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Regulation of Positive and Negative Selection and TCR Signaling During bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451936; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 1 Regulation of positive and negative selection and TCR signaling 2 during thymic T cell development by capicua 3 4 Soeun Kim1, Guk-Yeol Park1, Jong Seok Park1, Jiho Park1, Hyebeen Hong1, and 5 Yoontae Lee1,2,* 6 1Department of Life Sciences, Pohang University of Science and Technology 7 (POSTECH), Pohang, Gyeongbuk 37673, Republic of Korea 8 2Institute of Convergence Science, Yonsei University, Seoul 03722, Republic of Korea 9 10 *Correspondence to: Yoontae Lee, Room 388, POSTECH Biotech Center, 77 11 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, Republic of Korea, +82-54-279- 12 2354 (phone), +82-54-279-0659 (fax), [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451936; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 2 13 ABSTRACT 14 Central tolerance is achieved through positive and negative selection of thymocytes 15 mediated by T cell receptor (TCR) signaling strength. Thus, the dysregulation of the 16 thymic selection process often leads to autoimmunity. Here, we show that capicua 17 (CIC), a transcriptional repressor that suppresses autoimmunity, controls the thymic 18 selection process. Loss of CIC prior to T-cell lineage commitment impaired both 19 positive and negative selection of thymocytes. CIC deficiency attenuated TCR 20 signaling in CD4+CD8+ double-positive (DP) cells, as evidenced by a decrease in CD5 21 and phospho-ERK levels and calcium flux. We identified Spry4, Dusp4, Dusp6, and 22 Spred1 as CIC target genes that could inhibit TCR signaling in DP cells. Furthermore, 23 impaired positive selection and TCR signaling were partially rescued in Cic and Spry4 24 double mutant mice. Our findings indicate that CIC is a transcription factor required for 25 thymic T cell development and suggest that CIC acts at multiple stages of T cell 26 development and differentiation to prevent autoimmunity. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451936; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 3 27 INTRODUCTION 28 T cells play a crucial role in the adaptive immune system’s defense against external 29 invasion. To distinguish between self and non-self, T cells use their T cell receptors 30 (TCRs) to recognize peptide-loaded major histocompatibility complex (MHC) 31 molecules and respond to many types of antigens with an enormous TCR repertoire. 32 T cells with a specific TCR are generated in the thymus through a series of processes, 33 ranging from random rearrangement of TCR gene segments to selection processes 34 that entail apoptosis of inappropriate cells (Klein, Kyewski, Allen, & Hogquist, 2014). 35 T cell development in the thymus is tightly regulated to prevent the generation of 36 nonfunctional or self-reactive T cells. Progenitor cells from bone marrow (BM) become 37 CD4-CD8- double-negative (DN) cells and undergo a process called -selection, which 38 selects only T cells with a functional TCR chain. DN cells that have passed - 39 selection then become CD4+CD8+ double-positive (DP) cells and undergo positive 40 selection, which selects T cells that bind to self-peptide ligands loaded onto MHC (self- 41 pMHC) molecules. These CD4+CD8+ DP cells then develop into CD4+CD8- single- 42 positive (CD4+ SP) or CD4-CD8+ single-positive (CD8+ SP) T cells. Negative selection, 43 also known as clonal deletion, selectively removes T cells that bind with high affinity 44 to self-pMHC during the DP and SP stages. Because the intensity and duration of TCR 45 signaling based on TCR affinity to self-pMHC are the major determinants of selection 46 (Gascoigne, Rybakin, Acuto, & Brzostek, 2016), defects in the TCR signaling 47 component lead to abnormal T cell development and alteration of the TCR repertoire 48 (Guoping Fu et al., 2010; Sakaguchi et al., 2003). Impairment of thymic selection 49 caused by decreased TCR signaling destroys central tolerance, and consequently 50 induces autoimmunity accompanied by the expansion of the CD44hiCD62Llo activated bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451936; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 4 51 effector/memory T cell population (Guoping Fu et al., 2010; Sakaguchi et al., 2003; 52 Sommers et al., 2002). 53 Capicua (CIC) is an evolutionarily conserved transcriptional repressor that regulates 54 the receptor tyrosine kinase (RTK) signaling pathway in Drosophila and mammals 55 (Jiménez, Guichet, Ephrussi, & Casanova, 2000; Jiménez, Shvartsman, & Paroush, 56 2012). CIC is expressed as two different isoforms: long (CIC-L) and short (CIC-S), 57 which differ in their amino termini (Y. Lee, 2020). CIC recognizes specific octameric 58 DNA sequences (5ʹ-T(G/C)AATG(A/G)(A/G)-3ʹ) within its target gene promoter 59 regions and represses their expression (Kawamura-Saito et al., 2006; Shin & Hong, 60 2014; Weissmann et al., 2018). Several genomic and transcriptomic analyses have 61 identified CIC target genes, including ETV1, ETV4, ETV5, SPRY4, SPRED1, DUSP4, 62 and DUSP6, in various types of cells (Fryer et al., 2011; Weissmann et al., 2018; Yang 63 et al., 2017). Activation of the RTK/RAS/MAPK signaling pathway phosphorylates and 64 inactivates CIC via dissociation from target gene promoters, cytoplasmic translocation, 65 and/or proteasomal degradation (Jiménez et al., 2012; Keenan et al., 2020; Y. Lee, 66 2020). CIC also mediates the ERK-DUSP6 negative feedback loop to maintain ERK 67 activity within the physiological range in mammals (Ren et al., 2020). 68 We previously reported that murine CIC deficiency spontaneously induces 69 lymphoproliferative autoimmune-like phenotypes (S. Park et al., 2017). Hematopoietic 70 lineage cell-specific (Vav1-Cre-mediated knockout) and T cell-specific (Cd4-Cre- 71 mediated knockout) Cic-null mice commonly exhibit autoimmune-like symptoms 72 including hyperglobulinemia, increased serum anti-dsDNA antibody levels, and tissue 73 infiltration of immune cells, accompanied by increased frequency of CD44hiCD62Llo T 74 and follicular helper T (Tfh) cells in the spleen (S. Park et al., 2017). However, these 75 phenotypes were more severe in Cicf/f;Vav1-Cre mice than in Cicf/f;Cd4-Cre mice; bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451936; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 5 76 moreover, enlargement of secondary lymphoid organs was observed in Cicf/f;Vav1- 77 Cre mice but not in Cicf/f;Cd4-Cre mice (G. Park et al., 2020; S. Park et al., 2017). 78 These results indicated that deletion of Cic alleles in hematopoietic stem and 79 progenitor cells leads to more severe peripheral T cell hyperactivation and 80 autoimmunity than the Cd4-Cre-mediated Cic deletion in CD4+CD8+ DP thymocytes. 81 We also reported that enhanced peripheral T cell hyperactivation in Cicf/f;Vav1-Cre 82 mice relative to Cicf/f;Cd4-Cre mice was caused by CIC deficiency in T cells rather 83 than in other types of immune cells (G. Park et al., 2020), suggesting that this 84 abnormality could be caused by impaired control of early thymic T cell development in 85 Cicf/f;Vav1-Cre mice. It was reported that the frequency of CD4-CD8-CD44+CD25- DN1 86 cells was increased in adult stage-specific Cic-null mice (Tan et al., 2018). Taken 87 together, these studies suggest that CIC plays a crucial role in thymic T cell 88 development. 89 In this study, we found that CIC regulates thymic T cell development from the CD4- 90 CD8- DN stage and positive and negative selection of thymocytes, primarily during the 91 CD4+CD8+ DP stage. TCR signaling was significantly attenuated in DP cells of 92 Cicf/f;Vav1-Cre mice, thereby impairing both positive and negative selection in 93 Cicf/f;Vav1-Cre mice. We also identified Spry4, Dusp4, Dusp6, and Spred1 as CIC 94 target genes that could potentially contribute to the reduced TCR signaling strength 95 and impaired thymic selection processes in Cicf/f;Vav1-Cre mice. Our findings 96 demonstrate that CIC is a critical regulator of TCR signaling in DP cells and thymic T 97 cell development. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.11.451936; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 6 98 RESULTS 99 Changes in the frequencies of thymic T cell subsets over development in 100 Cicf/f;Vav1-Cre mice 101 To examine the role of CIC in thymic T cell development, we first evaluated the levels 102 of CIC in multiple subsets of developing thymocytes using homozygous FLAG-tagged 103 Cic knock-in (CicFLAG/FLAG) mice (S.
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