Id proteins suppress E2A-driven innate-like T cell development prior to TCR selection

Id proteins have been shown to promote the differentiation of conventional αβ and γδT cells, and to suppress the expansion of invariant Natural Killer T (iNKT) cells and innate-like γδNKT within their respective cell lineages. However, it remains to be determined whether Id proteins regulate lineage specification in developing T cells that give rise to these distinct cell fates. Here we report that in the absence of Id2 and Id3 proteins, E2A prematurely activates genes critical for the iNKT cell lineage prior to TCR expression. Enhanced iNKT development in Id3-deficient mice lacking γδ NKT cells suggests that Id3 regulates the lineage competition between these populations. RNA-Seq analysis establishes E2A as the transcriptional regulator of both iNKT and γδNKT development. In the absence of pre-TCR signaling, Id2/Id3 deletion gives rise to a large population of iNKT cells and a unique innate-like DP population, despite the block in conventional αβ T cell development. The transcriptional profile of these unique DP cells reflects enrichment of innate-like signature genes, including PLZF (Zbtb16) and Granzyme A (Gzma). Results from these genetic models and genome-wide analyses suggest that Id proteins suppress E2A-driven innate-like T cell programs prior to TCR selection to enforce predominance of conventional T cells.

we report that in the absence of Id2 and Id3 proteins, E2A prematurely activates genes critical 23 for the iNKT cell lineage prior to TCR expression. Enhanced iNKT development in Id3-deficient 24 mice lacking γ δ NKT cells suggests that Id3 regulates the lineage competition between these 25 populations. RNA-Seq analysis establishes E2A as the transcriptional regulator of both iNKT 26 and γ δ NKT development. In the absence of pre-TCR signaling, Id2/Id3 deletion gives rise to a 27 large population of iNKT cells and a unique innate-like DP population, despite the block in 28 conventional α β T cell development. The transcriptional profile of these unique DP cells reflects Introduction 34 The thymic output of a diverse and abundant population of conventional CD4 + and CD8 + α β T 35 cells constitutes the adaptive immune system that is necessary for a specific and effective 36 immune response to antigens. A smaller but significant population of unconventional T cells also 37 concomitantly develops in the thymus, with innate-like capabilities of mounting a non-specific 38 but rapid immune response to antigens. These innate-like T cells have garnered increasing 39 interest in lieu of their memory phenotype that can be harnessed in the context of allergies, 40 infections and tumors. They often possess limited TCR diversity that allows recognition of self-41 ligands presented by non-classical MHC molecules such as CD1 and MR1. [1] They are also 42 characterized by high levels of expression of the innate-like transcription factor, Promyelocytic 43 Zinc Finger (PLZF). [2][3][4][5][6] While the transcriptional programs that drive conventional CD4 + and 44 CD8 + T cell specification and development have been well characterized, little is known about 45 the innate-specific transcriptional programs upstream of PLZF that are responsible for the 46 divergence of innate-like T cells from conventional T cells. 47 Innate-like T cell populations include TCRαβ + Natural Killer T (NKT) cells, TCRγδ + NKT cells, 48 innate-like CD8 + T cells, CD8αα intraepithelial lymphocytes (IELs), and Mucosal-associated 49 invariant T (MAIT) cells. Invariant NKT (iNKT) cells are among the best characterized innate- 50 like T cells, which arise in parallel with conventional α β T cells. These cells are known to 51 stochastically express a canonical Vα14-Jα18 TCRα chain at the CD4 + CD8 + double positive 52 (DP) stage, which allows them to undergo TCR selection mediated by a CD1d molecule 53 expressed on other conventional DP thymocytes. [7,8] γ δ NKT cells are yet another population of 54 innate-like T cells that express a restricted Vγ1.1Vδ6.3 TCR. Both iNKT and γ δ NKT cells 55 express high levels of PLZF and readily produce effector cytokines like IL-4. [2,9] protein transcription factors E2A and HEB. Interestingly, Id proteins play opposite roles in the 58 development of conventional and innate-like T cells, such that they promote the former and 59 suppress the latter. In response to pre-TCR and TCR signals, Id proteins inhibit E protein activity 60 to play a critical role in promoting the differentiation and positive selection of conventional α β T 61 cells. [10][11][12] The Id-mediated inhibition is both necessary and sufficient to activate gene expression 62 programs critical for T cell maturation. In line with this, it has been shown that disruption of Id2 63 and Id3 impairs conventional α β T cell development beyond the TCR checkpoint [13] . Analogous 64 to α β T cell development, the function of Id3 in promoting conventional γ δ T cell development 65 has also been mapped downstream of the γ δ TCR. [14] In contrast, however, large populations of 66 iNKT, γ δ NKT and innate variant T FH cells have been observed in the same Id3-and Id2/Id3- 67 deficient animals, indicating a negative role for Id proteins in regulating innate-like T cell 68 development. [15][16][17][18][19][20][21] However, the mechanism that drives the development and expansion of these 69 innate-like T cell populations in Id-deficient mice is still elusive. Given the contradictory nature 70 of Id proteins in supporting conventional T cells and suppressing innate-like T cells, it is 71 reasonable to predict that Id proteins control innate-like T cell development through a distinct, or 72 additional mechanism, that is TCR signaling-independent. Id proteins have also been shown to 73 modulate E protein activity throughout T cell development. [10] Therefore, it remains to be 74 determined whether Id-mediated suppression of these innate-like T cells is limited to their 75 expansion upon selection and lineage commitment, or if it also influences their lineage 76 specification prior to TCR selection. 77 We first investigated the role of E proteins, particularly E2A, in iNKT cell development in the 78 absence of Id proteins. E2A ChIP-Seq revealed several genes that are important for iNKT cell development, such as Tcf7 [22] , Egr2 [23,24] and Bcl11b [25] , to be direct targets of E2A. This 80 suggested that Id proteins suppress E2A-driven iNKT-specific genetic programs. We then 81 examined any developmental competition between iNKT and γ δ NKT cells in Id3-deficient mice, 82 which have enhanced development of both these lineages. [20,21]  A second, independent round of RNASeq was done with DP (TCRγδ -CD4 + CD8 + ) cells sorted 166 from 5 weeks old WT (9 x 10 5 cells), pTαKO (4.5 x 10 5 cells), L-DKO (9 x 10 5 cells) and L-167 DKO pTαKO (9 x 10 5 cells) mice. 150 bp paired-end sequencing was done on the HiSeq2500 168 platform (Illumina). All subsequent data analysis was done using the steps outlined above. 169 RNA-Seq sequencing reads were first trimmed using Trimmomatic. [33] Read alignment was done 170 using Tophat and expression quantification was done using Cufflinks. [34]  cells. Other known interactions between these 111 genes were retrieved from GeneMania. [37] 83 196 of the 111 genes were also identified as E2A targets, which had E2A binding to the enhancer, 197 promoter, intragenic, intergenic or downstream regions of these genes, as annotated by Nebula. 198 These interactions, ChIP-Seq targets and gene expression patterns of the 111 genes were 199 represented as a network using Cytoscape3.4.0. [38] 200 Gene Set Enrichment Analysis (GSEA) 201 The GSEA [36,39]

Correlation analysis
were determined for all genes across six samples, including replicates of WT DP, pTαKO DP, L-212 DKO pTαKO DP and L-DKO DP, as derived from RNA-Seq analysis. Genes with both 213 coefficients greater than or equal to 0.7 were considered to be positively correlated, and those 214 with both coefficients less than or equal to -0.7 were considered to be negatively correlated with 215 Zbtb16 expression. Scatter plots were generated using a custom R script.  Figure 1a). [11] E2A also bound to the promoter and/or enhancer regions of several genes that have been 240 reported to be critical for iNKT cell development and function (Figure 1d). This included genes 241 such as Tcf7, Bcl11b, Sox4 and Gzma. [22,25,45] While direct regulation of PLZF (encoded by 242 Zbtb16) by E2A has been reported previously, [46] we found E2A binding to intragenic, rather  253 In the previous sections, we investigated the mechanisms that drive iNKT cell development in 254 Id2/Id3-double deficient mice. Interestingly, Id3-deficient mice have been described to support 255 the expansion of both γ δ NKT and iNKT cells. [19,47] We have also previously shown that varying 256 activity levels of E proteins can differentially influence lineage outcomes between γ δ NKT and 257 iNKT cells. [15] Additionally, recent publications have demonstrated the sharing of transcriptional 258 programs between these innate-like lineages, particularly between γ δ NKT and NKT2 cells. [48,49] 259 Given the transcriptional and functional similarities between these two innate-like populations, 260 and their expansion in Id-deficient mice, we wanted to examine if Id proteins suppressed these 261 lineages equally or differentially, particularly in the developmental window prior to TCR selection. We therefore tested lineage competition in Id3-deficient mice by eliminating γ δ 263 lineage development and expansion. 264 We found a significant increase in the iNKT population in Id3 -/-TCRδ -/mice as compared to Id3 -265 /mice (Figure 2a-c). There was a modest but significant increase in iNKT lineage-committed 266 stage 0 cells in Id3 -/-TCRδ -/mice as compared to Id3 -/mice (Figure 2d, e). It has been reported 267 that iNKT cells and γ δ NKT cells compete for a thymic niche, based on the reduction in iNKT 268 cells upon expansion of γ δ NKT cells. [50] In contrast, another study has reported that a reduction 269 in iNKT cells does not lead to a corresponding increase in γ δ NKT cells. [51] In order to separate and iNKT cells. Since the former arise at the DN3 stage and the latter at the DP stage, it also 277 indicates that an early lineage checkpoint may be shared between them. iNKT and Id3 -/γ δ NKT cells (Figure 3a, b). There were 1611 and 1865 genes dysregulated by at 289 least two fold in L-DKO iNKT and Id3 -/γ δ NKT cells respectively, as compared to their WT 290 controls (Figure 3c). Among these, more than 400 genes shared similar expression patterns in 291 the two cell types (Figure 3c, d). Pathway analysis of dysregulated genes revealed involvement 292 of several pathways, including IL2/STAT5, p53 and MTORC signaling (Figure 3e). We also 293 found enrichment of genes that were dysregulated in T-to-natural killer (ITNK) cells upon 294 Bcl11b deletion (Figure 3e). [52] 295

E2A drives a gene network that promotes iNKT and innate-like fate in the absence of Id
In order to specifically identify any dysregulation of innate-specific genes, we decided to filter 296 differentially expressed genes in L-DKO iNKT and/or Id3 -/γ δ NKT cells, i.e. a total of 3060 297 genes, against an innate-like gene signature. We first searched for the reference innate-like 298 signature genes from publicly available Immgen data. [53] We hypothesized that the genes that are  Table II). Groups 1 and 2 included "biased" genes that were upregulated or 320 downregulated by a significantly larger magnitude in one population as compared to the other. 321 On the other hand, group 3 included "common" genes that were significantly and similarly 322 upregulated or downregulated in both innate-like populations. These categories broadly represent 323 genetic programs that are potentially important for the lineage-specific development of iNKT 324 (group 1) and γ δ NKT cells (group 2), as well as for overall innate-like T cell specification (group 325 3). By combining known interactions between these genes with our RNA-Seq and ChIP-Seq 326 data, we created a network map with the three groups of genes demarcated (Figure 4c) completely lack iNKT cells. [54] We crossed pTα -/mice to L-DKO to generate Id2 f/f Id3 f/f LckCre + 342 pTα -/-(L-DKO pTα -/-) mice. 343 Despite the complete absence of iNKT cells in pTα -/mice, to our surprise, we found a robust 344 iNKT population in L-DKO pTα -/mice (Figure 5a-c). These iNKT cells also expressed high 345 levels of PLZF, which is closely associated with the innate-like lineage gene expression program 346 (Figure 5d). It is known that pTα -/mice have an increase in γ δ T cells, [27] and L-DKO pTα -/-347 mice showed a similar increase in the γ δ population compared to WT mice (Figure 5e). 348 However, the γ δ T cells in L-DKO pTα -/mice were predominantly Vγ1.1 + Vδ6.3 + and uniformly 349 upregulated PLZF, reflecting a specific increase in innate-like γ δ NKT cells in these mice 350 (Figure 5f-i). 351 As expected, L-DKO pTα -/mice still had a profound block in conventional α β T cell 352 development due to the lack of pre-TCR signaling (Figure 6a, b). Interestingly, despite the pre-development of DP cells (Figure 6a, c). Upon careful investigation, however, we found that 355 these DP cells upregulate PLZF (Figure 6d). These PLZF hi DP cells, however, did not recognize 356 the CD1d tetramer, indicating that they are not iNKT cells that aberrantly upregulate CD4 and 357 CD8. Total thymocytes from L-DKO pTα -/mice also displayed a prevalent innate-like 358 phenotype, as indicated by their PLZF expression pattern (Figure 6e)

Initiation of an innate-like T cell transcriptional program in the absence of Id proteins and
363 conventional T cell development 364 Our previous observation suggested that pre-TCR signaling and Id protein activity is necessary 365 to enforce conventional T cell fate, such that the absence of both gave rise to predominantly deficiency. [11] This suggests that several E2A targets identified in Id-deficient mice may also be 416 similarly regulated in WT mice to promote innate-like T cell development.
It has been shown that the effector programs of iNKT2 cells closely resemble that of γ δ NKT 418 cells, [49]  is regulated by Id proteins. [55] This data also challenges the stochastic TCR-driven iNKT In this study, we also uncovered a pre-TCR independent pathway for the development of iNKT 441 and innate-like T cells using our L-DKO pTα -/mice. Interestingly, the expanded innate-like 442 phenotype in the L-DKO pTα -/mice was not limited to only iNKT and γ δ NKT cells, but also a 443 novel innate-like PLZF hi DP population. The early induction of an innate-like transcriptional 444 program among all developing T cells in these mice with high E protein activity demonstrates 445 the necessity of Id proteins in suppressing innate-like fate choice. The expanded innate-like 446 populations in our Id-deficient mice allowed us to uncover the regulation of innate-like T cell 447 lineage development prior to TCR selection. However, the divergence of iNKT and innate-like T 448 cells from conventional T cells prior to TCR selection has also been proposed in other mouse 449 models with physiological levels of E protein activity. [55,61] It is likely that the depletion of Id 450 proteins unleashes the "early", pre-TCR-independent developmental program for iNKT and 451 other innate-like T cells, that can usually be observed in much smaller frequencies in other 452 studies. Consequently, we have also observed heterogeneous innate-like α β T cell lymphomas in 453 Id2/Id3 deficient mice that are derived from iNKT, CD1dTetor T FH cells. [16,62] 454 PLZF undoubtedly plays key roles in innate-like T cell development and effector function. 455 However, the transcriptional regulation of PLZF itself remains ambiguous. While some studies 456 have found strong agonistic signaling to be sufficient to induce PLZF expression, [5,24,63] others 457 have reported early and stable suppression of PLZF in conventional T cells, which can't be 458 reversed by TCR signaling alone. [64] The function of PLZF itself, in promoting innate-like 459 effector genes, has also been demonstrated to be independent of agonistic TCR signals. [65,66] 460 This implies that unveiling the transcriptional regulation of PLZF and lineage specification of