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Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua

Central tolerance is achieved through positive and negative selection of thymocytes mediated by T cell receptor (TCR) signaling strength. Thus, dysregulation of the thymic selection process often leads to autoimmunity. Here, we show that Capicua (CIC), a transcriptional repressor that suppresses autoimmunity, controls the thymic selection process. Loss of CIC prior to T-cell lineage commitment impairs both positive and negative selection of thymocytes. CIC deficiency attenuated TCR signaling in CD4+CD8+ double-positive (DP) cells, as evidenced by a decrease in CD5 and phospho-ERK levels and calcium flux. We identified Spry4, Dusp4, Dusp6, and Spred1 as CIC target genes that could inhibit TCR signaling in DP cells. Furthermore, impaired positive selection and TCR signaling were partially rescued in Cic and Spry4 double mutant mice. Our findings indicate that CIC is a transcription factor required for thymic T cell development and suggests that CIC acts at multiple stages of T cell development and differentiation to prevent autoimmunity.

CD5 and phospho-ERK levels and calcium flux. We identified Spry4, Dusp4, Dusp6, 26 and Spred1 as CIC target genes that could inhibit TCR signaling in DP cells. 27 Furthermore, impaired positive selection and TCR signaling were partially rescued in 28 Cic and Spry4 double mutant mice. Our findings indicate that CIC is a transcription 29 factor required for thymic T cell development and suggests that CIC acts at multiple 30 stages of T cell development and differentiation to prevent autoimmunity.

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T cells play a crucial role in the adaptive immune system's defense against external 33 invasion. To distinguish between self and non-self, T cells use their T cell receptors 34 (TCRs) to recognize peptide-loaded major histocompatibility complex (MHC) 35 molecules and respond to many types of antigens with an enormous TCR repertoire. 36 T cells with a specific TCR are generated in the thymus through a series of 37 processes, ranging from random rearrangement of TCR gene segments to selection   After obtaining the results shown in Figure 1D, we wondered why the frequency of 158 DP cells was comparable between WT and Cic f/f ;Cd4-Cre mice because Cic alleles 159 were supposed to be deleted in DP cells by Cd4-Cre (P. P. Lee et al., 2001). 160 Western blotting for CIC in multiple developing thymic T cell subsets from WT,       Figure 5D). We also observed a moderate decrease in calcium influx in 265 DP cells from Cic f/f ;Cd4-Cre mice ( Figure 5D), suggesting that CIC sensitively 266 regulates TCR activation-induced calcium influx in DP thymocytes. To further 267 evaluate CIC regulation of TCR signaling in DP thymocytes, we assessed the 268 activation of key TCR signaling cascade components in DP cells of WT, Cic f/f ;Cd4- 269 Cre, and Cic f/f ;Vav1-Cre mice after TCR stimulation by treatment with anti-CD3 and 270 anti-CD4. Among the components tested, including ZAP-70, PLC, ERK, JNK, and 271 p38, phospho-ERK levels were significantly decreased in DP cells from Cic f/f ;Vav1- 272 Cre mice compared to those from WT and Cic f/f ;Cd4-Cre mice ( Figure 5E and F).

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Together, these results demonstrate that CIC deficiency attenuates TCR signaling by 274 inhibiting calcium influx and ERK activation, especially in DP thymocytes.     Furthermore, similar to the results in Figure 5A, CD5 levels were most strongly 296 reduced in DP thymocytes derived from Cic f/f ;Vav1-Cre BM cells ( Figure 6E).

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As an alternative approach to examine T cell intrinsic function of CIC in the 298 regulation of thymic T cell development and selection processes, we generated and 299 analyzed the proximal Lck promoter (pLck)-driven Cre-mediated T cell-specific Cic   Figure 7A).

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The expression of all four genes was also upregulated in CD4 + and CD8 + SP   Figure 7E). These data imply that Spry4 derepression might critically contribute to 371 attenuated TCR signaling in CIC-deficient DP thymocytes.   Our previous study showed that the frequency of thymic CD4 + and CD8 + SP cells      To create a mixed BM chimera, 1 × 10 6 BM cells from each donor were mixed and 531 injected intravenously into C57BL/6 recipient mice that had been irradiated (10 Gy). To measure Nur77 expression, freshly isolated thymocytes at 1 × 10 7 cells/ml were 538 incubated with plate-coated anti-CD3 (5 g/ml) and anti-CD28 (10 g/ml) for 2 h, 539 followed by staining of surface markers (CD4, CD8, and TCR) and intracellular 540 staining of Nur77. Samples were analyzed using an LSRII Fortessa flow cytometer 541 or CytoFLEX LX. Data were analyzed using FlowJo software.