The role of the LAT-PLC-gamma1 interaction in T regulatory cell function

Abstract

The interaction between the linker for activation of T cells (LAT) with PLC-gamma1 is important for TCR-mediated Ca(2+) signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells, implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect, we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wild type LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling, proliferation, and IL-2 production. Furthermore, the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3(+) natural Treg cells were present in these mice after deletion, they were unable to suppress the proliferation of conventional T cells. Our data indicate that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4(+)CD25(+) regulatory T cells.

Figure 1. T cell development in ERCreLAT^f/m mice

(a-b) CD4 and CD8 expression on thymocytes (upper panel) and splenocytes (lower panel) from untreated (a) and tamoxifen-treated (b) ERCreLAT^f/m and ERCreLAT^f/+ littermates. (c) Efficient deletion of LAT after tamoxifen treatment. GFP expression on CD4⁺ T cells was analyzed. The shaded area represents CD4⁺ T cells from a C57BL/6 mouse. (d) Expression of CD4 and Foxp3 on GFP⁺ cells from the lymph nodes of ERCreLAT^f/m and ERCreLAT^f/+ littermates 4-5 days after tamoxifen treatment. Data shown are representative of three mice of each genotype.

Figure 2. Defective TCR-mediated signaling and function in mature ERCreLAT^f/m T cells

Analysis of T cells from ERCreLAT^f/m and ERCreLAT^f/+ mice 4-5 days after tamoxifen injections. (a) TCRβ expression on ERCreLAT^f/m (solid line) and ERCreLAT^f/+ (dotted line) in GFP⁺CD4⁺ lymph node cells. Shaded area represents TCRβ expression on B cells. (b)Western blot analysis showing total tyrosine phosphorylation in ERCreLAT^f/m and ERCreLAT^f/+ activated T cells after anti-CD3 stimulation. Data shown are a representative of 3 independent experiments. (c) Western blots showing phosphorylation of PLC-γ1 and Erk1/2. (d) Calcium flux in ERCreLAT^f/m (gray line) and ERCreLAT^f/+ (black line) GFP⁺CD4⁺ cells in response to TCR stimulation. (e) IL-2 production in ERCreLAT^f/m (filled bars) and ERCreLAT^f/+ (empty bars) CD4⁺ cells. The figure is a representative of 4 independent experiments. (f) Proliferation in ERCreLAT^f/m (filled bars) and ERCreLAT^f/+ (empty bars) CD4⁺ cells.

Figure 3. Recapitulation of the LATY136F phenotype in ERCreLAT^f/m mice 3 weeks after deletion of floxed LAT

Analysis of ERCreLAT^f/m and ERCreLAT^f/+ mice after 3-4 weeks of tamoxifen treatment. (a) CD4 and CD8 expression on GFP⁺ thymocytes from ERCreLAT^f/m and ERCreLAT^f/+ mice (upper panel). CD25 and CD44 expression profile on DN thymocytes (lower panel). (b) Total numbers of CD4⁺, CD8⁺ and B220⁺ cells in the spleens of ERCreLAT^f/m and ERCreLAT^f/+ mice. The figure shown is one representative of 5 mice analyzed. (c) CD4 vs CD8 (upper panel) and CD62L vs CD44 surface expression in GFP⁺CD4⁺ splenocytes. (d) Intracellular IL-4 expression in GFP⁺CD4⁺ splenocytes after P+I stimulation. The shaded area represents unstimulated controls. (e) Surface CD25 and intracellular Foxp3 expression in GFP⁺CD4⁺ lymph node cells.

Figure 4. The presence of hyper-activated ERCreLAT^f/m T cells in the periphery of reconstituted LAT^−/− mice

Analysis of LAT^−/− mice reconstituted with ERCreLAT^f/m or ERCreLAT^f/+ T cells and treated with tamoxifen for 4-5 weeks. (a) Total numbers of CD4⁺, CD8⁺ and B220⁺ cells in the spleen of tamoxifen treated LAT^−/− recipients reconstituted with ERCreLAT^f/m or ERCreLAT^f/+ T cells. (b) CD4 and CD8 expression on splenocytes. 3 mice of each genotype were analyzed. Data shown is a representative of one of 4 such experiments. (c) TCRβ expression on GFP⁺ CD4⁺ cells in LAT^−/− recipients that received cells from either ERCreLAT^f/m (solid line) or ERCreLAT^f/+ (dotted line). The shaded area represents a negative control. (d) IFN-γ, IL-2 and IL-4 production in GFP⁺CD4⁺ cells after P+I stimulation. Unstimulated controls (shaded area) and stimulated cells (solid line) are depicted. (e) B220 and MHC Class II expression on lymph node cells (top panel). IgD and IgM expression on B220⁺ cells (bottom panel). (f) Blood serum levels of IgE, IgG₁ and IgM from LAT^−/− recipients injected with T cells from ERCreLAT^f/m (squares) and ERCreLAT^f/+ (diamonds) mice.

Figure 5. Impaired suppressive function of ERCreLAT^f/mCD4⁺Foxp3⁺ cells

(a) CD25 and Foxp3 expression in CD4⁺GFP⁺ cells in ERCreLAT^f/m and ERCreLAT^f/+ mice 4 days after tamoxifen treatment (upper panel) and in LAT^-/- mice reconstituted with ERCreLAT^f/m and ERCreLAT^f/+ T cells after 4 weeks of maintained tamoxifen treatment (lower panel). 3 mice were analyzed for each genotype. The figure shown is one representative of 4 experiments performed. (b) Total number of cells from the mesenteric, inguinal, brachial and axillary lymph nodes (left panel) and the spleen (right panel) of ERCreLAT^f/m and ERCreLAT^f/+ mice were analyzed 4 days after tamoxifen treatment. (c) In vitro suppression assay using various ratios of CD4⁺CD25⁻ responder cells (R) to CD4⁺CD25⁺ suppressor cells (S) from ERCreLAT^f/m and ERCreLAT^f/+ mice 4-5 days after tamoxifen treatment. Proliferation of wildtype Thy1.1⁺CD4⁺CD25^- responder cells is shown as indicated by CFSE dilution. The figure shown is one representative of 3 experiments performed. (d) Expression of IL-10 and TGFβ in ERCreLAT^f/m and ERCreLAT^f/+ CD4⁺CD25⁺ T cells after one hour of anti-CD3 stimulation. (e) CTLA-4 expression on GFP⁺CD4⁺Foxp3⁺ cells from the lymph nodes of ERCreLAT^f/m mice (solid line) and ERCreLAT^f/+ mice (dashed line) 4-5 days after tamoxifen treatment. GFP⁺CD4⁺Foxp3^- cells were used as a control (filled area).