL2 measured by ELISA. (F) Luminex analysis of serum antibody reactivity against F4 epitope on COL2; wild-type peptide R-R with unmodified arginine residues, Cit-R with citrullinated arginine residues, and hLys with hydroxylated lysine modifications. * p < 0.05, and ** p < 0.01.