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. 2015 Jul 15;195(2):695-705.
doi: 10.4049/jimmunol.1403187. Epub 2015 Jun 1.

The Importance of IL-6 in the Development of LAT-Mediated Autoimmunity

Sarah A O'Brien  1 , Minghua Zhu  1 , Weiguo Zhang  2
Affiliations

The Importance of IL-6 in the Development of LAT-Mediated Autoimmunity

Sarah A O'Brien et al. J Immunol. .

Abstract

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is highly tyrosine phosphorylated upon engagement of the TCR. Phosphorylated LAT binds Grb2, Gads, and phospholipase C (PLC)γ1 to mediate T cell activation, proliferation, and cytokine production. T cells from mice harboring a mutation at the PLCγ1 binding site of LAT (Y136F) have impaired calcium flux and Erk activation. Interestingly, these T cells are highly activated, resulting in the development of a lymphoproliferative syndrome in these mice. CD4(+) T cells in LATY136F mice are Th2 skewed, producing large amounts of IL-4. In this study, we showed that the LATY136F T cells could also overproduce IL-6 due to activated NF-κB, AKT, and p38 pathways. By crossing LATY136F mice with IL-6-deficient mice, we demonstrated that IL-6 is required for uncontrolled T cell expansion during the early stage of disease development. Reduced CD4(+) T cell expansion was not due to a further block in thymocyte development or an increase in the number of regulatory T cells, but was caused by reduction in cell survival. In aged IL-6(-/-) LATY136F mice, CD4(+) T cells began to hyperproliferate and induced splenomegaly; however, isotype switching and autoantibody production were diminished. Our data indicated that the LAT-PLCγ1 interaction is important for controlling IL-6 production by T cells and demonstrated a critical role of IL-6 in the development of this lymphoproliferative syndrome.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest

Figures

Figure 1
Figure 1. Increased production of IL6 by LATY136F T cells
(A) Increased IL4, IL5, IL13, IL6, and TNFα production by real-time PCR analysis of cytokine transcripts in LATm/m CD4+ T cells compared to WT. Relative expression of cytokine RNA was normalized to β-actin. (B) Intracellular staining of IL6. WT and LATm/m splenocytes were stimulated with PMA and ionomycin for 4 hours prior to intracellular staining. (C) The effect of pharmacological inhibitors on cytokine production. LATm/m splenocytes were incubated with different inhibitors or DMSO in the presence of IL2 for 48 hours prior to stimulation with PMA and ionomycin and intracellular staining. Significance differences in cytokine production with various inhibitors (PD98059 for MEK1/2, SB203580 for p38, LY294002 for PI3K, trifluoroacetate for Akt1/2, and QNZ for NF-κB) is compared to DMSO treated cells. (D–E) Activation of signaling pathways in LATm/m T cells. Whole cell lysates from WT and LATm/m T cells (D) or LATm/m and IL-6−/−LATm/m T cells (E) without activation were analyzed by Western blotting with antibodies as indicated. Data are representative of 3–5 individual experiments. Mice used were 8–10 weeks old. Two-tailed t test; *, p<0.05, **, p<0.005.
Figure 2
Figure 2. The effect of IL6 on LATY136F T cell homeostasis and development
(A) Spleens from 6 wk-old WT, LATm/m, IL-6−/−, and IL-6−/−LATm/m mice. (B) Spleen weight and the numbers of CD4+ T cells from 4 to 6 wk-old mice. (C) Representative FACS plots of splenocytes. The expression of CD44 and CD62L was pre-gated on CD4+ cells. For intracellular staining of IFNγ and IL4, cells were stimulated with PMA and ionomycin for 4 hours. (D) Analysis of thymocyte development in 4 wk-old mice. (E) The numbers of different subsets of thymocytes. (F) IL6 deficiency had no effect on Treg cells in 6 wk-old IL-6−/−LATm/m mice. FoxP3 expression in CD4+ T cells was analyzed by intracellular staining. Data are representative of 3–5 individual experiments. Two-tailed t test; **, p<0.005. ***, p<0.001.
Figure 3
Figure 3. Myd88 in not required for IL6 production in LATm/m T cells
(A) IL6 production. Splenocytes from WT, LATm/m, Myd88−/−, and Myd88−/−LATm/m mice were stimulated for 4 hours with PMA and ionomycin before intracellular staining. (B) Spleen weight and the numbers of CD4+ T cells. (C) FACS analysis of splenocytes. Cells were pre-gated on CD4+ cells for CD44 vs. CD62L expression. Data are representative of 3 individual experiments. All data were obtained from the analysis of 4–6 wk-old mice. Two-tailed t test; *, p<0.05, ***, p<0.001.
Figure 4
Figure 4. IL6 deficiency on LATY136F T cell survival
(A) T cell proliferation. Ki67 intracellular staining was pre-gated on CD4+ T cells. (B) Splenocytes from 4–6 week old mice were stained with 5 µM CFSE and stimulated with plate-bound anti-CD3 (2C11) or PMA and ionomycin for 24 hours. Cells were cultured for additional 48 hours, harvested, and analyzed by FACS. The solid black line represents stimulated cells, and the shaded histogram represents unstimulated cells. (C). 3×106 CD4+ T cells were sorted, stained with CFSE , and transferred into LAT−/− host mice. Mice were sacrificed 6 days later and CFSE dilution in CD4+ T cells was analyzed by FACS.
Figure 5
Figure 5. IL6 deficiency on LATY136F T cell survival
(A) The percentage of live (Annexin V−7AAD−) CD4+ T cells. (B) Analysis of BCL2 expression in CD4+ T cells by intracellular staining. (C) IL6Rα surface expression on CD4+ T cells. WT and IL6−/− (solid line); LATm/m and IL6−/−LATm/m (dashed line). The numbers labeled in the FACS plots represent geometric mean florescent intensity. (D–E) pSTAT3 activation. Splenocytes were either treated with IL6 (1µg/ml) for 20 minutes or left untreated before pSTAT3 staining. Mice were used at 6 wks of age. Data are representative of 3 independent experiments. Two-tailed t test; *, p<0.05, **, p<0.005, ***, p<0.001.
Figure 6
Figure 6. The development of an autoimmune syndrome in aged IL6−/−LATm/m mice
(A) Spleen weight, splenocyte number, and CD4+ T cell number. WT, LATm/m, IL6−/−, and IL6−/−LATm/m mice were 3 months old. (B) FACS analysis of splenic CD4 and CD8 expression. (C) MHC class II, IgD, and IgM expression on CD19+B220+ B cells from LATm/m and IL6−/−LATm/m mice. (D) Serum levels of IgM, IgG1, IgE, and anti-dsDNA antibodies. (E) Autoantibody production. NIH3T3 cells were incubated with sera from different mice followed by anti-mouse IgG-FITC. (F) IL21 mRNA levels from whole lymph nodes. RNAs were isolated from inguinal lymph nodes in Trizol reagent using a Beadbug homogenizer, and 5µg RNA was used for reverse-transcription. cDNAs were normalized to β-actin. (G) Long-term survival of LATm/m and IL6−/−LATm/m mice. The numbers indicated the average age of mice that either died or had to be sacrificed. Data are representative of 3–5 independent experiments. Two-tailed t test; *, p<0.05, ***, p<0.001.
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