Mislocalization of SLP-76 leads to aberrant inflammatory cytokine and autoantibody production

Abstract

Central and peripheral tolerance is required to prevent immune responses to self-antigens. We now present a mouse model in which wild-type (WT) SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) has been constitutively targeted to the membrane, where CD4+ T cells become spontaneously dysregulated and develop an inflammatory phenotype. Mice bearing membrane-targeted SLP-76 (MTS) have a partial T-cell lymphopenia and impaired signaling though the mature T-cell receptor. The CD4+ T cells that develop in these mice possess an activated-like phenotype and are skewed toward the inflammatory T(H)1 and T(H)17 lineages. MTS mice also spontaneously develop autoantibodies at an early age. To rule out abnormal thymic selection as the sole cause of the MTS phenotype, we expressed WT SLP-76 along with the MTS followed by deletion of the WT allele in peripheral T cells. The peripheral MTS-expressing T cells demonstrate skewed cytokine responses when transferred into lymphopenic hosts. Thus, the abnormal effector T-cell phenotype still occurs in the presence of preserved central and peripheral tolerance, suggesting that diminished T-cell receptor signaling can promote skewed T-cell responses.

Figure 1

Peripheral CD4⁺ T cells in MTS mice are greatly decreased in number, possess an activated-like phenotype, and have impaired TCR signaling. (A) Total number of cells obtained from the spleen and peripheral LNs of WT (□) and MTS (▩) mice and percentages of lymphocyte subsets as determined by flow cytometry. Data are mean ± SEM and are representative of more than 5 independent experiments using 2 or 3 mice per group. Significance was determined using the 2-tailed Student t test. **P < .01. ***P < .001. (B) Surface expression of activation markers of WT (solid line) and MTS (shaded) mice. Histograms are gated on CD4⁺ T cells. Data are representative of more than 5 independent experiments using 2 or 3 mice per group. (C) Intracellular staining for the incorporation of BrdU in WT and MTS mice. Histograms are gated on CD4⁺ T cells. Data are representative of 2 independent experiments using 2 or 3 mice per group. (D) Purified peripheral T cells were stimulated for indicated lengths of time with a TCR cross-linking antibody, lysed, and blotted for phospho-PLC-γ1, total PLC-γ1, phospho-SLP-76, total SLP-76, and phospho-ERK. Data are representative of 3 independent experiments. (E) Peripheral cells from WT (black line) and MTS (gray line) mice were loaded with the calcium indicator Indo-1 and analyzed for their ability to flux calcium in response to CD3/CD4 cross-linking and ionomycin at the indicated time points. Histograms are gated on CD4⁺ T cells. Data are representative of 4 independent experiments using 1 or 2 mice per group. (F) Splenocytes isolated from either WT (black line) or MTS (gray line) mice were cultured in the presence of the indicated stimulus and stained for the early activation markers CD25 and CD69. Percentages are taken from positive gates set on the total CD4⁺ T-cell population. Data are mean ± SEM and are representative of 2 independent experiments. P + I indicates PMA plus ionomycin.

Figure 2

MTS mice have peripheral CD4⁺ T cells that are skewed toward the inflammatory T_H1 and T_H17 lineages and develop spontaneous autoantibody production. (A) Cells were stimulated with PMA plus ionomycin for 4 hours, and cytokine production was assessed by intracellular staining for inflammatory cytokines IFN-γ, IL-2, IL-17A, and IL-17F. Populations shown are gated on CD4⁺ T cells. Data are representative of at least 5 independent experiments of 1 to 3 mice per group. (B) RNA isolated from unstimulated purified T cells of WT (□) and MTS (▩) mice were analyzed for mRNA encoding ROR-γt (rorc) and T-bet (tbx21) by real-time PCR analysis. Data are mean ± SEM and are representative of 3 independent experiments of 2 or 3 mice per group. (C) Sera isolated from WT (●) and MTS () mice were analyzed for the presence of anti-dsDNA antibodies and antinuclear antibodies (IgG). Horizontal lines represent averages. Significance was determined using the Mann-Whitney test. ***P < .001. Images were obtained with a Nikon Eclipse E600 microscope (40× objective and 1:40 serum dilution) and captured with a Nikon DXM 1200 camera. Images were processed with IP Labs Scientific Image Processing (Scanalytics Inc).

Figure 3

MTS mice have normal percentages of peripheral CD4⁺ T_regs, which are able to efficiently suppress WT effector T-cell proliferation. (A) Spleen and LNs from WT and MTS mice were stained for the presence of intracellular FoxP3. Populations are gated on CD4⁺ T cells. Data are representative of 3 independent experiments of 2 or 3 mice per group. (B) Sorted CD4⁺CD25⁺ WT (black line) or MTS (gray lines) T cells were cultured in the presence of stimulated CD4⁺CD25⁻ WT effector cells, and proliferation was measured by tritiated thymidine incorporation. Data are mean ± SEM and are representative of 3 independent experiments.

Figure 4

MTS/ΔSLP CD4⁺ T cells do not initially possess an activated-like phenotype. (A) Diagram of constructs that permit selective induction of the MTS mutation via tamoxifen treatment. (B) Western blot analysis of total SLP-76 protein in purified T cells isolated from mice of various genotypes (left) and expression of YFP protein in CD4⁺ T cells 2 days after tamoxifen treatment. Populations are gated on CD4⁺ T cells. ΔSLP denotes a floxed WT allele of SLP-76, which was deleted after tamoxifen treatment. (C) Surface expression of activation markers using flow cytometry of +/ΔSLP (solid line) and MTS/ΔSLP (shaded) mice 2 days after tamoxifen treatment. Histograms are gated on YFP⁺CD4⁺ T cells. Data are representative of 5 independent experiments using 2 or 3 mice per group. (D) Intracellular staining for the incorporation of BrdU in +/ΔSLP and MTS/ΔSLP mice. Histograms are gated on YFP⁺CD4⁺ T cells. Data are representative of 2 independent experiments using 2 mice per group.

Figure 5

MTS/ΔSLP CD4⁺ T cells have impaired TCR signaling and are not poised to produce pro-inflammatory cytokines. (A) Purified peripheral T cells were stimulated for indicated lengths of time with a TCR cross-linking antibody, lysed, and blotted for phospho-PLC-γ1, total PLC-γ1, phospho-SLP-76, total SLP-76, and phospho-ERK. Data are representative of 3 independent experiments. (B) Peripheral cells from +/ΔSLP (black line) and MTS/ΔSLP (gray line) mice were loaded with the calcium indicator Indo-1 and analyzed for their ability to flux calcium in response to CD3/CD4 cross-linking and ionomycin at the indicated time points. Histograms are gated on YFP⁺CD4⁺ T cells. Data are representative of 3 independent experiments using 1 or 2 mice per group. (C) Splenocytes isolated from either +/ΔSLP (black line) or MTS/ΔSLP (gray line) mice were cultured in the presence of the indicated stimulus and stained for the early activation markers CD25 and CD69. Percentages are taken from positive gates set on the total YFP⁺CD4⁺ T-cell population. Data shown are representative of 3 independent experiments. P + I indicates PMA plus ionomycin. (D) Peripheral cells isolated from +/ΔSLP and MTS/ΔSLP mice after tamoxifen treatment were stimulated with PMA plus ionomycin and stained for the presence of intracellular inflammatory cytokines. Populations are gated on YFP⁺CD4⁺ T cells. Data are representative of 5 independent experiments using 2 or 3 mice per group.

Figure 6

Transfer to a lymphopenic host reveals dysregulated responses of MTS/ΔSLP CD4⁺ T cells independent of thymic development. (A) Diagram of selective induction of the MTS mutation followed by sorting and adoptive transfer into lymphopenic hosts. (B) Surface expression of activation markers after adoptive transfer of YFP⁺CD4⁺ +/ΔSLP (solid line) or MTS/ΔSLP (shaded) T cells. Histograms are gated on YFP⁺CD4⁺ T cells. Data are representative of 2 independent experiments using 3 mice per group. (C) After adoptive transfer, peripheral cells were stimulated with PMA plus ionomycin and stained for the presence of intracellular inflammatory cytokines. Populations are gated on YFP⁺ CD4⁺ T cells. Data are mean ± SEM and are representative of 3 independent experiments using 3 mice per group. (D) Staining for the intracellular incorporation of BrdU. Histograms are gated on YFP⁺CD4⁺ T cells. (E) Total number of recovered YFP⁺CD4⁺ T cells from RAG^−/− recipients. Data are mean ± SEM and are representative of 1 experiment using 3 mice per group. Significance was determined using the 2-tailed Student t test. *P < .05