Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes

The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. This altered development process resulted in significant reductions in CD4 and CD8 single positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαβ+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of selection process to ensure normal thymocyte development.

5 development. For example, studies of mice lacking Vav-1 have shown that T cell development is partially 69 blocked at pre-TCR β selection and is strongly blocked in both positive and negative selection [19][20][21]. Mice 70 lacking both isoforms of Rac1 and Rac2 show defects in pre-TCR β-selection at DN thymocytes and positive 71 selection of DP thymocytes [22,23]. Mice lacking Pak2 show defects in pre-TCR β-selection of DN 72 thymocytes, positive selection of DP thymocytes, and maturation of SP thymocytes [24]. 73 74 Fam49b has been identified as an inhibitor of TCR signaling through binding with active Rac-1/2 in 75 Fam49b-KO Jurkat T cells [25]. Those studies showed that lack of Fam49b led to hyperactivation of Jurkat T 76 cells following TCR stimulation, as measured by the enhancement of CD69 induction, Rac-PAK axis signaling, 77 and cytoskeleton reorganization [25]. Since TCR signaling strength controls thymocyte development, we 78 hypothesized that Fam49b would be critical for thymocyte development in vivo and investigated this using a 79 novel knockout mouse line. Here, we found the Fam49b was dispensable for positive selection but was required 80 to prevent overly robust elimination of thymocytes at the negative selection stage, thus identifying Fam49b as a 81 critical regulator of negative selection. 82

Generation of Fam49b-KO mice and Fam49a-KO mice 84
To assess the role of Fam49b in T cell development, we generated Fam49b-KO mice by creating a premature 85 stop codon in exon 6 of the Fam49b locus using CRISPR/Cas9 (Fig. 1A). Fam49a is a homologous protein that 86 is ~80% identical to Fam49b that has also been suggested to be involved in lymphopoiesis in zebrafish [26]. We 87 generated Fam49a-KO mice in a similar manner by creating a stop codon in exon 7 of the Fam49a locus 88 (Fig.1B). Immunoblot of spleen tissues confirmed that Fam49a or Fam49b expression was undetectable in 89 Fam49a-KO mice or Fam49b-KO mice respectively in contrast to wild type (WT, C57BL/6J) mice (Fig. 1C). 90 Real-time RT-PCR analysis of flow cytometry-sorted WT thymocytes subsets showed Fam49b is expressed 91 broadly throughout thymic development, whereas Fam49a was mainly expressed in mature T cells ( Fig. 1D and 92 Supplementary Fig. 1). The expression of Fam49a was not detectable in WT thymocytes (Fig. 1D). Both 93 Fam49a-KO and Fam49b-KO mice were fertile and did not show any apparent abnormalities. 94 95

Defective T cell development in Fam49b-KO mice, but not Fam49a-KO mice. 96
Flow cytometry analysis of cells isolated from lymph nodes showed that the frequency and number of 97 peripheral CD4 + T cells and CD8 + T cells were significantly reduced in Fam49b-KO mice ( Fig. 2A) compared 98 to WT and Fam49a-KO mice. Notably, reduction in the number of CD8 + T cells was greater than that of CD4 + 99 T cells. As a result. the ratio of CD4 + T cells over CD8 + T cells was increased in Fam49b-KO mice (Fig. 2B). In 100 contrast, Fam49a-KO mice resembled WT mice in terms of T cell number and CD4/CD8 composition. 101 Peripheral T cells in naïve mice can be divided into native and memory T subpopulation, which can be 102 distinguished based on the expression of adhesion molecule CD62L and CD44. Assessment of the phenotype of 103 peripheral T cells indicated that the reduction in T cell numbers was mainly due to a reduced number of naïve 104 (CD44 lo CD62L + ) CD4 + and CD8 + T cells in Fam49b-KO mice, while the size of the memory population was 105 unchanged (Fig. 2C). Again, little difference was observed between Fam49a-KO and WT mice in terms of the 106 phenotype of peripheral T cell subsets. 107 108 To further investigate if the decrease in naïve peripheral T cells subset in Fam49b-KO mice was due to defects 109 of T cell development, we analyzed the surface expression of CD4 and CD8 on thymocytes. The frequencies of 110 CD4 SP and CD8 SP cells were reduced and the ratios of CD4 SP to CD8 SP thymocytes were increased in 111 Fam49b-KO mice thymi ( Fig. 2D and Supplementary Fig. 2). These data indicate that Fam49b deficiency 112 7 leads to impaired thymocyte development for both CD4 + and CD8 + T cells, with a more marked impact on the 113 CD8 + T cell population. In contrast, loss of Fam49a showed little, if any, impacts on T cell numbers and  114  cellularity in periphery, or T-cell thymic development. Given a lack of any phenotypic changes in Fam49a-KO  115 mice T cells, together with an absence of Fam49a expression in thymus (Fig. 1D) 3A) and increased 125 ratio of peripheral CD4 + T over CD8 + T was observed in Fam49b-KO chimera mice compared to WT chimera 126 mice (Fig. 3B). The Fam49b-KO thymocytes developed in WT thymic environment are like those developed in 127 the germline Fam49b-KO environment in terms of both thymocyte and peripheral lymphocyte phenotypes. 128 Therefore, the effect of Fam49b mutation on T cell development is predominantly due to thymocyte intrinsic 129 functions. 130 131 Next, we sought to determine which step of T cell development was altered in Fam49b-KO mice. We thus 132 subdivided thymocytes into four stages based on the differential expression of TCRβ and CD69 expression ( Fig.  133 3C and Supplementary Fig. 2) [27]. The proportion of stage 1 thymocytes (TCRβ lo CD69 -), which include the 134 DN and pre-selection DP cells, was similar between WT and Fam49b-KO mice. The percentage of stage 2 135 thymocytes (TCRβ int CD69 + ), which represent transitional DP undergoing TCR-mediated positive selection, was 136 significantly higher in the Fam49b-KO mice. The proportion of late stage thymocytes including the post-137 positive selection (TCRβ hi CD69 + ) and the mature thymocytes (TCRβ hi CD69 -) was markedly decreased (Fig  138  3C). Consistent with our observation in periphery, the increased ratio of CD4 SP to CD8 SP was observed 139 among the late stage thymocytes (TCRβ hi CD69 + and TCRβ hi CD69 -) in Fam49b-KO mice (Fig 3D). These data 140 show that the post-positive-selection process is impaired in Fam49b-KO thymocyte. 141 142 We further distinguished the pre-and post-positive selection populations by expression of cell surface TCRβ and 143 CD5 ( Fig. 3E and Supplementary Fig. 3) [27]. These markers define a developmental progression: stage 1 144 (TCRβ lo CD5 lo ) represents the pre-selection phase of DP thymocytes, and Stage 2 (TCRβ lo CD5 int ) are cells 145 initiating positive selection. Stage 3 (TCRβ int CD5 hi ) represents thymocytes in the process of undergoing 146 positive selection, and Stage 4 (TCRβ hi CD5 hi ) consists primarily of post-positive selection SP thymocytes. We 147 observed that all the early phase populations (TCRβ lo CD5 lo , TCRβ lo CD5 int , TCRβ int CD5 hi ) increased 148 significantly in proportion in Fam49b-KO, whereas the post-positive selection SP thymocytes (TCRβ hi CD5 hi ) 149 were markedly decreased (Fig. 3E). Similarly, an increased ratio of CD4 SP to CD8 SP was observed in the 150 post-positive selection population (TCRβ hi CD5 hi ) in Fam49b-KO thymocytes (Fig. 3F). This phenotype was 151 further verified by the observation of lower percentage of mature SP CD24 lo TCRβ hi cells in Fam49b-KO mice 152 compared with WT mice (Fig. 3G). Taken  whereas the frequencies of cells to be eliminated through death by neglect were similar between Fam49b-KO 168 and WT mice (Fig. 4A).

170
Negative selection can occur in the thymic cortex as DP thymocytes are undergoing positive selection or in the 171 thymic medulla after positive selection [29]. To determine whether loss of Fam49b led to increased deletion in 172 the cortex or medulla, we stained the thymocytes for CCR7, which marks medullary thymocytes and is the 9 receptor for the medullary chemokines CCL19/21 [28,30]. The frequencies of cleaved-caspase3 + CCR7cells 174 and cleaved-caspase3 + CCR7 + cells were significantly increased in the Fam49b-KO mice, suggesting that more 175 thymocytes were eliminated through clonal deletion in both the cortex and medulla of Fam49b-KO thymus as 176 compared with WT thymus (Fig. 4A). 177 178 Next, to determine if TCR-signal strength in Fam49b-KO thymocyte was increased, we assessed the surface 179 expression of CD5 and CD69, two surrogate markers for TCR-signal strength [31,32]. We found that both CD5 180 and CD69 expressions were upregulated on Fam49b-KO DP thymocytes, but not on CD4 SP and CD8 SP 181 thymocytes (Fig. 4B), suggesting Fam49b-KO DP thymocytes had received stronger TCR signaling than the 182 WT thymocytes. We next investigated the TCR-signaling strength of Fam49b-KO peripheral T cells by 183 measuring IL-2 production in response to anti-CD3ε stimulation. Peripheral T cells were purified from spleen 184 and lymph nodes from WT or Fam49b-KO mice and were stimulated in anti-CD3ε Ab coated plates for 3 days. 185 We observed that IL-2 production was strongly elevated in Fam49b-KO CD4 + CD25and CD8 + T cells 186 compared to WT T cells (Fig. 4C). Despite the high IL-2 production of these cells, proliferation of both 187 CD4 + CD25and CD8 + Fam49b-KO T cells in response to anti-CD3ε stimulation in vitro were similar to that of 188 WT T cells (Supplementary Fig. 5 decreased from 60% in WT mice to 30% in Fam49b-KO mice, whereas the frequency of liver iNKT cells was 200 unaffected (Fig. 5A). The frequency of Treg among lymph node CD4 + T cells increased slightly from 16% to 201 20% in lymph nodes in Fam49b-KO mice, though the absolute number of Treg was ~80% of the number in WT 202 mice. Enhanced frequency of Treg seems to be a result of greater reduction of total CD4 + T cells compared to 203 Treg ( Fig. 5A and Supplementary Fig. 6A). 204

205
Gut IEL T lymphocytes are extremely heterogenous, and based on the differentiation mechanisms, can be 206 subdivided into two major subpopulations including natural intraepithelial lymphocytes (natural IELs) and 207 induced intraepithelial lymphocytes (induced IELs) [35]. Natural IELs home to gut immediately after thymic 208 maturation. They are TCRγδ + and TCRαβ + T cells that can be either CD8αα + or CD8αα − . In contrast, induced 209 IELs arise from conventional peripheral CD8αβ + TCRαβ + T cells and are activated post-thymically in response 210 to peripheral antigens. The two populations can be distinguished by the expression of CD5; natural IELs are 211 CD5and induced IELs CD5 + . Based on our observation of the dramatic loss of CD8αα + TCRαβ + IELs in 212 Fam49b-KO mice, we postulated that other IEL subsets might be altered as well. Fam49b-KO mice showed a 213 substantial reduction of natural IELs, including both the TCRγδ + IELs as well as CD8αα + TCRαβ + IELs ( Fig.  214 5B), whereas the relative frequencies of induced IELs (CD8αβ + TCRαβ + IELs) were increased ( Fig. 5C and 215 Supplementary Fig. 6B). These results suggest that Fam49b is involved in shaping the agonist-selected 216 unconventional T cell populations and that Fam49b deficiency leads to substantial loss of the natural IELs, 217 including CD8αβ + TCRαβ + IELs and TCRγδ + IELs. 218

219
Development of T cells is critically dependent on the strength of signaling through the TCR that lead to positive 220 or negative selection [36,37]. However, the roles of additional intracellular proteins and signaling pathways 221 that regulate TCR signaling strength in the thymus have not been fully elucidated. Here, by studying the thymic 222 development of T cells in Fam49b-KO mice, we report that Fam49b finetunes thymic selection by negatively 223 regulating TCR signal-strength in the thymus and is essential for normal thymocyte development. Mice 224 deficient in Fam49b developed severe T cell lymphopenia due to enhanced TCR-signaling in DP thymocytes. In 225 Fam49b-KO thymus, post-positively selected population was significantly reduced, while generation of DN or 226 immature DP thymocytes was mostly unaffected. We further confirmed that the loss of post-positive selection 227 thymocytes in Fam49b-KO mice was due to enhanced clonal deletion instead of death by neglect. Antibodies and reagents.