LAT-mediated signaling in CD4+CD25+ regulatory T cell development

Abstract

Engagement of the T cell receptor for antigen (TCR) induces formation of signaling complexes mediated through the transmembrane adaptor protein, the linker for activation of T cells (LAT). LAT plays an important role in T cell development, activation, and homeostasis. A knock-in mutation at Tyr136, which is the phospholipase C (PLC)-gamma1-binding site in LAT, leads to a severe autoimmune disease in mice. In this study, we show that CD4+CD25+ T reg cells that expressed Foxp3 transcription factor were nearly absent in both thymus and peripheral lymphoid organs of LAT(Y136F) mice. This defect was not a result of the autoimmune environment as LAT(Y136F) T reg cells also failed to develop in healthy LAT-/- mice that received mixed wild-type and LAT(Y136F) bone marrow cells. Moreover, adoptive transfer of normal CD4+CD25+ T reg cells protected neonatal LAT(Y136F) mice from developing this disease. These T reg cells effectively controlled expansion of CD4+ T cells in LAT(Y136F) mice likely via granzymes and/or TGF-beta-mediated suppression. Furthermore, ectopic expression of Foxp3 conferred a suppressive function in LAT(Y136F) T cells. Our data indicate that the LAT-PLC-gamma1 interaction plays a critical role in Foxp3 expression and the development of CD4+CD25+ T reg cells.

Figure 1.

Disruption of the LAT–PLC-γ1 interaction leads to the absence of CD4⁺CD25⁺ regulatory T cells in the periphery. (A) FACS analysis of CD4, CD8, and CD25 expression on thymocytes and splenocytes from 6-wk-old WT and LAT^Y136F mice. (B) Quantitative real-time PCR analysis of Foxp3 expression in thymocytes and splenocytes. Total RNAs were prepared from 2 × 10⁵ CD4⁺ SP thymocytes and CD4⁺ splenocytes sorted by FACS. The levels of Foxp3 RNA were normalized with β-actin RNA. The data are representative of three independent experiments. (C) Expression of Foxp3 on CD4⁺ thymocytes and splenocytes from 6-wk-old WT and LAT^Y136F mice by intracellular staining with an anti-Foxp3 antibody.

Figure 2.

CD25 and Foxp3 expression in LAT^Y136F T cells in young mice and mice with mixed bone marrow chimeras. (A) FACS analysis of CD4, CD8, CD25, and Foxp3 in thymocytes and splenocytes from 24-d-old WT and LAT^Y136F mice. (B) Irradiated LAT^−/− mice were reconstituted with mixed bone marrow cells from Thy1.2⁺ LAT^Y136F mice (3.0 × 10⁶ cells) and congenic B6 Thy1.1⁺ mice (1.5 × 10⁶ cells) after T cell depletion. 6 wk after reconstitution, thymocytes and splenocytes from these mice were analyzed. Cells were stained with APC anti-CD4, Texas red anti-CD25, PE-Cy5 anti-CD8, PE anti-Thy1.2 PE, biotin anti-Thy1.1, and strepavidin PE-Cy7 and FITC anti-Foxp3. FACS plot shown is a representative of analysis of six mice.

Figure 3.

Neonatal adoptive transfer of normal CD4⁺CD25⁺ regulatory T cells prevents the lymphoproliferative syndrome in LAT^Y136F mice. Neonatal Thy1.2⁺ LAT^Y136F mice were adoptively transferred with 2–3 × 10⁵ CD4⁺CD25⁺ or CD4⁺CD25⁻Thy1.1⁺ T cells and analyzed 7 wk later. (A) Photographs of spleens and lymph nodes from 7-wk-old WT, LAT^Y136F, LAT^Y136F received CD4⁺CD25⁺, and LAT^Y136F received CD4⁺CD25⁻ T cells. This picture is a representative of six experiments. The numbers of splenocytes in these mice were 9.3 × 10⁷, 34.9 × 10⁷, 9.8 × 10⁷, and 31.0 × 10⁷, respectively. (B) FACS analysis of donor cell engraftment. splenocytes were stained with FITC anti-Thy1.1 or FITC anti-Thy1.2, PE anti-CD25, and APC anti-CD4. The percentage of cells in the gated region is shown in the dot plot. FACS plot shown is a representative of five independent experiments.

Figure 4.

Analysis of host-derived cells in LAT^Y136F mice after adoptive transfer of CD4⁺CD25⁺ T cells. (A) FACS analysis of thymocytes and splenocytes from untreated and treated LAT^Y136F mice 7 wk after injection. (B) Expression of CD25, TCR-β, CD62L, and CD44 in host-derived Thy1.2⁺ CD4⁺ T cells from control and treated LAT^Y136F mice. Numbers in the dot plots or histograms indicate the percentage of cells within the designated gate. The shaded area in the histogram represents FACS staining of samples with isotype control antibodies. FACS plot shown is a representative of five independent experiments.

Figure 5.

Effect of adoptive transfer of normal CD4⁺CD25⁺ T cells into LAT^Y136F mice. (A) Immunohistochemistry staining of frozen sections of spleen (a–c), liver (d–f), and fluorescence staining of kidney (g–i). Frozen sections were prepared from the spleens, livers, and kidneys of 7- to 9-wk-old untreated controls and treated LAT^Y136F mice. Spleen and liver sections were stained with anti-B220 (blue) and anti–TCR-β (red). Kidney sections were stained with anti–mouse IgG-FITC (green) for glomeruli. Spleen and kidney photographs were taken at ×10 magnification. Liver photographs were taken at ×100 magnification. (B) Anti–double-stranded DNA antibody titers in control untreated and LAT^Y136F mice treated with CD4⁺CD25⁺ regulatory T cells. (C) Decrease in hyperactivated B lymphocyte numbers (B220⁺MHCII^hi and B220⁺IgM^lo) in treated LAT^Y136F mice. (D) Reduced IgG1 production. IgG1 from serum samples of 7-wk-old WT, LAT^Y136F, and treated LAT^Y136F mice were subjected to serial dilution and quantitated by ELISA. (E) Expression of Foxp3, granzyme A, granzyme B, and TGF-β. Thy1.2⁺ T cells (CD4⁺CD25⁺ and CD4⁺CD25⁻) were freshly isolated from normal C57BL/6 mice by FACS sorting. Thy1.1⁺ T cells were sorted from LAT^Y136F mice received adoptive transfer of CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells from Thy1.1⁺ mice, respectively. Amounts of cDNAs used RT-PCR were normalized by the level of β-actin. Data in B and D represent mean ± SD of serum samples from five groups of mice.

Figure 6.

Expression of Foxp3 confers suppressive function in LAT^Y136F CD4⁺CD25⁻ T cells. (A) Retroviral constructs and retroviral transduction efficiency of CD4⁺LAT^Y136F cells before neonatal injection. Retrovirally transduced cells were stained with fluorescent-conjugated APC anti-CD4 and PE anti–TCR-β, PE anti-GITR, and PE anti-CD25 antibodies, respectively. Gated GFP⁺CD4⁺ cells were analyzed for expression of TCR-β, GITR, and CD25. (B) Representative spleens and lymph nodes of 6-wk-old untreated WT, LAT^Y136F mice and treated LAT^Y136F mice injected with either 2 × 10⁵ T cells expressing GFP alone or GFP and Foxp3. The picture shown is a representative of four experiments. (C) FACS analysis of CD4 and CD8 expression on splenocytes of recipient origin.