The Tec Kinase ITK Regulates Thymic Expansion, Emigration, and Maturation of γ δ NKT Cells

This information is current as Catherine C. Yin, Ok Hyun Cho, Katelyn E. Sylvia, Kavitha of September 29, 2021. Narayan, Amanda L. Prince, John W. Evans, Joonsoo Kang and Leslie J. Berg J Immunol 2013; 190:2659-2669; Prepublished online 1 February 2013;

doi: 10.4049/jimmunol.1202531 Downloaded from http://www.jimmunol.org/content/190/6/2659

Supplementary http://www.jimmunol.org/content/suppl/2013/02/01/jimmunol.120253 Material 1.DC1 http://www.jimmunol.org/ References This article cites 50 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/190/6/2659.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Tec Kinase ITK Regulates Thymic Expansion, Emigration, and Maturation of gd NKT Cells

Catherine C. Yin, Ok Hyun Cho, Katelyn E. Sylvia, Kavitha Narayan, Amanda L. Prince, John W. Evans, Joonsoo Kang, and Leslie J. Berg

The Tec family tyrosine kinase, Itk, regulates signaling downstream of the TCR. The absence of Itk in CD4+ T cells results in impaired Th2 responses along with defects in maturation, cytokine production, and survival of iNKT cells. Paradoxically, Itk2/2 mice have spontaneously elevated serum IgE levels, resulting from an expansion of the Vg1.1+Vd6.3+ subset of gd T cells, known as gd NKT cells. Comparisons between gd NKT cells and ab iNKT cells showed convergence in the pattern of cell surface marker expression, cytokine profiles, and gene expression, suggesting that these two subsets of NKT cells undergo similar differentiation programs. Hepatic gd NKT cells have an invariant TCR and are derived predominantly from fetal progenitors that expand in the thymus during the first weeks of life. The adult thymus contains these invariant gd NKT cells plus a heterogeneous population of Downloaded from Vg1.1+Vd6.3+ T cells with diverse CDR3 sequences. This latter population, normally excluded from the liver, escapes the thymus and homes to the liver when Itk is absent. In addition, Itk2/2 gd NKT cells persistently express high levels of Zbtb16 (PLZF) and Il4, genes that are normally downregulated in the most mature subsets of NKT cells. These data indicate that Itk signaling is required to prevent the expansion of gd NKT cells in the adult thymus, to block their emigration, and to promote terminal NKT cell maturation. The Journal of Immunology, 2013, 190: 2659–2669. http://www.jimmunol.org/ 2 2 he Tec family tyrosine kinase, Itk, regulates signal trans- One unexpected phenotype observed in Itk / mice was the duction in T cells via the TCR. In the absence of Itk, T cells spontaneous elevation in levels of serum IgE (10, 11). On the basis 2 2 T have defects in phospholipase C-g1 activation, calcium of the data that indicated that Itk / conventional CD4+ T cells mobilization, MAPK phosphorylation, and AP-1 and NFAT acti- were unable to generate Th2 effector responses (12) and the fact 2 2 vation following stimulation (1). As a consequence, Itk plays a that Itk / ab TCR+ iNKT (ab iNKT) cells were also defective critical role in ab T cell development, influencing both positive in producing IL-4 (13, 14), the source of type 2 cytokines respon- and negative selection. During thymic development, conventional sible for promoting B cell isotype switching to IgE was initially CD4+ and CD8+ T cells are the predominant populations that arise. unclear. However, recent studies showed that the hyper-IgE syn-

2 2 by guest on September 29, 2021 These cells develop with a naive phenotype (CD62Lhi/CD44lo) and drome seen in Itk / mice was dependent on gd TCR+ T cells (15). 2 2 require a lengthy process of activation, proliferation, and differ- Subset analysis of gd TcellsinItk / mice identified the Vg1.1+ entiation prior to exhibiting effector cell characteristics (2–5). In Vd6.3+ subset, expressing the transcription factor PLZF (hereafter contrast, thymic development in Itk2/2 mice produces populations referred to as V6 or gd NKT cells) (16–18), as greatly increased in 2 2 of both CD4+ and CD8+ T cells that resemble memory cells and number in Itk / mice. Functional studies showed that these cells exhibit innate effector functions (6–9). Thus, TCR signaling via Itk secreted high levels of Th2 cytokines (15). In addition to the shared does not simply regulate the efficiency of conventional CD4+ and expression of PLZF linking gd to ab NKT cells (19, 20), tran- CD8+ T cell maturation but also maintains the appropriate balance scriptome analysis substantiated a common molecular program of naive versus innate T cell subsets. among these two cell lineages (21). Elegant studies have demonstrated that the adult thymus con- tains a mixed population of gd NKT cells. One subpopulation originates from fetal progenitors, undergoes substantial expansion Department of Pathology, University of Massachusetts Medical School, Worcester, in early neonatal life, and localizes to the liver; these cells pre- MA 01655 dominantly express an invariant TCR sequence that is character- Received for publication September 10, 2012. Accepted for publication January 4, ized by the absence of junctional diversity, consistent with their 2013. fetal/neonatal origin. In contrast, a second subpopulation is derived This work was supported by National Institutes of Health Grants AI084987 and AI083505 (to L.J.B.) and CA100382 (to J.K.). The ImmGen Project is supported from adult precursors and remains as a largely thymic resident by National Institutes of Health Grant AI072073. Core resources supported by the population (22–24). Diabetes Endocrinology Research Center (Grant DK32520) were also used. ab iNKT cells are an innate subset of ab T cells that reside in The sequences presented in this article have been submitted to the National Center the thymus, spleen, and liver, are rapid producers of cytokines for Biotechnology Information Gene Expression Omnibus Web site (http://www.ncbi. nlm.nih.gov/geo) under accession number GSE15907. such as IFN-g, IL-4, and IL-10 (25, 26), and predominantly ex- press an invariant TCR (27). ab iNKT cells develop in the thymus Address correspondence and reprint requests to Dr. Leslie J. Berg, Department of + + Pathology, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: leslie. from CD4 CD8 double-positive precursors and are dependent on [email protected] the presence of the nonclassical MHC class I molecule CD1d (28– The online version of this article contains supplemental material. 30). Following their initial positive selection, ab iNKT cell pre- Abbreviations used in this article: DAVID, Database for Annotation, Visualization, cursors undergo additional stages of differentiation in the thymus. and Integrated Discovery; Id3, inhibitor of DNA binding 3; KEGG, Kyoto Encyclo- Starting with the earliest detectable population (CD24 (HSA)+, pedia of Genes and Genomes; WT, wild-type. TCR+CD44loNK1.1lo; stage 0), ab iNKT cells proceed to down- lo Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 regulate CD24 (stage 1) and then upregulate CD44 (CD24 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202531 2660 Itk REGULATES gd NKT CELL MATURATION

CD44hiNK1.1lo; stage 2). Finally, at the terminal maturation stage, Sample preparation for microarray analysis the cells upregulate the IL-2/IL-15R b-chain (CD122) and NK lo hi hi Samples were prepared for microarray analysis as described previously (21). cell receptors, such as NK1.1 (CD24 CD44 NK1.1 ; stage 3). Briefly, thymocytes were pooled from 4 to 30 mice and enriched for gd Transition through these stages is also accompanied by a change T cells by depletion of CD8+ cells with magnetic beads and an autoMACS. 4 4 in cytokine profile. At the intermediate stage, ab iNKT cells are Cells were then stained and sorted with a FACSAria (∼2 3 10 –3 3 10 . potent producers of IL-4 but make little IFN-g; this phenotype cells; 99% pure) directly into TRIzol (Invitrogen). Independent triplicates were sorted unless indicated otherwise (complete sorting details available correlates with expression of high levels of PLZF. As they tran- from the ImmGen Project). Population labels correlate with ImmGen sition to stage 3, ab iNKT cells produce abundant amounts of (http://immgen.org) populations as follows: mature WT thymic V6 (MatV6. 2 2 IFN-g but less IL-4, and PLZF expression decreases (31). WT) and T.gd.vg1+vd6+24alo.Th; total V6 cells from Itk / thymus 2/2 + 2 Previous studies have shown that Itk2/2 mice have reduced (TotalV6. Itk ), T.gd.vg1+vd6+.Th.ITKko; mature WT CD4 CD8 thymocytes (MatCD4), T.4SP24-.Th; mature WT CD42CD8+ thymocytes numbers of ab iNKT cells and that those present have a defect in (MatCD8), T.8SP24-.Th; WT thymic stage 0–1 iNKT cells (NKT.442 maturation (13, 14, 32). Although gd T cells develop in the thy- NK1.12 [sorted in duplicate]), NKT.44-NK1.1-.Th; WT thymic stage 2 2 mus, they develop from CD4 CD8 progenitors rather than from 2 iNKT cells (NKT.44+NK1.12), NKT.44+NK1.1-.Th; and WT thymic + + stage 3 iNKT cells (NKT.44+NK1.1+), NKT.44+NK1.1+.Th. Note that all CD4 CD8 double-positive thymocytes (22, 33, 34), and their 2/2 lo thymic selection requirements are not known. Thus, the exact V6 cells in Itk mice are CD24 . Microarray data are available on the Gene Expression Omnibus Web site (http://www.ncbi.nlm.nih.gov/geo), extent of overlap in gd and ab iNKT cell developmental pro- accession number GSE15907. grams, and the impact of TCR signaling in gd NKT cell differ- entiation is unclear. In this study, we show that CD24lo gd NKT Tcrd cloning and sequencing thymocytes follow a maturational sequence composed of the three Total RNA was isolated from WT and Itk2/2Vg1.1+Vd6.3+ liver gd T cells. Downloaded from 2 2 stages ascribed to ab iNKT cells. Itk2/2 gd NKT cells are im- RNA was also isolated from WT-4Get and Itk / -4Get mature (HSAlo) + + + 2 + + paired in their maturation, leading to increased export of PLZFhi Vg1.1 Vd6.3 thymic gd T cells that were GFP CD122 , GFP CD122 , and GFP2CD122+. Reverse transcription of 0.2 mg RNA was performed IL-4–producing gd NKT cells from the thymus. TCRd sequence with qScript DNA Supermix (Quanta). TCRd V region sequences were analysis indicated that, unlike the gd NKT cells in livers of wild- amplified from cDNA by PCR using published primers (37). Amplified type (WT) mice that are exclusively derived from fetal progeni- sequences were cloned into pCR2.1-TOPO TA vector by TOPO TA cloning 2/2 tors, Itk hepatic gd NKT cells include a subset derived from kit (Invitrogen), and independent clones were subjected to DNA se- http://www.jimmunol.org/ adult progenitors. These data indicate that Itk normally functions quencing using both forward and reverse primers. To reduce sequencing error, only sequences confirmed in both directions were included in the to prevent the expansion, as well as the export, of adult-origin gd alignment analysis. In addition, rearrangements that resulted in out-of- NKT cells and that Itk plays a parallel role in the functional and frame junctions were also excluded from the final data set. phenotypic maturation of ab and gd NKT cells. Data analysis and visualization Materials and Methods Two-tailed nonparametric Mann–Whitney tests were performed using Prism (GraphPad software). Cell preparation and sorting for microarray Mice analysis were performed as previously described by ImmGen standard 2/2 operating procedures (see supplemental notes 1 and 2 in Ref. 21). RNA Itk mice (35) are on the C57BL/6 strain. 4Get mice (36) were crossed to by guest on September 29, 2021 Itk2/2 mice to obtain Itk2/2-4Get mice. MHC22/2 and b2m2/2 mice were processing and microarray analysis with the Affymetrix MoGene 1.0 ST obtained from The Jackson Laboratory or Taconic Laboratories and are on array were performed at the ImmGen processing center (by ImmGen a C57BL/6 background. C57BL/6 mice were used as controls. Mice were standard operating procedures; http://www.immgen.org/index_content. used between 2 and 3 mo of age and were maintained at the University of html). Microarray data were analyzed and visualized as described previ- Massachusetts Medical School under specific pathogen-free conditions in ously (21). Briefly, data were analyzed with modules of the GenePattern accordance with institutional animal care and use committee guidelines. genomic analysis platform. ConsolidatedProbeSets, a custom GenePattern module written by S. Davis (Harvard Medical School), was used for the Cell preparations, Abs, and flow cytometry consolidation of multiple probe sets into a single mean probe-set value for each gene. Genes with differences in regulation were identified with the To isolate lymphocytes from the liver, livers were first perfused with 5 ml Multiplot module of GenePattern through the use of the average of all PBS through the portal vein, followed by collagenase digestion of minced replicates. Genes were considered to be regulated differently if they dif- liver. Lymphocytes were then isolated by Percoll gradient centrifugation. fered in expression by .2-fold, had a coefficient of variation among The following Abs were purchased from BD Pharmingen: rat anti-mouse replicates ,0.5, had a Student t test p , 0.05, and had a mean expression IgG1-FITC, Vd6.2/6.3-PE, CD49a-Alexa Fluor 647, IFN-g-AlexaFluor value .120 in at least one subset in the comparison. Heat maps were 700, and Ly49F-biotin (bio). NKG2A/C/E-FITC, TCRd-PE-Cy5, TCRd- generated by hierarchical clustering (HierarchicalClustering module of PerCp eFluor 710, CD122-PerCp eFluor 710, IL-4-PE-Cy7, Streptavidin GenePattern) of data on the basis of gene (row) and subset (column) with (Strep)-PE-Cy7, CD49a-Alexa Fluor 647, CD244.2-Alexa Fluor 647, the Pearson correlation for distance measurement. Data were log trans- TCRb-allophycocyanin eFluor 780, and CD24-eFluor 450 were purchased formed and clustered with pairwise complete linkage. Data were centered from eBioscience. CD8a-PE-Texas Red was purchased from Invitrogen on rows before visualization with the HeatMapViewer module of Gene- Molecular Probes. CD1d-PBS57-PE and CD1d-PBS57 allophycocyanin Pattern. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway tetramers were provided by the National Institute of Allergy and Infectious analysis was used for functional classifications through the Database for Diseases Tetramer Facility. Vg1.1 Ab was a gift from L. Puddington Annotation, Visualization, and Integrated Discovery (DAVID) portal. (University of Connecticut Health Center, Farmington, CT) and conjugated to Alexa Fluor 647 with the Invitrogen Molecular Probes Alexa Fluor 647 Protein Labeling kit or to biotin with the FluoReporter Mini-Biotin-XX Results Protein Lableing kit also from Invitrogen Molecular Probes. Vg1.1-FITC gd NKT cells in the spleens of Itk2/2 mice display an immature was purchased from BioLegend. The following Abs were purchased from phenotype Santa Cruz Biotechnology: PLZF (D-9) and normal mouse IgG. Cells (1 3 106–4 3 106 events) were collected on a LSRII (BD Biosciences) flow We have previously shown that the absolute number and propor- cytometer. Data were analyzed using FlowJo software (Tree Star). tion of peripheral gdT cells are increased in Itk2/2 mice. This In vitro T cell activation defect can be attributed to a specific subset of gdT cells expressing the Vg1.1+Vd6.3+ TCR (hereafter referred to as V6) (Fig. 1A) (15). 2/2 WT and Itk thymocytes were stimulated as described previously As numerous similarities between V6 cells and ab iNKT cells (15). Cells were surface stained for anti-TCRd, anti-Vg1.1+, anti- + have been observed, we performed a side-by-side comparison of Vd6.3 , or CD1d-PBS57 tetramer, fixed, and permeabilized using 2/2 fixation/permeabilization kit (eBioscience) and stained for IL-4, IFN-g, ab iNKT cells and V6 cells in the spleens of both WT and Itk 2 2 and PLZF. mice. Consistent with previous reports, ab iNKT cells from Itk / The Journal of Immunology 2661

FIGURE 1. Splenic gd NKT cells in Itk2/2 mice have increased expression of PLZF and IL-4 mRNA. Splenocytes from WT and Itk2/2 mice or WT-4Get and Itk2/2-4Get mice were stained and ana- lyzed by flow cytometry. (A) Total spleno- cytes were analyzed for TCRd (left panels), and positive cells were gated on and examined for Vg1.1 and Vd6.3 ex- pression (second panels). V6 cells were further examined for HSA expression (third panels). iNKT cells were identified by staining with CD1d tetramer and anti- TCRb (fourth panels). iNKT cells further examined for HSA expression (right Downloaded from panels). (B) HSA2 V6 gd T cells and iNKT cells in the spleen were analyzed for expression of CD44, CD122, CD4, and intracellular PLZF. The mean fluo- rescent intensity of total PLZF in V6 cells is 395 6 41 for WT and 733 6 24 for Itk2/2. Dot plots show representative http://www.jimmunol.org/ data; compilations of data from of all ex- periments are shown in the graphs below. Each data point represents one mouse, and the black bars indicate the means. Statistically significant differences are shown with p values. (C)HSA2 V6 gd T cells and iNKT cells from WT-4Get and Itk2/2-4Get mice were stained and ana- lyzed for CD44 versus GFP, CD122 ver- sus GFP, and CD4 versus GFP expression. by guest on September 29, 2021 Data are representative of at least two independent experiments. (D)Compila- tions of data show absolute numbers of PLZF+ cells, and percentages and abso- lute numbers of GFP+ cells within the HSA2 V6 gd T cell or iNKT cell pop- ulation. Statistically significant differ- ences are shown with p values.

mice were reduced ∼2-fold in both numbers and percentages (13, To address the potential of these cells to produce cytokine, we 14, 32). To focus on the later stages of NKT cell maturation, we first used IL-4 reporter mice (4Get (36)), which express GFP analyzed CD24lo subsets of V6 cells and ab iNKT cells and from a bicistronic IL-4 mRNA. For these experiments, we found that the majority of these cells expressed the maturation compared the proportion of V6 and iNKT cells expressing GFP markers CD44 and CD122. Furthermore, as previously described in the presence or absence of Itk. As shown in Fig. 1C, the vast for ab iNKT cells (19, 20), maturation of WT V6 cells is as- majority of V6 cells from the spleens of Itk2/2 mice were GFP+, sociated with downregulation of CD4 and PLZF (Fig. 1B). Ex- compared with V6 cells from WT controls. Furthermore, the amination of the CD24lo V6 population from Itk2/2 mice revealed cell surface phenotype of GFP+ V6 cells from 4Get mice cor- a drastically altered phenotype when compared with WT controls. related with that of PLZF-expressing V6 cells in both WT and Although the majority of Itk2/2 V6 cells expressed CD44 and Itk2/2 mice (Fig. 1B, 1C). Interestingly, analysis of 4Get mice CD122, similar to WT cells, they also aberrantly retained ex- showed that the Itk2/2 iNKT cell population included fewer pression of PLZF and CD4, indicating incomplete maturation cytokine-producing cells than the WT iNKT cell subset, again (Fig. 1B). A similar trend, although less dramatic, was ob- consistent with previous published data (13, 14, 32). Taken to- served when comparing WT versus Itk2/2 iNKT cells in the gether, these data indicate that the majority of V6 cells from the spleen, as previously reported, including modestly impaired spleens of Itk2/2 mice display a more immature phenotype than upregulation of CD122 and downregulation of CD4 on Itk2/2 their WT counterparts, a phenotype shared with Itk2/2 iNK- iNKT cells (14). T cells. 2662 Itk REGULATES gd NKT CELL MATURATION

Increased proportions of PLZF- and CD4-expressing gd 2A). A comparison of WT liver cells with those from Itk2/2 mice NKT cells in the livers of Itk2/2 mice showed that iNKT cells were similar; in contrast, Itk2/2 liver V6 Unlike conventional T cells, NKT cells preferentially home to the cells had a substantial increase in the proportion that expressed PLZF and CD4 (Fig. 2A). Consistent with these data, an increased spleen and liver following thymic egress; furthermore, ab 2/2 iNKT cells continue to mature in the liver relative to their phe- proportion of Itk liver V6 cells also constitutively expressed notype in the spleen (38). To determine whether a similar change IL-4 mRNA (Fig. 2B). 2/2 These data suggested that peripheral gd NKT cell maturation was occurring with V6 cells and whether Itk liver V6 cells 2/2 were also impaired in this maturation, we examined liver was impaired in Itk mice. Alternatively, we considered the possibility that adult Itk2/2 mice might be generating unusually NKT cells. As shown in Fig. 2, we found that fewer WT ab + iNKT cells and WT V6 cells in the liver expressed high levels of large numbers of PLZF V6 cells in the thymus and that ongoing PLZF compared with these populations in the spleen (Figs. 1B, emigration of these cells was contributing substantially to the V6 population in the liver. This is in contrast to what is seen in WT mice, where the V6 cells found in the adult liver are predomi- nantly derived from fetal/neonatal progenitors (22, 39). To address this latter possibility, we first examined the rearranged TCRd gene sequences in V6 cells from the livers of WT and Itk2/2 mice. As expected, liver V6 cells from WT mice exhibited extremely lim- ited diversity in their TCRd sequences, with 100% of the

sequences identical to the canonical sequences previously found in Downloaded from V6 cells isolated from the fetal thymus and distinguished by their absence of the Dd1 and N region nucleotides (22). In contrast, TCRd sequences from Itk2/2 liver V6 cells included a fraction of sequences that were clearly of adult origin, in addition to those with the canonical TCRd chain rearrangements (Fig. 2C, Sup-

plemental Fig. 1). These data indicate that, in the absence of Itk, http://www.jimmunol.org/ adult-derived thymic V6 cells are able to emigrate to the liver.

Altered thymic development of gd NKT cells in Itk2/2 mice To address the changes in V6 cell development that might account for the presence of adult-derived gd NKT cells in the liver of Itk2/2 mice, we examined thymocytes from WT and Itk2/2 mice and characterized iNKT cell and V6 cell populations. As previously observed, the percentage of thymic iNKT cells in Itk2/2 mice was decreased compared with WT (Fig. 3A) (13, 14, 32). In contrast, by guest on September 29, 2021 Itk2/2 thymi contained a substantial increase in the proportion of gd T cells, an increase that was largely accounted for by expanded numbers of V6 cells (Fig. 3A). Several stages of ab iNKT cell differentiation have been characterized based on a variety of cell surface markers, cyto- kines, and transcription factors. One notable example is the up- regulation of CD44 and CD122, which indicate a transition to the terminal maturation stage (40, 41). Similar to ab iNKT cells, HSAlo V6 cells upregulate CD44 while concurrently upregulating CD122 (Fig. 3B). Analysis of Itk2/2 thymic V6 cells indicated modest changes in the percentages of CD44+ and CD122+ cells compared with WT V6 cells, leading to a reduction in the pro- portion of CD44+Itk2/2 V6 cells that had upregulated CD122. One difference between ab iNKT cells and V6 cells was the expression of CD4. Whereas WT ab iNKT cells remained largely + FIGURE 2. gd NKT cells in the livers of Itk2/2 have increased PLZF CD4 in the thymus as they matured, WT thymic V6 cells and IL-4 mRNA expression. Lymphocytes were isolated from livers of WT downregulated CD4 coincident with the upregulation of CD44 and Itk2/2 mice or WT-4Get and Itk2/2-4Get mice and were analyzed by and CD122. Strikingly, the majority of V6 cells in the thymus of 2/2 + flow cytometry. (A)WTandItk2/2 HSA2 V6 gd T cells and iNKT cells Itk mice remained CD4 (Fig. 3B). Taken together, these data 2 2 were examined for CD4 versus PLZF expression. Dot plots show repre- suggest that gd NKT cells in Itk / mice have a modest defect in sentative data, and the graphs show a compilation of data from all experi- the transition to terminal maturation, similar to the impaired ments. Statistically significant differences are shown with p values. (B)WT- maturation seen with thymic Itk2/2 iNKT cells (Fig. 3B) (14). 2/2 2 4Get and Itk -4Get HSA V6 gd T cells and iNKT cells were analyzed A major transcription factor important in the development of ab for expression of CD4 and GFP. Dot plots show representative data, and the iNKT cells is PLZF (19, 20). As reported, PLZF levels decline as ab graphs show compilations of data from all experiments. Statistically sig- iNKT cells mature; thus, the iNKT cells expressing the highest levels nificant differences are shown with p values. (C) Sorted V6 gd T cells from WT and Itk2/2 livers were analyzed for in-frame sequences of Vd6andJd1 of CD44 or CD122 express less PLZF than their less mature coun- junctions. The graph indicates the percentage of sequences corresponding to terparts (Fig. 3B) (19, 20). A parallel pattern of expression was ev- fetal-derived rearrangements or adult-derived rearrangements. The total ident for thymic V6 cells (Fig. 3B), as has also recently been reported number of sequences included in the analysis is indicated above each bar. (42). For V6 cells, those expressing the highest levels of PLZF Actual sequences are shown in Supplemental Fig. 1. expressed less CD44 and CD122 than the PLZFint subset. As shown, The Journal of Immunology 2663 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. gd NKT cells in the thymus of Itk2/2 mice have the characteristics of less mature ab iNKT cells. Thymocytes from WT and Itk2/2 mice or WT-4Get and Itk2/2-4Get mice were stained and analyzed by flow cytometry. (A) Total thymocytes were analyzed for TCRb versus TCRd (left panels), and TCRd-positive cells were gated on and examined for Vg1.1 and Vd6.3 expression (second panels). V6 cells were further gated on HSA (third panels). iNKT cells were identified by staining with CD1d tetramer and anti-TCRb (fourth panels). iNKT cells were further gated on HSA expression (right panels). (B) HSA2 V6 gd T cells and iNKT cells in the thymus were analyzed for the expression of CD44, CD122, CD4, and intracellular PLZF. Dot plots show representative data; compilations of data from of all experiments are shown in the graphs below. Statistically significant differences are shown with p values. (C) HSA2 V6 gd T cells and iNKT cells from WT-4Get and Itk2/2-4Get mice were stained and analyzed for CD44 versus GFP, CD122 versus GFP, and CD4 versus GFP expression. Data are representative of at least two independent experiments. (D) PLZFhi and PLZFint V6 gd T cell and iNKT cell populations were gated as shown in (B), and absolute numbers were calculated (left two panels). The percentages and absolute numbers of GFP+ cells in the HSA2 V6 gd T cell and iNKT cell populations are shown (right two panels). Statistically significant differences are shown with p values. we observed an overall increase in the total numbers of both PLZFhi also CD4 negative. Itk2/2 thymic V6 cells showed a similar pat- and PLZFint V6 populations among Itk2/2 thymocytes (Fig. 3D). tern; however, the proportion of cells retaining high levels of GFP Another feature of ab iNKT cell terminal maturation is a re- was dramatically increased compared with WT (Fig. 3C, 3D). duction in IL-4 production (32). To determine whether a similar pattern is observed for V6 cells, we analyzed thymocytes from Cytokine production by gd NKT cells correlates with PLZF WT- and Itk2/2-4Get mice. V6 cells from WT mice showed expression graded GFP expression, such that the lowest levels of GFP cor- As ab iNKT cells undergo terminal maturation, they switch from related with the highest expression of maturation markers, CD44 being single IL-4 producers to being dual producers of both IL-4 and CD122. In addition, GFP-negative WT thymic V6 cells were and IFN-g, a change that correlates with reduced PLZF expression 2664 Itk REGULATES gd NKT CELL MATURATION

(19, 20, 43). To determine whether V6 cells expressing high compared with WT CD24lo V6 cells, and 124 genes were down- versus intermediate levels of PLZF showed a similar pattern of regulated (Fig. 5A, Supplemental Table 1). Using the DAVID cytokine production, we stimulated WT and Itk2/2 thymocytes analysis program, we identified several KEGG pathways that with PMA and ionomycin and examined IFN-g and IL-4 pro- appeared to be disregulated in Itk2/2 V6 cells (Fig. 5B). These duction. Similar to ab iNKT cells, V6 cells were capable of pathways were involved in cytokine signaling, some of which are producing both cytokines upon stimulation, although the propor- required for the development of T cells, as well as effector tion of V6 cells producing IFN-g and IL-4 simultaneously is less functions, including molecules frequently associated with ab than seen for ab iNKT cells. Surprisingly, there was little dif- iNKT and NK cells. To confirm these findings at the protein level, ference in the frequency of total Itk2/2 V6 cells capable of pro- we examined the expression of several NK cell receptors that can ducing IFN-g and/or IL-4 compared with WT V6 cells (Fig. 4A). mediate effector function in ab iNKT cells. As shown, this We then examined cytokine production in cells stained for PLZF analysis revealed that Itk2/2 V6 cells have decreased or absent expression. As can be seen in Fig. 4B and 4C, only the PLZFint expression of CD122, NK1.1, 2B4, Ly49E/F, NKG2A/C/E, and subset of iNKT cells was capable of producing IFN-g, whereas CD94 compared with WT V6 cells (Fig. 6A, 6B). As acquisition both PLZFhi and PLZFint iNKT cells produced IL-4. A similar of NK cell receptors occurs at the final stage of ab iNKT cell pattern was seen for V6 cells and was shared between WT and Itk2/2 maturation (30, 32, 44), these data suggest that this developmental thymocytes. These results indicate that PLZFhi cells in Itk2/2 program is conserved for gd NKT cells as well. mice are functionally competent, despite their delayed progression Given the similarity between ab iNKT cells and V6 cells, we toward terminal maturation. then examined the relationship between Itk2/2 V6 cells and several

2 2 isolated subsets of iNKT cells as well as conventional CD4 and Downloaded from The gene signature of Itk / gd NKT cells resembles immature CD8 thymocytes, using the ab iNKT gene signature list generated WT iNKT cells previously (21). For this analysis, we compared the list of genes that The data presented thus far suggest that the terminal maturation of were upregulated in ab iNKT cells versus naive conventional CD4 V6 cells is impaired or delayed in the absence of Itk. To examine and CD8 thymocytes, along with genes that were downregulated in this issue at a molecular level, we performed gene expression ab iNKT cells versus CD4 and CD8 thymocytes, to the 124 genes 2/2 microarray analysis. V6 cells were isolated from the thymus of that were downregulated in Itk V6 cells relative to WT V6 cells. http://www.jimmunol.org/ 2/2 Itk mice, and their gene expression profile was compared with Approximately 40% of the genes (51 genes) that were decreased in that of multiple populations of WT thymocytes. As shown in Fig. Itk2/2 versus WT V6 cells are shared with the subset of genes 2/2 5, 71 genes were upregulated by $2-fold in Itk V6 cells normally upregulated in ab iNKT cells relative to conventional CD4 and CD8 T cells (Fig. 5C). In contrast, only ∼5% of the genes (six genes) that were decreased in Itk2/2 V6 cells are common to the genes normally downregulated in ab iNKT cells. These data indicate that the WT V6 cells are similar to WT ab iNKT cells and further that Itk2/2 V6 cells are lacking a subset of the mature ab iNKT cell signature. Using cluster analysis and comparing the 51 by guest on September 29, 2021 genes that WT ab iNKT and Itk2/2 V6 cells share in Fig. 5C, the heat map shows that WT HSAlo V6 cells are similar to mature WT ab iNKT cells, whereas the Itk2/2 V6 cells are most similar to immature (NK1.12)WTab iNKT cells (Fig. 5D, Supplemental Table 2). Taken together with the phenotypic analysis, these data confirm that Itk2/2 V6 cells are impaired in their terminal matu- ration in the thymus. Itk2/2 thymic gd NKT cells are derived from both fetal and adult progenitors Previous studies have demonstrated that peripheral V6 cells in WT mice arise from fetal progenitors that expand in number in the neonatal thymus and migrate out to populate peripheral tissues (22, 24). In contrast, the V6 cells found in adult Itk2/2 liver in- cluded a subset that was clearly derived from adult progenitors. These findings suggested that Itk might be required to specifically limit the numbers of adult progenitor–derived V6 cells in the thymus; for example, Itk might be required to promote a TCR- dependent cell death signal, akin to the negative selection of conventional T cells. If this were the case, we would predict that FIGURE 4. Levels of PLZF expression correlate with IFN-g production thymic V6 cells in adult Itk2/2 mice would be greatly enriched for 2/2 by gd NKT cells. Thymocytes from WT or Itk mice were stimulated the adult progenitor–derived population. In addition, we consid- with 10 ng/ml PMA and 2 mg/ml ionomycin for 5 h and then stained for hi int/lo 2 ered the possibility that PLZF versus PLZF V6 cells might intracellular IL-4, IFN-g, and PLZF. Dot plots show gated HSA V6 represent distinct V6 subsets. For instance, it was possible that thymocytes or CD1d tetramer–positive cells. (A) Nonstimulated (top) and PLZFint/lo cells represented the continued presence of fetal pro- stimulated (middle) cells were analyzed for IFN-g versus IL-4 production. genitor–derived V6 cells expressing the invariant TCR d sequence, The graph (bottom) shows a compilation of data indicating the percentage hi of double IL-4+IFNg+–producing cells among WT V6 compared with whereas the PLZF cells might represent V6 cells developing in iNKT cells. Statistical significance is shown with p values. (B and C) the adult thymus from adult progenitors. Stimulated cells were analyzed for IL-4 versus PLZF expression (B)or To address these issues, we analyzed three populations of V6 2 2 IFN-g versus PLZF expression (C). cells from the thymus of WT and Itk / mice. To facilitate the The Journal of Immunology 2665 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Gene expression microarray analysis indicates impaired terminal maturation of Itk2/2 gd NKT cells. (A) Scatter plot showing expression of consolidated probe sets by CD24(HSA)lo V6 gd T cells in thymocytes from adult WT and Itk2/2 mice. Nomenclature is according to ImmGen (http:// immgen.org): MatV6.WT = CD24(HSA)lo V6 gd T cells from adult WT thymus; TotalV6.Itk2/2 = total V6 gd T cells from adult Itk2/2 thymus, which are 95–98% CD24lo. Each dot represents one gene (mean of all probe sets and mean of replicates); red and blue indicate genes with expression increased or decreased, respectively, by .2-fold (p , 0.05, Student t test; coefficient of variation , 0.5; mean expression value . 120 in one subset); numbers in parentheses above plot indicates total number of these genes. The numbers within the plot indicate the number of upregulated (red) or downregulated (blue) genes in the comparison. Data are from three independent experiments with 4–30 mice each. (B) The 124 genes increased in MatV6.WT versus TotalV6. Itk2/2 cells were classified into functional pathways using the KEGG analysis program (DAVID). The top five pathways enriched in the data set are shown along with the number of genes, the gene names, the p value, and the Benjamini p value for each pathway. (C) Venn diagram (Figure legend continues) 2666 Itk REGULATES gd NKT CELL MATURATION

FIGURE 6. Decreased surface expression of NK cell markers on Itk2/2 V6 cells. (A) Thymocytes from WT and Itk2/2 mice were stained for expression of NK1.1 along with CD122, CD44, and CD4. (B) Thymocytes from WT and Itk2/2 mice were stained for expression of NK cell markers CD49a (ITGA1), 2B4 (CD244), Ly49E/F (KLRA5/6), NKG2A/C/E (KLRC1/2/3), CD94 (KLRD1), and CD122. Dot plots show gated CD24loV6+ cells. Results are representative of two experiments with at least three mice per group. Downloaded from

isolation of distinct subsets, we used WT-4Get and Itk2/2-4Get tained a mixture of cells of both origins. As a result, these data thymocytes and sorted GFPhiCD1222 (stage 1), GFPintCD122+ support the conclusion that V6 stages 1→2→3 represent a devel- 2 + (stage 2), and GFP CD122 (stage 3) populations, taking ad- opmental lineage of gd NKT cells. http://www.jimmunol.org/ vantage of the fact that GFP expression is correlated with PLZF expression in V6 cells (see Fig. 3). TCRd sequence analysis in- Discussion dicated the presence of a highly diverse repertoire in each of the To understand the basis for the gd T cell–dependent hyper-IgE subsets and in both WT and Itk2/2 mice (Supplemental Fig. 1). syndrome that develops in the absence of Itk, we performed Furthermore, a significant proportion of the sequences from both a detailed analysis gd NKT cells in Itk2/2 mice. Our previous WT and Itk2/2 thymocytes of each subset used Dd1 and contained studies demonstrated that this gd T cell subset, expressing the N region additions, indicating that these adult thymic V6 cells Vg1.1/Vd6.3 (V6) TCR and the transcription factor PLZF, was were not derived from fetal progenitors. Nonetheless, the invariant highly expanded in Itk2/2 mice and could secrete large amounts TCRd sequence found in WT liver V6 cells was detected in each of type II cytokines, such as IL-4 and IL-13 (15). Using a com- by guest on September 29, 2021 of the adult thymic V6 subsets, confirming the persistence of these bination of phenotypic, functional, and molecular analysis of V6 fetal-derived cells in the adult thymus, as reported previously (22). cells in the thymus, spleen, and liver, we have determined A detailed inspection of the data from WT thymocytes showed a number of important features of these cells. First, we show that that, among the more mature V6 subsets (CD122+ stages 2 and 3), the increased V6 population in Itk2/2 mice is generally accounted there was a reduced proportion of these fetal progenitor–derived for by cells expressing high levels of PLZF and constitutively sequences relative to their abundance in the less mature GFPhi expressing IL-4 mRNA. Second, based on a comparison with CD1222 subset, stage 1 (p , 0.0001 and p = 0.002, respectively; iNKT cells, we conclude that Itk2/2 V6 cells do not fully mature Fig. 7). This finding indicated selection against this invariant TCR and cannot transit to the stage associated with high level IFN-g during the maturation of adult thymic V6 cells. Although overall production. Third, we provide evidence that Itk2/2 V6 cells the pattern of TCRd sequences among adult thymic Itk2/2 V6 arising from adult progenitors are migrating to the liver, a process cells was similar to that seen in the WT adult thymus, we did that is not permitted for WT V6 cells. Taken together, these observe an apparent lack of selection against the invariant fetal findings reveal alterations in the V6 gd NKT cell population progenitor–derived sequence as Itk2/2 V6 cells mature. These present in Itk2/2 mice could account for the hyper-IgE syndrome data indicated that the numerical increase in V6 cells in the Itk2/2 seen in these mice. thymus was not due to a preferential expansion of adult progen- We found a number of striking parallels between V6 cells and itor–derived cells but instead was the result of expansion of both iNKT cells that suggest a common developmental program shared fetal- and adult-derived V6 cell subsets. Furthermore, these data between these two cell lineages. Recently, we found that mature demonstrated that, in the WT thymus, V6 subsets distinguished by WT thymic V6 cells and thymic iNKT cells express a common high or low PLZF expression and/or CD122 expression were not NKT cell gene expression signature (21). By examining changes derived from different progenitors; instead, each population con- in cell surface marker expression, PLZF levels, and cytokine

comparing the list of genes that were increased or decreased in iNKT (NKT.44+NK1.1+) cells versus MatCD4 (CD24lo) and MatCD8 (CD24lo) single- positive thymocytes (identified previously in Ref. 21) with the list of 124 genes downregulated in TotalV6.Itk2/2 versus MatV6.WT cells. Roughly 40% of the genes decreased in TotalV6.Itk2/2 versus MatV6.WT cells are normally increased in NKT.44+NK1.1+ cells versus mature single-positives. (D) Heat map showing relative expression of the 51 genes identified in (C) that were decreased in TotalV6.Itk2/2 versus MatV6.WT cells and increased in NKT.44+ NK1.1+ cells versus MatCD4 and MatCD8 single-positive thymoctyes in populations of NKT and MatCD4 cells from WT mice and V6 cells from WT or Itk2/2 mice. Data were log transformed, centered by gene row, and hierarchically clustered by gene and subset. The clustering dendrogram for samples is shown. Color-coding reflects relative expression levels of a given gene in each subset and does not provide information about absolute expression levels of each gene. The Journal of Immunology 2667

study showed that this competition was acting on the most mature NK1.1+PLZFlo subset of each NKT cell population (14). As our data indicate that it is predominantly the NK receptor–negative PLZFhi population of gd NKT cells that is increased in Itk2/2 mice, it seems unlikely that the observed expansion is caused by a reduced number of Itk2/2 ab iNKT cells. Instead, we suggest that the increased number of Itk2/2 gd NKT cells, as well as their impaired terminal maturation, is due to defective TCR signaling in the absence of Itk. For ab iNKT cells, TCR signaling has been shown to be essential for their maturation to the NK1.1+ stage, because this developmental progression fails to occur in the absence of Zap-70 (45). For V6 cells, enforced Vg1.1/Vd6.3 TCR transgene enhanced the generation of PLZF+ FIGURE 7. The expanded population of gd NKT cells in the thymus of 2 2 NKT cells, indicating that TCR signaling provides a selective Itk / mice includes cells derived from both fetal and adult progenitors. The graph shows the relative proportions of in-frame TCR Vd6and cue (16). Although comprehensive analysis of progressive gd Jd1 junctions in three subsets of sorted thymocytes from WT-4Get and NKT cell maturation in mice with mutations in TCR signaling Itk2/2-4Get mice that derive from fetal versus adult progenitors. Pop- components has not been performed, our data demonstrate that ulations analyzed were CD1222GFP+ (stage 1), CD122+GFP+ (stage 2), stage 1 V6 cells do not efficiently mature when Itk signaling is 2 and CD122+GFP (stage 3). The total number of sequences included in the impaired. In aggregate, these findings support the hypothesis that Downloaded from analysis is indicated above each bar. Statistically significant differences qualitatively distinct TCR signaling occurs at multiple maturation were determined using the exact binomial test of goodness-of-fit, and p checkpoints for both ab and gd NKT cells. values are shown. At the right, the gating strategy for isolation of each For gd T cells, TCR-dependent maturation processes are not thymocyte subset is shown. restricted to the NKT subset, because the maturation of other gd T cell subsets has been shown to be modulated by TCR signaling.

profiles, we now show that gd NKT cells can be subdivided into Studies of a natural population of nonclassical MHC class I T10/ http://www.jimmunol.org/ populations nearly identical to the stage 1–3 subsets of iNKT cells 22–specific gd T cells have revealed several interesting features of (27, 41, 45). This comparison indicates that the most mature gdTCR signaling–mediated thymic selection processes. First, the subset of V6 cells in the thymus are PLZFint, express markers of presence of the cognate ligand for this gd subset in WT C57BL/6 activation (CD44, CD122, and a host of NK receptors), down- mice reduces the numbers of T22 tetramer–binding cells, indica- regulate CD4, and are dual producers of IFN-g and IL-4. Fur- tive of a negative selection process. Second, mice lacking T10/22 thermore, our TCRd sequence analysis showed that both IL-4+ still generate the tetramer+ gd T cells, but these cells are deficient PLZFhi and IL-4/IFNg+PLZFint subsets contain a mixture of in- in IFN-g production, whereas the tetramer+ cells found in WT variant fetal-derived and heterogeneous adult-derived TCRd gene mice are primarily IFN-g producers. These data indicate that rearrangements. This result supports a model of a common de- intrathymic TCR signaling promotes the generation of gd effector by guest on September 29, 2021 velopmental program for these two gd NKT cell subpopulations. cells fated for IFN-g production (46). Third, examination of the A major unresolved issue is the underlying cause of increased cell surface phenotype of gd TCR+ thymocytes shows an inverse numbers of more immature PLZFhi V6 cells in Itk2/2 mice. One correlation between CD122 expression and expression of the possibility is that competition exists between gd NKT cells and gut-homing chemokine receptor CCR9, such that cells expressing ab iNKT cells, and Itk2/2 V6 cell numbers are increased because high levels of CD122 express little CCR9. Importantly, in mice of a reduction in ab iNKT cells in these mice. However, a recent lacking T10/T22, all T22 tetramer+ gd TCR+ thymocytes express

FIGURE 8. A model of V6 development in WT and Itk2/2 mice. In WT mice (left panel), V6 cells initially develop from fetal precursors (pink) and expand in the thymus during the first 2 wk after birth (22). These cells undergo a maturation program similar to ab iNKT cells before emigrating to the periphery, with a preference for homing to the liver (bold pink). As WT mice age, a second wave of V6 cells develops from adult precursors (blue) and matures prior to emigrating to peripheral lymphoid organs. A small number of fetal-derived and adult-derived V6 cells are present and continuously maturing in the thymus of adult mice. Similar to WT V6 cells, Itk2/2 V6 cells (right panel) also initially develop from fetal precursors (pink) and preferentially home to the liver; however, increased numbers of V6 cells are found in the absence of Itk. A second wave of adult precursor-derived Itk2/2 V6 cells also develops in increased numbers (blue). Itk2/2 V6 cells have a defect in terminal maturation and retain high expression of PLZF and IL-4 relative to WT V6 cells. In addition, Itk2/2 V6 cells arising from adult precursors are able to emigrate to the liver (green). E20, Embryonic day 20. 2668 Itk REGULATES gd NKT CELL MATURATION high levels of CCR9, whereas in WT mice expressing the self- mice that show expanded populations of PLZF+ innate T cells, ligand, a majority of these cells are CCR92 and CD122+. These such as Id32/2 mice. In the case of Itk2/2 mice, altered regulation findings provide a clear precedent for intrathymic TCR signaling– of the Lin28b pathway is unlikely to fully account for the increased mediated maturation of gd T effector subsets and support our number of V6 cells in the adult Itk2/2 thymus, because we see conclusion that Itk2/2 gd NKT cells escape negative selection, do a concomitant decrease in the numbers of ab iNKT cells. Thus, not efficiently upregulate CD122, and fail to downregulate CCR9 although there are likely distinct progenitor-specific programs because of impaired TCR signaling. Our efforts to define a po- controlling fetal versus adult NKT cell differentiation, Itk-de- tential TCR-dependent ligand for V6 cells in the thymus have, to pendent TCR signaling is an arbiter of the size of NKT cell pop- date, been largely unsuccessful. We find that V6 cells are not ulations and is required for their terminal maturation. dependent on classical or nonclassical MHC class I or class II molecules for their development (Supplemental Fig. 2). None- Acknowledgments theless, additional studies of genetically modified mice support the We thank Regina Whitehead and Sharlene Hubbard for technical assistance. conclusion that V6 cell development is strongly influenced by We also thank members of the ImmGen core team (M. Painter, J. Ericson, TCR signaling. Specifically, mice deficient for Kruppel-like factor and S. Davis) for help with data generation and processing and eBioscience, 2 (47) or inhibitor of DNA binding 3 (Id3) (17, 48), as well as Affymetrix, and Expression Analysis for support of the ImmGen Project. mice expressing a mutant form of the SLP-76 adaptor protein (SLP76-Y145F) that is unable to bind to ITK (17, 49), have been Disclosures analyzed. In each of these cases, the mice exhibit a similar phe- 2 2 The authors have no financial conflicts of interest. notype to Itk / mice, characterized by a dramatic increase in the Downloaded from numbers of thymic V6 cells. Taken together, these data are con- sistent with the model that gd NKT cells normally undergo neg- References ative selection because of robust TCR signaling in the thymus. 1. Andreotti, A. H., P. L. Schwartzberg, R. E. Joseph, and L. J. Berg. 2010. T-cell signaling regulated by the Tec family kinase, Itk. Cold Spring Harb. Perspect. 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WT liver V6+ cells (n=17) (9) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E H I G G I R A T D K (1) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC  A L W E H I G G I R A T D (6) GCT CTC TGG GAG CT T ATCGGAGGGATACGAG CT ACC GAC AAA A L W E L I G G I R A T D K

(1) GCT CTC TGG GAG CT T ATCGGAGGGATACGAG CT A L W E L I G G I R A

Itk-/- liver V6+ cells (n=14)

(4) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E H I G G I R A T D K

(3) GCT CTC TGG GAG CTG G TCGGAGGGATACGAG CT ACC GAC AAA  A L W E L V G G I R A T D K

(3) GCT CTC TGG GAG CTG G C CGGAGGGATACGAG CT ACC GAC AAA A L W E L A G G I R A T D

  (4) GCT CTC TGG GAG CTG G TCCCATAT ATGGC CCTTT ATCGGAGGGATACG GCA ACC GAC AAA A L W E L V P Y M A L Y R R D T A T D K

WT-4Get thymocyte stage 1 (CD122-GFP+; n=28)

(4) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E H I G G I R A T D K

(5) GCT CTC TGG GAG CTG G TCGGAGGGATACGAG CT ACC GAC AAA  A L W E L V G G I R A T D K (2) GCT CTC TGG GAG CTG G TCGGAGGGATACGAG CT A L W E L V G G I R A

(5) GCT CTC TGG GAG CTG G C CGGAGGGATACGAG CT ACC GAC AAA A L W E L A G G I R A T D K

(2) GCT CTC TGG G GCCT A ATCGGAGG AATAT CT ACC GAC AAA A L W G L I G G I S T D K (1) GCT CTC TGG GAG CTG G T TGGC TT GGAGGG TCCAG CT ACC GAC AAA A L W E L V G L E G P A T D K (1) GCT CTC TGG GAG CTG G CCT CGGAGGG CCCA ACC GAC AAA

A L W E L A P E G P T D K

(1) GCT CTC TGG GAG CTG G AG GG GGAT ATCGGAGGGATAC CTG CT ACC GAC AAA

A L W E L E G D I G G I P A T D K (1) GCT CTC TGG GAG CTG AAG GG AGAT ATCGGAGGGAT TCCCG CT ACC GAC AAA

A L W E L K G D I G G I P A T D K

(1) GCT CTC TGG GAG CT C CAT ATCGGAGGGATACGAG CTT CC GAC AAA   A L W E L H I G G I R A S D K (1) GCT CTC TGG GAG C CAG ATAT C GGG G CT ACC GAC AAA A L W E P D I G A T D K

(1) GCT CTC TGG GAG C CCT AT ATCGGAGGGATA GTCTTCT CT ACC GAC AAA ALWEP Y IGGI V FS T D K (1) GCT CTC TGG GAG TGGC CGGAGGGATACGAG CTTAGG ACC GAC AAA A L W E W P E G Y E L R T D K

(1) GCT CTC TGG GAG CT TGAT ATCGGAGGGATACG TCCCG CT ACC GAC AAA

A L W E L D I G G I R P A T D K (1) GCT CTC TGG GAG CT T ATCGGAGGGATACG GGGTTCCG CT ACC GAC AAA

A L W E L I G G I R G S A T D K

WT-4Get thymocyte stage 2 (CD122+GFP+; n=25)

(1) GCT CTC TGG GAG C GT ATCGGAGGGATACGAG CT A L W E R I G G I R A  (1) GCT CTC TGG GAG C AG ATCGGAGGGATACGAG CT ACC GAC AAA A L W E Q I G G I R A T D K

(2) GCT CTC TGG GAG CTG G TCGGAGGGATACGAG CT ACC GAC AAA A L W E L V G G I R A T D K

(1) GCT CTC TGG GAG C AT ATCGGAGGGATACG GG CT ACC GAC AAA A L W E H I G G I R A T D K

(1) GCT CTC TGG GAG CTG G C CGGAGGGATA ACGG CT ACC GAC AAA   A L W E L A G G I T A T D K (1) GCT CTC TGG GAG TTCG AGGGATACGAG CTTG CC GAC AAA A L W E F E G Y E L A D K (1)( GCT CTC TGG GAG C AGT ATGGCA GAAT ATCGGAGGGAT CC GAC AAA A L W E Q Y G R I S E G S D K (1)( GCT CTC TGG GAG C ATGGCATAT CGTCT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E H G I S S I G G I R A T D K (1)( GCT CTC TGG GAG C ACAT ATGGCAT ATCGGAGGGATACGA ACC GAC AAA

A L W E H I W H I G G I R T D K (1)( GCT CTC TGG GAG C ACCTT ATCGGAGGGATACGAG CCCCGTA T ACC GAC AAA

A L W E H L I G G I R A P Y T D K (1)( GCT CTC TGG GAG C AT ATGGCATAT GA CGGAGGGATACGAG CC GAC AAA

A L W E H M A Y D G G I R A D K (1)( GCT CTC TGG GAG CTG G ACGGA ATGGCATAT T GGATACGAG CT ACC GAC AAA

A L W E L D G M A Y W I R A T D K (1)( GCT CTC TGG GAG CTG G CGAGAGGCAT ATCGGAGGGATACG GGGGAT CC GAC AAA A L W E L A R G I S E G Y G G S D K (1)( GCT CTC TGG GAG CTG G AGGGA CGGAGGGATACG C CC GAC AAA   A L W E L E G R R D T P D K (1)( GCT CTC TGG GAG C CTCAT ATCGGAGGGATACGAG CTC CC GAC AAA A L W E P H I G G I R A P D K (1)( GCT CTC TGG GAG C CTCAT ATCGGAGGGATAC CCTCCC CC GAC AAA

A L W E P H I G G I P S P D K (2)( GCT CTC TGG GAG CTG G GGGGA GG T ATCGGAG CC ACC GAC AAA A L W E L G G G I G A T D K

(2)( GCT CTC TGG GAG CTG G GGG CGGAGGGATACG GGG CT ACC GAC AAA

A L W E L G A E G Y G A T D K (1)( GCT CTC TGG GAG CTG G CGGCCCT ATA TGGCCCTC CT ACC GAC AAA A L W E L A A L Y G P P T D K

(1)( GCT CTC TGG GAG CTG G GG GGAGGGATACGAG CCC CT ACC GAC AAA A L W E L G G G I R A P T D K (1)( GCT CTC TGG GAG CT AGGG GGC TTCTT ATCGGAGGGATAC C CC GAC AAA A L W E L G G F L S E G Y P D K (1)( GCT CTC TGG GAG CT C TAT ATCGGAGGGATACGAG CTC CC GAC AAA A L W E L Y I G G I R A P D K

WT-4Get thymocyte stage 3 (CD122+GFP-; n=22)

(3) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC AAA  A L W E H I G G I R A T D K (2) GCT CTC TGG GAG CTG G TCGGAGGGATACGAG CT ACC GAC AAA A L W E L V G G I R A T D K

(1) GCT CTC TGG GAG CTG G C CGGAGGGATACGA A CT ACC GAC AAA

A L W E L A G G I R T T D K (1) GCT CTC TGG GAG CTG G GGGATA CT ACC GAC AAA

A L W E L G D T T D K (1) GCT CTC TGG GAG CTG AC CGGAGGG GTACGGT CT A L W E L T G G V R S (1) GCT CTC TGG GAG T TCGG T ACC GAC AAA ALWE G TDK

(1) GCT CTC TGG GAG C CTCAT ATCGGAGGGATACGAG CTC CC GAC AAA

A L W E P H I G G I R A P D K (1) GCT CTC TGG GAG C C ATGGCA ACTCC GAGGGATACGAG CT ACC GAC AAA A L W E P W Q L R G I R A T D K (1) GCT CTC TGG GAG CTG G GGTTGGG TGG CCCCCAT ATCGGAGGG GT C GAC AAA A L W E L G L G G P H I G G V D K

(1) GCT CTC TGG GAG CTG G AG GG GGAT ATCGGAGGGATAC CTG CT ACC GAC AAA A L W E L E G D I G G I P A T D K   (1) GCT CTC TGG GAG TGG CGGAGGGATAC CCCTC CT ACC GAC AAA A L W E W R R D T P P T D K (1) GCT CTC TGG GA CAAT ATCGGAGGGATACG GG CT ACC GA A L W D N I G G I R A T (1) GCT CTC TGG GA CCAGGT ATGGC CCCCACC CGGAGGGATACGAG CT ACC GAC AAA A L W D Q V W P P P G G I R A T D K (1) GCT CTC TGG GAG CT T ATCGGAGGGATACGAG CTCCCC CT ACC GAC AAA A L W E L I G G I R A P P T D K

(1) GCT CTC TGG GAG CTG G GG GGAGGGATACGAG CCC CT ACC GAC AAA A L W E L G G G I R A P T D K (1) GCT CTC TGG GAG CTG G CGGGG CGGAGGGATA CCCCCG CT ACC GAC AAA A L W E L A G R R D T P A T D K

(1) GCT CTC TGG GAG CTG TAT ATCGGAGGG GTTG CT ACC GAC AAA A L W E L Y I G G V A T D K (1) GCT CTC TGG GAG CTG TAT ATCGGAGGGATACGAG CTA CT ACC GAC AAA A L W E L Y I G G I R A T T D K (1) GCT CTC TGG GAG CT CCG CGGAGGGATACGAG CT ACC GAC AAA A L W E L R G G I R A T D K Itk-/--4Get thymocyte stage 1 (CD122-GFP+; n=25)

(4) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E H I G G I R A T D K (1) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC A L W E H I G G I R A T (1) GCT CTC TGG GAG C GT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E R I G G I R A T D K  (1) GCT CTC TGG GAG CTG G TCGGAGGGATACGAG CT ACC GAC AAA A L W E L V G G I R A T D K

(1) GCT CTC TGG GAG CTG ATCGGAGGGATACGAG CT ACC GAC AAA A L W E L I G G I R A T D K

(2) GCT CTC TGG GAG CTG G C CGGAGGGATACGAG CT A L W E L A G G I R A

(1) GCT CTC TGG GAG CT TGAT ATCGGAGGGATACGAG CT A L W E L D I G G I R A (2) GCT CTC TGG GAG CTG G GGGATACG G CT ACC GAC AAA A L W E L G D T A T D K (1) GCT CTC TGG GAG CTG G T GGG TC CT ACC GAC AAA A L W E L V G P T D K (1) GCT CTC TGG GAG CT TCATCG AGGGATACGAG CT ACC GAC AAA A L W E L H R G I R A T D K (1) GCT CTC TGG GAG CTG G TCCTCTA CGGAG CT ACC GAC AAA A L W E L V L Y G A T D K (1) GCT CTC TGG GAG CTG G CATAT ATCGGAGGGATAC CC GAC AAA A L W E L A Y I G G I P D K   (1) GCT CTC TGG GAG CTG G ATGGC CCTT GGATCGGGGC A ACC GAC AAA A L W E L D G P W I G A T D K (1) GCT CTC TGG GAG CTC G AGCCGG TGGCATAT ATGTGG CGGAGGGATACGAG A ACC GAC AAA A L W E L E P V A Y M W R R D T R T D K (2) GCT CTC TGG GAG TTCAAT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E F N I G G I R A T D K (1) GCT CTC TGG GAG GAAT ATCGGAGGGGT G CT ACC GAC AAA A L W E E Y R R G A T D K (1) GCT CTC TGG GAG C CCTAT ATCGGAGGGATA GTCTTCT CT ACC GAC AAA A L W E P Y I G G I V F S T D K (1) GCT CTC TGG GAG CTG TCAT ATGGC ATAGGGGGGATACGAG CT ACC GAC AAA A L W E L S Y G I G G I R A T D K (1) GCT CTC TGG GAG CT ATGGCAT ATCGGAGGGATACGAG AAC CC GAC AAA A L W E L W H I G G I R E P D K

Itk-/--4Get thymocyte stage 2 (CD122+GFP+; n=33)

(9) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E H I G G I R A T D K

(6) GCT CTC TGG GAG CTG G TCGGAGGGATACGAG CT ACC GAC AAA  A L W E L V G G I R A T D K

(1) GCT CTC TGG GAG CT T ATCGGAGGGATACGAG CT ACC GAC AAA A L W E L I G G I R A T D K

(1)( GCT CTC TGG GAG CTG ATCGGAGGGATA TATG CT ACC GAC AAA A L W E L I G G I Y A T D K

(1)( GCT CTC TGG GAG CTG G C CGGAGGGATAC CCG CT ACC GAC AAA A L W E L A G G I P A T D K

((2) GCT CTC TGG GAG CT CGC CGGAGGGATACG GG CT ACC GAC AAA A L W E L A G G I R A T D K ((1) GCT CTC TGG GAG C CGGAT ATCGGAGGGATACGAG CT A L W E P D I G G I R A (2)( GCT CTC TGG GAG CTG G AT ATCGGAGGG T CT ACC GAC AAA A L W E L D I G G S T D K ((1) GCT CTC TGG GAG CTG G AAA GAGGGATACGAG CT ACC GAC AAA   A L W E L E R G I R A T D K (1)( GCT CTC TGG GAG CTG G AGGGGG AT ATCGGAGGGATACCTG CT ACC GAC AAA

A L W E L E G D I G G I P A T D K

(2)( GCT CTC TGG GAG C TC CAT ATCGGAGGGATACGAG CT GCC GAC AAA’ A L W E L H I G G I R A A D K

(2)( GCT CTC TGG GAG C TG TAT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E L Y I G G I R A T D K (1)( GCT CTC TGG GA CCAG ATAT GGCCCCCACC CGGAGGGATACGAG CT ACC GAC AAA A L W D Q I W P P P G G I R A T D K (2)( GCT CTC TGG GAG CTG G TCCCAGCC GAG CTCG CC GAC AAA A L W E L V P A E L A D K (1)( GCT CTC TGG GAG CT CGGAGGGATACCCC CT G CT ACC GCC AGA A L W E L G G I P P A T A R Itk-/--4Get thymocyte stage 3 (CD122+GFP-; n=18)

(8) GCT CTC TGG GAG C AT ATCGGAGGGATACGAG CT ACC GAC AAA  A L W E H I G G I R A T D K (3) GCT CTC TGG GAG CTG G C CGGAGGGATACGAG CT ACC GAC AAA A L W E L A G G I R A T D K

(1) GCT CTC TGG GAG T GGAGGGATACGAG CC CC GAC AAA A L W E W R D T S P D K (1) GCT C C AT ATCGGAGGGATACG GAA A L W E L P Y R

(2) GCT CTC TGG GAG CT CCCAT ATCGGAGGGATACG GAA A L W E L P Y R R D T E   (1) GCT CTC TGG GAG CT C T ATAT GGCCCCT ATCGGAGGGATACGAG CT ACC GAC AAA A L W E L Y M A P I G G I R A T D K

(1) GCT CTC TGG GAG C CAG ATAT C GGG G CT ACC GAC AAA A L W E P D I G A T D K (1) GCT CTC TGG GAG C CTTCT ATCGGAGGGATACGA AT C GAC AAA A L W E P S I G G I R I D K

Supplemental Figure 1: Sequence analysis of Vδ6-Jδ1 junctions in γδNKT cells from the

-/- liver and thymus of WT and Itk mice.

At the top, the germline sequences are shown. Below, the junctional regions of Vδ6 (variable

region), Dδ1 and 2 (diversity region) and Jδ1 (joining region) rearrangements in liver and

thymus subsets are shown. Only rearrangements resulting in in-frame junctions are included.

The frequency (left) indicates the number of independent clones with each sequence. Junctions

were characterized as fetal or adult based on the absence or presence of Dδ1 sequences and N-

region nucleotides, respectively. In addition, sequences classified as fetal were confirmed based

on comparison to those reported by Grigoriadou, et al (1) as being derived from isolated fetal V6

cells.

1. Grigoriadou, K., L. Boucontet, and P. Pereira. 2003. Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors. J Immunol 171: 2413–2420. A B WT 2m-/- 105 10 12

104

p=0.0151 # V6 T cells # V6 T cells

HSA 8 62 31 41 PLZF 103 WT 2m-/- WT MHC2-/- 105 11 28

104 # V6 T cells 49 33

HSA 8 11 PLZF 103 WT MHC2-/-

Supplemental Figure 2. Neither2m-dependent MHC class I molecules nor MHC class II molecules are not required for the development of V6 cells. (A) Thymocytes from WT, 2m-/- and MHC2-/- V6 cells were examined for HSA versus PLZF expression. Dot-plots show representative data. (B) Graphs show a compilation of total thymic V6 cells in WT, 2m-/- and MHC2-/- mice. Statistically significant differences are shown with p values. Data are representative of at least 2 independent experiments. -/- Table 1: List of differentially expressed genes between WT and Itk mature V6 thymocytes. -/- List of the 195 genes that were increased or decreased in Itk V6 thymocytes compared to WT CD24lo V6 thymocytes. Nomenclature is according to immgen.org: MatV6.WT= CD24(HSA)lo V6 -/- γδ T cells from adult WT thymus; TotalV6.Itk-/-, total V6 γδ T cells from adult Itk thymus, which are 95-98% CD24lo. Genes were classified as being increased if the mean expression value (MEV) was changed by 2-fold or more in pair wise comparisons with a coefficient of variation among replicates <0.5, t-test P-value<0.05, and MEV>120 in at least one population. For each gene, the MEV in each population is shown, along with the fold change (FC, ratio of MEVs) and t-test P- values. Fractional FC values indicative of decreased expression were converted to whole numbers (-1/FC). Table was sorted by the FC in ascending order.

Name MatV6.WT Total V6.Itk-/- FC Total V6.Itk-/-/MatV6 t-test Total V6.Itk-/-/MatV6 Klra5 606.58 11.54 -52.56 0.0010557 Klrc1 575.27 28.56 -20.14 0.0000060 Klra6 252.79 25.40 -9.95 0.0016892 Fcer1g 757.52 76.45 -9.91 0.0001625 Klrc2 314.20 31.90 -9.85 0.0024922 Lrrc6 180.94 20.28 -8.92 0.0213632 Ccl5 726.60 91.14 -7.97 0.0374020 Klrd1 631.61 82.92 -7.62 0.0000130 Blk 981.73 129.00 -7.61 0.0000098 Itga1 191.72 27.91 -6.87 0.0011449 Klrk1 457.32 66.83 -6.84 0.0012523 Cd244 262.96 38.81 -6.78 0.0136412 Klrb1f 421.95 62.47 -6.75 0.0002129 Ncr1 142.58 21.59 -6.60 0.0106565 Jun 545.81 90.48 -6.03 0.0276376 Ccr10 667.55 115.88 -5.76 0.0005923 Nkg7 1413.22 257.11 -5.50 0.0010667 Xcl1 406.83 77.05 -5.28 0.0002279 Epha3 142.61 28.22 -5.05 0.0343729 Myo1f 713.09 143.31 -4.98 0.0007780 Slamf7 356.80 72.42 -4.93 0.0000429 Styk1 274.89 61.21 -4.49 0.0000092 Slc9a7 174.81 39.14 -4.47 0.0002986 Sulf2 229.41 51.77 -4.43 0.0029345 Klrb1a 179.61 43.37 -4.14 0.0006197 Spp1 128.78 31.26 -4.12 0.0204007 Il21 161.52 39.73 -4.07 0.0076207 Ccr8 760.04 191.17 -3.98 0.0002209 Klrb1b 170.35 43.26 -3.94 0.0001500 Neurl3 257.75 65.94 -3.91 0.0002747 Ifng 167.13 44.39 -3.77 0.0087229 Il18rap 617.53 164.24 -3.76 0.0003828 Trps1 135.33 36.80 -3.68 0.0001248 Klra9 427.69 116.40 -3.67 0.0000355 A430084P05Rik 168.29 45.95 -3.66 0.0000021 Spock2 381.08 105.12 -3.63 0.0038692 Cd7 1718.07 477.23 -3.60 0.0000656 Stat4 418.30 117.76 -3.55 0.0001689 Klra10 433.45 124.98 -3.47 0.0000105 Name MatV6.WT Total V6.Itk-/- FC Total V6.Itk-/-/MatV6 t-test Total V6.Itk-/-/MatV6 Vdr 946.32 274.60 -3.45 0.0000512 Serpina3f 129.97 37.75 -3.44 0.0059606 Il2rb 2382.43 727.38 -3.28 0.0011186 Lag3 241.59 73.97 -3.27 0.0001228 Ahr 332.79 103.61 -3.21 0.0003202 Dennd4a 311.74 97.54 -3.20 0.0000671 Tm6sf1 661.31 211.55 -3.13 0.0018068 Rnu3b1 3624.65 1174.16 -3.09 0.0007692 Lat2 187.27 61.24 -3.06 0.0003966 Snord118 221.24 73.72 -3.00 0.0108502 Itga4 1075.93 359.05 -3.00 0.0033366 Cpne7 285.40 95.91 -2.98 0.0019574 Arl4d 274.38 94.09 -2.92 0.0091520 H19 196.42 67.36 -2.92 0.0298369 Fasl 178.06 63.37 -2.81 0.0003674 Lyn 223.84 79.74 -2.81 0.0007855 Fam20a 290.79 104.86 -2.77 0.0000514 Lpar6 1178.25 427.59 -2.76 0.0011545 Gpr177 194.75 70.93 -2.75 0.0007785 Plscr1 368.91 134.80 -2.74 0.0014697 Cmah 137.25 50.43 -2.72 0.0082426 Maged1 127.54 47.00 -2.71 0.0023434 Ikzf3 1046.56 386.01 -2.71 0.0015563 Rnu3a 1028.04 381.58 -2.69 0.0262510 Il10ra 130.51 48.55 -2.69 0.0008816 F2r 1152.13 429.83 -2.68 0.0002247 Cx3cr1 239.56 90.49 -2.65 0.0084698 Scpep1 191.05 73.02 -2.62 0.0000780 Emp3 750.95 289.51 -2.59 0.0008284 Sox4 314.39 122.27 -2.57 0.0018326 Srgap3 144.14 56.99 -2.53 0.0070167 Hist1h2bg 305.89 121.55 -2.52 0.0004475 Slc15a2 148.60 59.43 -2.50 0.0010909 Tbx21 494.19 197.80 -2.50 0.0002969 Ly6c2 1082.55 435.77 -2.48 0.0003159 Gzmb 633.86 255.90 -2.48 0.0008128 Nr4a1 616.33 248.83 -2.48 0.0006337 Fosb 168.15 68.19 -2.47 0.0479280 Samd3 269.60 110.31 -2.44 0.0001055 Sccpdh 282.67 116.33 -2.43 0.0008605 Tmem176b 278.99 114.90 -2.43 0.0004377 Ikzf2 835.81 344.31 -2.43 0.0012362 Xdh 390.25 161.42 -2.42 0.0002561 2010002N04Rik 146.93 60.90 -2.41 0.0017150 Itgae 1372.08 570.78 -2.40 0.0003836 A430093F15Rik 567.11 236.90 -2.39 0.0131224 Thbs1 236.69 99.04 -2.39 0.0000006 Cysltr2 138.97 58.38 -2.38 0.0011334 I730030J21Rik 632.35 266.79 -2.37 0.0004160 Angptl2 263.64 112.22 -2.35 0.0000469 Lgals1 3635.99 1551.19 -2.34 0.0000039 Name MatV6.WT Total V6.Itk-/- FC Total V6.Itk-/-/MatV6 t-test Total V6.Itk-/-/MatV6 Trf 510.17 218.57 -2.33 0.0021964 Arap3 439.53 189.63 -2.32 0.0012150 Lass6 144.92 63.03 -2.30 0.0027767 Anxa6 2366.86 1040.20 -2.28 0.0006272 Emp1 206.61 90.97 -2.27 0.0011383 Nrp1 317.76 140.73 -2.26 0.0061755 Crim1 411.38 183.74 -2.24 0.0003377 Sidt1 397.81 179.59 -2.22 0.0023735 Spry2 845.57 381.82 -2.21 0.0007383 Ifngr1 757.30 343.46 -2.20 0.0010733 Rxra 360.48 163.55 -2.20 0.0048260 Ubash3b 660.65 304.03 -2.17 0.0000687 Ms4a4b 685.57 318.49 -2.15 0.0030471 Gpr114 453.28 210.77 -2.15 0.0090368 H2-Q6 1405.72 655.29 -2.15 0.0000005 Atp5d 1706.72 799.55 -2.13 0.0000852 Cd1d1 357.90 168.53 -2.12 0.0021637 Gpr52 132.88 62.71 -2.12 0.0216561 Maf 795.65 375.64 -2.12 0.0009779 Tagap 830.13 392.10 -2.12 0.0016755 Prkcb 405.55 191.74 -2.12 0.0030816 Ehd4 266.80 126.90 -2.10 0.0000278 Tirap 402.75 193.28 -2.08 0.0025335 Tns4 146.50 70.36 -2.08 0.0064425 Rnu1b1 538.16 258.71 -2.08 0.0055281 5830411N06Rik 444.51 214.56 -2.07 0.0056417 Prex1 879.49 424.61 -2.07 0.0008968 Klf2 591.97 291.58 -2.03 0.0004505 Adamts10 452.36 223.25 -2.03 0.0043571 Otud1 292.70 144.89 -2.02 0.0000232 Tnfsf10 519.72 258.72 -2.01 0.0012965 Dad1 1040.71 518.59 -2.01 0.0005191 Slc25a38 393.85 196.41 -2.01 0.0012226 Rorc 206.50 103.16 -2.00 0.0002276 1110002B05Rik 792.39 1589.13 2.01 0.0184379 Rps3a 2281.02 4591.84 2.01 0.0015101 Pttg1 205.19 413.76 2.02 0.0002329 Cdkn1b 292.66 591.26 2.02 0.0304524 BC002163 156.48 318.13 2.03 0.0016174 Akap6 189.94 387.76 2.04 0.0001164 AI747699 162.62 334.80 2.06 0.0025647 Gm10785 67.18 139.14 2.07 0.0033010 Mpp6 281.16 585.54 2.08 0.0000206 Gm7239 141.52 296.50 2.10 0.0394281 Gm13194 68.68 145.16 2.11 0.0025320 Rpl35a 1196.90 2554.14 2.13 0.0024201 Hectd2 112.45 242.97 2.16 0.0330727 St8sia1 222.18 482.05 2.17 0.0001005 Gm9197 994.12 2202.35 2.22 0.0001031 Impdh2 294.13 652.69 2.22 0.0000299 2010016I18Rik 118.21 263.02 2.23 0.0112077 Name MatV6.WT Total V6.Itk-/- FC Total V6.Itk-/-/MatV6 t-test Total V6.Itk-/-/MatV6 Gm7693 62.91 141.15 2.24 0.0059760 Rnf7 103.18 232.59 2.25 0.0192755 Gm8936 206.21 465.75 2.26 0.0152089 Hist1h1b 1318.93 3015.55 2.29 0.0117301 Gm6134 331.75 758.86 2.29 0.0000140 Grm4 53.33 123.55 2.32 0.0005304 BC005685 407.02 945.25 2.32 0.0053203 Spc25 160.39 374.62 2.34 0.0075292 Tex2 427.33 999.45 2.34 0.0001571 Fam169b 94.11 220.45 2.34 0.0000470 Robo1 57.14 134.01 2.35 0.0007106 B930036N10Rik 210.75 500.86 2.38 0.0071539 Vegfa 74.56 178.74 2.40 0.0057285 Rps19 548.43 1319.58 2.41 0.0000366 Fam46c 118.20 284.78 2.41 0.0013337 Plce1 50.35 121.60 2.42 0.0011939 Sgk3 246.65 607.50 2.46 0.0009173 D10Ertd322e 57.58 143.80 2.50 0.0017007 Gm6983 90.93 227.19 2.50 0.0045592 LOC641050 250.49 626.57 2.50 0.0008188 Cox6a1 552.67 1403.36 2.54 0.0000628 Utp3 181.54 461.32 2.54 0.0000084 Hc 69.27 176.93 2.55 0.0022111 Aldh2 147.82 379.00 2.56 0.0016297 Ccr2 521.37 1362.91 2.61 0.0008710 Smpdl3b 54.67 143.30 2.62 0.0001684 Lrig1 138.28 364.03 2.63 0.0000121 LOC625360 150.83 398.74 2.64 0.0009480 Pde7b 59.64 162.00 2.72 0.0007030 Trim36 247.95 673.74 2.72 0.0000485 Tmie 130.23 371.28 2.85 0.0000807 Themis 162.95 467.02 2.87 0.0000768 Nrn1 49.83 144.73 2.90 0.0143701 Atp8a2 269.36 790.60 2.94 0.0025624 Adh1 583.07 1767.54 3.03 0.0034993 Ass1 510.21 1693.13 3.32 0.0025969 Gm12643 346.02 1167.90 3.38 0.0028830 Rps6ka2 86.03 291.21 3.38 0.0000609 Rpl22l1 191.16 694.60 3.63 0.0032522 Gm10673 84.39 315.49 3.74 0.0278930 Ctla2a 109.64 413.21 3.77 0.0190028 Slc15a1 109.04 425.47 3.90 0.0043181 Gadl1 279.47 1170.05 4.19 0.0013129 Asns 72.29 312.27 4.32 0.0008115 Atp6v0d2 34.53 149.56 4.33 0.0073007 Fam84a 68.19 313.77 4.60 0.0012338 Aqp9 110.77 520.86 4.70 0.0004120 Cd4 311.11 1624.03 5.22 0.0006427 Lztfl1 356.46 1917.36 5.38 0.0000484 Ccr9 379.30 2109.04 5.56 0.0000886 Gpnmb 203.37 1236.47 6.08 0.0000103 Name MatV6.WT Total V6.Itk-/- FC Total V6.Itk-/-/MatV6 t-test Total V6.Itk-/-/MatV6 Art2b 347.96 2138.62 6.15 0.0000559 Tdgf1 139.25 926.29 6.65 0.0001251 1810011H11Rik 104.81 863.26 8.24 0.0002451

-/- Supplemental Table 2: Itk V6 thymocytes are similar to early stage NKT cells.

List of the 51 genes identified in Figure 5C that were decreased in Itk-/- V6 thymocytes versus WT CD24lo V6 cells and increased in NKT.44+NK1.1+ cells versus MatCD4 and MatCD8 single positive thymoctyes. Nomenclature is according to immgen.org: MatV6.WT= CD24(HSA)lo V6 γδ T cells from adult WT thymus; -/- TotalV6.Itk-/-, total V6 γδ T cells from adult Itk thymus, which are 95-98% CD24lo; NKT.44+NK1.1+, CD44+NK1.1+ thymic iNKT cells; NKT.44-NK1.1-, CD44-NK1.1- thymic iNKT cells; MatCD4, CD24lo CD4+8- thymocytes; MatCD8, CD24lo CD4-8+ thymocytes. For each gene, the MEV in each population is shown, along with the fold change (FC, ratio of MEVs) and t-test P-values for select populations. Fractional FC values indicative of decreased expression were converted to whole numbers (-1/FC). Table was sorted by the FC in NKT.44+NK1.1+ vs. NKT.44-NK1.1- in descending order. Increased FCs are in red, and decreased FC are in blue. Only those FC that had an accompanying t-test P-value<0.05 were color coded.

FC FC NKT.44+ NKT.44- Total NKT.44+ NKT.44+NK1.1+/ MatV6.WT/ Name NK1.1- NK1.1- V6.Itk-/- MatCD4 ImmV6.WT NK1.1+ MatV6.WT NKT.44-NK1.1- Total V6.Itk-/- Gzmb 263.52 59.63 55.90 35.89 53.79 3690.25 633.86 62.50 -11.34 Klra9 183.62 36.80 116.40 14.88 20.31 2048.93 427.69 55.56 -3.67 Klra10 186.67 29.15 124.98 11.28 19.54 1327.23 433.45 45.45 -3.47 Klra5 101.50 44.49 11.54 9.61 12.89 1711.04 606.58 38.46 -52.56 Styk1 111.64 45.74 61.21 45.24 47.87 1697.79 274.89 37.04 -4.49 Klrc1 130.69 44.84 28.56 20.23 31.17 1424.19 575.27 32.26 -20.14 Itga1 51.89 25.94 27.91 24.79 21.61 591.77 191.72 22.73 -6.87 Klra6 90.35 34.96 25.40 16.39 23.13 619.46 252.79 17.86 -9.95 Xcl1 176.18 77.30 77.05 55.18 166.14 1303.42 406.83 16.95 -5.28 Fasl 184.63 66.38 63.37 55.58 46.53 1008.42 178.06 15.15 -4.93 Slamf7 192.86 87.09 72.42 37.99 84.65 1309.85 356.80 15.15 -2.81 Klrc2 87.01 70.41 31.90 20.25 29.30 949.16 314.20 13.51 -9.85 Klrd1 217.81 78.10 82.92 22.27 56.34 961.66 631.61 12.35 -7.62 Myo1f 563.94 161.47 143.31 78.90 276.54 1852.20 713.09 11.49 -4.98 Klrk1 121.80 87.51 66.83 19.13 23.52 966.89 457.32 10.99 -6.84 Fcer1g 101.12 105.67 76.45 38.62 357.52 1035.02 757.52 9.80 -9.91 Ccl5 169.33 98.25 91.14 98.04 154.38 930.92 726.60 9.43 -7.97 Dennd4a 239.29 160.97 97.54 115.89 150.21 1431.71 311.74 8.93 -3.20 Il10ra 62.11 52.75 48.55 141.87 363.33 446.93 130.51 8.47 -2.69 Klrb1f 130.38 41.67 62.47 29.22 46.70 292.94 421.95 7.04 -6.75 Ly6c2 782.17 260.58 435.77 234.46 172.49 1711.63 1082.55 6.58 -2.48 Samd3 277.45 99.95 110.31 32.64 34.21 640.24 269.60 6.41 -2.44 Crim1 268.01 195.00 183.74 457.31 276.51 1229.05 411.38 6.29 -2.24 Ifngr1 711.05 482.87 343.46 813.36 909.23 2998.52 757.30 6.21 -2.20 Klrb1b 115.96 42.22 43.26 30.15 30.45 253.19 170.35 5.99 -3.94 Cd244 53.62 56.66 38.81 44.51 82.91 336.02 262.96 5.92 -6.78 Sulf2 69.77 75.00 51.77 52.67 70.43 432.60 229.41 5.78 -4.43 Il18rap 786.26 241.39 164.24 47.52 54.57 1357.44 617.53 5.62 -3.76 Tbx21 575.14 335.66 197.80 69.89 99.37 1877.19 494.19 5.59 -2.50 Arl4d 220.58 133.31 94.09 62.78 62.30 691.39 274.38 5.18 -2.92 Cd7 1280.02 774.48 477.23 783.08 817.55 3825.89 1718.07 4.95 -3.60 Serpina3f 52.08 40.76 37.75 32.14 41.72 175.78 129.97 4.31 -3.44 Il2rb 1723.56 1284.87 727.38 684.43 2299.61 4968.56 2382.43 3.86 -3.28 A430084P05Rik 71.78 51.08 45.95 35.62 63.50 167.25 168.29 3.28 -3.66 Ahr 328.16 182.64 103.61 104.19 923.03 592.53 332.79 3.25 -3.21 Lpar6 1060.02 553.08 427.59 740.23 887.38 1786.63 1178.25 3.23 -2.76 H2-Q6 740.39 885.13 655.29 427.18 376.41 2510.40 1405.72 2.83 -2.15 Lyn 93.49 86.44 79.74 43.15 238.16 228.27 223.84 2.64 -2.81 Spry2 313.62 296.88 381.82 57.65 572.98 775.98 845.57 2.61 -2.21 Trf 260.06 220.26 218.57 68.80 215.24 562.94 510.17 2.56 -2.33 Srgap3 108.10 75.43 56.99 45.39 128.21 188.20 144.14 2.49 -2.53 Cysltr2 145.21 67.45 58.38 16.35 82.75 163.27 138.97 2.42 -2.38 Tnfsf10 221.19 210.02 258.72 126.64 384.06 442.01 519.72 2.11 -2.01 Arap3 176.37 187.67 189.63 88.27 190.49 383.85 439.53 2.04 -2.32 Lat2 64.84 65.44 61.24 48.83 237.97 125.49 187.27 1.92 -3.06 Cpne7 101.71 76.28 95.91 68.70 65.72 140.51 285.40 1.84 -2.98 F2r 959.80 550.47 429.83 150.66 214.43 908.59 1152.13 1.65 -2.68 Ehd4 204.87 147.56 126.90 77.87 282.90 221.90 266.80 1.50 -2.10 Maf 520.84 421.32 375.64 134.46 174.88 469.98 795.65 1.12 -2.12 Tirap 182.08 147.07 193.28 50.91 67.98 124.10 402.75 -1.19 -2.08 Blk 635.73 504.69 129.00 65.88 297.87 163.01 981.73 -3.13 -7.61