Ikaros Null Mice Display Defects in T Cell Selection and CD4 versus CD8 Lineage Decisions

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1 This information is current as of February 23, Ikaros Null Mice Display Defects in T Cell Selection and CD4 versus CD8 Lineage Decisions Julie A. Urban and Susan Winandy J Immunol 2004; 173: ; ; References Subscriptions Permissions Alerts This article cites 33 articles, 17 of which you can access for free at: Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD Copyright 2004 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology Ikaros Null Mice Display Defects in T Cell Selection and CD4 versus CD8 Lineage Decisions 1 Julie A. Urban and Susan Winandy 2 Previous evidence suggested that the hemopoietic-specific nuclear factor Ikaros regulates TCR signaling thresholds in mature T cells. In this study, we test the hypothesis that Ikaros also sets TCR signaling thresholds to regulate selection events and CD4 vs CD8 lineage determination in developing thymocytes. Ikaros null mice were crossed to three lines of TCR-transgenic mice, and positive selection, negative selection, and CD4 vs CD8 lineage decisions were analyzed. Mice expressing a polyclonal repertoire or a MHC class II-restricted TCR transgene exhibited enhanced positive selection toward the CD4 lineage. Moreover, in the absence of Ikaros, CD4 development can occur with decreased thresholds of TCR signaling. In addition, CD4 single-positive thymocytes were detected in MHC class I-restricted TCR-transgenic Ikaros null mice. To assess the role of Ikaros in negative selection, we analyzed deletion of T cells induced by conventional Ag or by endogenous superantigen. Surprisingly, negative selection was impaired in Ikaros null thymocytes despite evidence of high levels of TCR signal and no intrinsic defect in apoptosis ex vivo. To our knowledge, these data identify Ikaros as the first nuclear factor that plays a critical role in regulating negative selection as well as CD4 vs CD8 lineage decisions during positive selection. The Journal of Immunology, 2004, 173: Development of functional T cells from committed progenitors is driven by TCR-mediated events, positive selection and negative selection, which occur in the thymus (1). Positive and negative selection occur at the immature CD4 CD8 or double-positive (DP) 3 stage of T cell development, and both rely upon TCR specificity for self-mhc:self-peptide. Positive selection is mediated by interaction of TCR with self-ag presented by cortical thymic epithelial cells. It is postulated that there is a threshold of affinity for this interaction that results in the delivery of survival and differentiation signals. If the affinity/avidity of TCR:MHC/self-peptide interaction falls below this threshold, the DP thymocyte dies by apoptosis. This process ensures that only thymocytes that express TCRs that are able to recognize Ag in the context of self-mhc are allowed to develop. In addition, concomitant with positive selection, CD8 or CD4 lineage fate is determined by whether the TCR expressed on the DP thymocyte interacts with Ag in the context of MHC class I or MHC class II, respectively (2). In addition, strength of the TCR signal has been proposed to be important in this decision, with a stronger signal promoting the CD4 lineage decision (3, 4). Negative selection, in contrast, ensures that T cells which express TCRs that are able to interact with self-mhc/self-peptide with very high affinity/avidity are eliminated by apoptosis, since they would pose the threat of autoimmunity. Negative selection, Department of Microbiology-Immunology, Northwestern University, Feinberg School of Medicine, Chicago, IL Received for publication April 6, Accepted for publication July 26, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Biomedical Research Support Program Award from the Howard Hughes Medical Institute. J.A.U. is supported by Public Health Service Grant 5 T32 AI Address correspondence and reprint requests to Dr. Susan Winandy, Department of Microbiology-Immunology, Northwestern University, Feinberg School of Medicine, 320 East Superior Street, Morton 6-639, Chicago, IL address: s-winandy@northwestern.edu 3 Abbreviations used in this paper: DP, double positive; CD40-L, CD40 ligand; SP, single positive; Tg, transgenic; Mtv, mammary tumor virus. like positive selection, is mediated by interaction of TCRs on the surface of DP thymocytes with self-mhc/self-peptide. However, there are three crucial differences. First, the cells that present Ag responsible for mediating negative selection are thymic medullary epithelial cells and thymic dendritic cells (5, 6). Second, negative selection is the result of higher affinity-avidity interactions than those that result in positive selection. Third, it is believed that negative selection may also depend upon interaction of costimulatory molecules on the surface of the thymocyte with their ligands on the surface of the APCs. The definitive identity of these costimulatory molecules has remained elusive, although CD28 and CD40 ligand (CD40-L) have been proposed as candidates (7 11). Much progress has been made toward identification of cytoplasmic signaling pathways initiated by TCR complex engagement that play central roles in positive and negative selection. In particular, intact ERK and calcineurin pathways are essential for positive selection, whereas JNK and p38 signaling pathways are uniquely involved in negative selection (5, 12, 13). Much less progress has been made toward defining the critical nuclear factors whose functions are regulated by these pathways, leading to changes in gene expression resulting in survival, differentiation, or apoptosis. These nuclear factors interpret the signaling thresholds that differentiate between low- and high-affinity/avidity interactions. Therefore, they play a central role in determining whether a DP thymocyte will undergo positive or negative selection, as well as whether they will attain the CD4 or CD8 fate. One candidate for a nuclear protein that plays an important role in T cell development through regulation of selection events is the Ikaros protein. Ikaros is expressed almost exclusively in cells of the hemopoietic and lymphoid lineages and is expressed at its highest levels in developing thymocytes and mature T cells. Ikaros null mice display multiple defects in T cell development (14). First, all waves of fetal T cell development are absent. Postnatally, a reduced number of TCR T cells differentiate in the thymus (3- to 9-fold fewer than Ikaros wild-type thymi). However, differentiation is abnormal, resulting in an increased percentage of CD4 single-positive (SP) thymocytes. Moreover, mature Ikaros null T cells, as well as those with reduced levels of Ikaros activity, are hyperresponsive to TCR signaling (14 16). This phenotype has Copyright 2004 by The American Association of Immunologists, Inc /04/$02.00

3 The Journal of Immunology 4471 been linked to reduced thresholds of signaling required for proliferation, suggesting that Ikaros sets thresholds of activation for mature T cells (16). We hypothesize that Ikaros may also set thresholds of TCR signaling during T cell development in the thymus. If this is the case, defects in the TCR-mediated selection events, positive and negative selection, should be observed in the absence of Ikaros activity. In this report, we demonstrate that thresholds of TCR signaling strength required for positive selection are lowered in Ikaros null mice, resulting in enhanced positive selection toward the CD4 lineage. We also document a defect in negative selection in Ikaros null thymocytes. Surprisingly, although Ikaros null thymocytes are receiving high levels of TCR signal, negative selection does not occur. Therefore, this report defines Ikaros as the first nuclear factor involved in regulating negative selection as well as CD4 vs CD8 lineage decisions during positive selection. Materials and Methods Mice Ikaros null mice of the H-2 b haplotype (C57BL/6 Sv129) were crossed to F5 (kind gift from K. Georgopoulos, Massachusetts General Hospital, Charlestown, MA) and H-Y TCR-transgenic (Tg) (The Jackson Laboratory, Bar Harbor, ME) mice. Ikaros null mice were also crossed onto the H-2 d haplotype and DO11.10 TCR Tg (kind gift from Dr. S. Miller, Feinberg School of Medicine, Northwestern University, Chicago, IL). All TCR Tg mice were crossed to Rag1 / to prevent expression of endogenous TCR. For endogenous superantigen experiments, Ikaros null mice were backcrossed to BALB/c mice for at least five generations. Genotypes were analyzed by PCR analysis using tail DNA as previously described (14). Mice were analyzed between 3 and 4 wk of age using age-matched controls. Mice were bred and maintained in the Northwestern University Center for Comparative Medicine (Chicago, IL). All animal studies were approved by Northwestern University s Animal Care and Use Committee. Antibodies The following Abs were used for flow cytometry analysis: anti-cd4 (GK1.5), anti-cd8 (53-6.7), anti-tcr (H57-597), anti-cd5 (53-7.3), anti- CD69 (H1.2F3), anti-cd40-l (MR1), anti-v 8.1/8.2 (MR5; BD Biosciences, Mountain View, CA), anti-v 12 (Mr11-1; BD Biosciences), and anti-v 3 (KJ25). All Abs were purchased from ebioscience (San Diego, CA) unless otherwise stated. Abs were used as allophycocyanin, FITC, or PE conjugates. Cell preparation, staining, and flow cytometry Lymphocyte suspensions were made from thymus and spleen by dissociation between two frosted slides in RPMI 1640/10% FCS/500 U/ml each of penicillin and streptomycin/50 M 2-ME (RPMI 1640 complete). RBC were lysed in RBC lysis buffer (0.144 M NH 4 Cl/0.017 M Tris, ph 7.65) for 5 min at room temperature. Cells were washed and resuspended in PBS/2% FCS/1 mm EDTA and plated in microwell staining plates at cells/well. FcRs were blocked by incubating with rat serum and anti- CD16/CD32 (ebioscience) before staining. Directly conjugated Abs were added to cells and incubated on ice for min, then washed and fixed in 75 l of 2% paraformaldehyde. Fixed cells were analyzed h later on a FACSCalibur (BD Biosciences) flow cytometer. Analyses were performed on CellQuest Pro software. CD40-L expression assays Flat-bottom 96-well plates were coated with 75 l of anti-cd3 and anti- CD28 at 10 g/ml each in PBS or PBS alone overnight at 4 o C. Plates were washed three times with medium before plating cells. Freshly isolated thymocytes were cultured at cells/well in 200 l of RPMI 1640 complete in the presence or absence of plate-bound anti-cd3 plus anti- CD28 Abs for 4hat37 o C. Cells were then stained with fluorochromeconjugated Abs against CD4, CD8, and CD40-L, followed by analysis on a FACSCalibur (BD Biosciences) flow cytometer. Ex vivo negative selection assays Freshly isolated thymocytes were cultured at cells/well in 200 l of RPMI 1640 complete in flat-bottom 96-well plates in the presence or absence of plate-bound anti-cd3 and anti-cd28 Abs for 24 h at 37 o C. Plates were coated with 75 l of anti-cd3 and anti-cd28 at 10 g/ml each in PBS or PBS alone overnight at 4 o C. Plates were washed three times with medium before plating cells. Cells were then stained with fluorochrome-conjugated Abs against CD4 and CD8. In addition, to detect apoptotic cells, the Annexin V PE Apoptosis Detection Kit I (BD Biosciences) was used. Cells were stained according to the manufacturer s protocol. They were then analyzed on a FACSCalibur (BD Biosciences) flow cytometer. Results Increased proportion of CD4 SP thymocytes develop in Ikaros null thymuses Previous studies have shown that an increased percentage of CD4 T cells are observed in the thymi of Ikaros null mice (14). In our hands, the Ikaros null thymus contained a 3- to 4-fold greater proportion of CD4 SP T cells accompanied by a corresponding decrease in the DP population (Fig. 1, A and C). The increased proportion of CD4 T cells is not the result of abnormal proliferation since Ikaros null thymocytes show similar cell cycle profiles as their wild-type counterparts (data not shown). No intermediate phenotype is observed in Ikaros null heterozygotes (Ikaros / ), indicating that 50% of Ikaros activity is sufficient to retain normal CD4 vs CD8 ratios. The increased proportion of SP thymocytes is unique to the CD4 lineage since the proportion of CD8 SP cells in Ikaros null thymi is similar to that observed in wild-type and Ikaros / thymi (Fig. 1, A and C). A comparison of absolute numbers of thymocyte populations in Ikaros null and Ikaros wild-type mice reveals that all are decreased in the Ikaros null thymus, with the least difference in absolute numbers observed in the CD4 SP subset (Fig. 1D). The increased proportion of CD4 SP thymocytes was also observed when Ikaros null Rag1 / mice were backcrossed onto the DO11.10 TCR Tg background and the H-2 d haplotype. The DO11.10 TCR is a MHC class II-restricted TCR specific for chicken OVA peptide in the context of I-A d that exclusively directs development of CD4 T cells (17). Because the Rag1 gene product is necessary for TCR rearrangement, thymocytes can express only the DO11.10 TCR. DO11.10, Ikaros null Rag1 / thymi contain an average 5- to 6-fold higher percentage of CD4 SP cells than their Ikaros wild-type counterparts (Fig. 1, B and C). Taken together, these data provide evidence that with either a polyclonal or a MHC class II-restricted TCR repertoire, more efficient positive selection toward the CD4 lineage is observed in the absence of Ikaros activity. Ikaros null CD4 SP thymocytes are mature and functional To determine whether Ikaros null CD4 SP thymocytes are mature, we compared the expression of maturation markers on the surfaces of Ikaros null CD4 SP thymocytes to their DP counterparts. Expression of TCR, CD5, and CD69 are up-regulated upon TCR engagement as cells transit from the DP to the SP stage and, therefore, are markers of successful positive selection (2, 18, 19). There is an up-regulation of TCR and CD5 on Ikaros null CD4 SP thymocytes relative to their DP counterparts as observed in Ikaros wild-type thymocytes (Fig. 1E). Up-regulation of CD69 is also observed although the percentage of CD4 SP thymocytes expressing this maturation marker at any given time is less than that observed in wild-type thymi, perhaps due to the transient nature of CD69 up-regulation as compared with that of TCR and CD5 (Fig. 1E). Significantly, however, the ratio of splenic to thymic CD4 T cells is not statistically different in Ikaros null vs wild-type mice (Ik /, ; Ik null, ), suggesting that equivalent proportions of CD4 SP thymocytes are being exported to the periphery. Similar results were obtained with the DO11.10, Ikaros null Rag1 / CD4 SP thymocytes (data not shown). This is

4 4472 IKAROS NULL MICE DISPLAY DEFECTS IN T CELL SELECTION CD40-L expression upon TCR stimulation has been used as a test of helper activity in CD4 T cells, and, therefore, is a measure of functionality (22, 23). Ikaros null and DO11.10, Ikaros null Rag1 / thymocytes and their Ikaros wild-type counterparts were stimulated with plate-bound anti-cd3 plus anti-cd28 and, 4 h later, CD40-L expression on the surface of thymocyte subsets was assessed. As expected, Ikaros null as well as Ikaros wild-type DP and CD8 SP T cells showed no up-regulation of CD40-L (data not shown). In contrast, within the Ikaros null thymocyte populations, an increased ratio of CD4 SP thymocytes to total thymocytes displayed CD40-L up-regulation compared with their wild-type counterparts (Fig. 2). Taken together, these data support the idea that the overall percentage of mature, functional CD4 T cells is increased in the absence of Ikaros (Fig. 2B). FIGURE 1. Increased percentage of CD4 SP thymocytes in Ikaros null mice represent mature CD4 T cells. Thymocytes from 3- to 4-wk-old Ikaros null (Ik / ), Ikaros heterozygous (Ik / ), and wild-type (Ik / ) mice were stained with anti-cd4-allophycocyanin, anti-cd8 -PE, and either anti-tcr -FITC, anti-cd5-fitc, or anti-cd69-fitc. A, The percentage of DP, CD4 SP, and CD8 SP cells are compared for Ik /,Ik /, and Ik / mice (A) as well as those mice on the DO11.10 /Rag / background (B). C, Averages and SEM for the groups in A and B are listed in the table. The percentage of T cell subsets in each set of thymi is given. The values are the mean SEM of 2 15 animals for each genotype., A value of p 0.05 and, value of p 0.08, as compared with Ik /.Ik / and Ik / are not significantly different. Statistics were determined using twotailed t tests. D, Absolute numbers of thymocyte subsets in Ikaros null and wild-type mice. Average numbers of thymocytes ( 10 6 ) that fall into the double-negative, DP, CD4, and CD8 subpopulations are shown for 3- to 4-wk-old Ikaros wild-type ( / ; n 10) and Ikaros null (n 15) mice. The values shown are mean SEM. E, Representative experiment comparing maturation markers on DP (shaded) and CD4 SP (black line) thymocytes in Ik / (left) and Ik / (right) mice. Dot plots indicate CD4 vs CD8 profiles of live cells and numbers in quadrants are percentage of live cells. Boxes within quadrants indicate the gating of DP and CD4 SP cells for the histograms. Numbers on the histograms represent the percentage of CD4 SP cells within the respective gate. NA, Not applicable. strong evidence that the increase in percentage of CD4 SP thymocytes in the Ikaros null thymi is indeed attributable to increased maturation toward the CD4 lineage in the absence of Ikaros activity. The functionality of Ikaros null and wild-type CD4 SP thymocytes was also compared. Activated CD4 T cells transiently upregulate surface expression of CD40-L, which is required for costimulation between B and T cells (20, 21). Up-regulation of Ikaros null CD4 SP thymocytes express reduced levels of CD5 Levels of CD5 expression on the surface of T cells have been shown to parallel the avidity of the positively selecting TCR: MHC/self-peptide interaction. It has been shown that a strong TCR signal induces higher levels of CD5 surface expression than does a weaker TCR signal (24). We observed that although levels of CD5 cell surface expression are similar on Ikaros null, Ikaros /, and Ikaros wild-type DP and CD8 SP thymocytes, they are lower on Ikaros null CD4 T cells relative to their Ikaros wild-type and Ikaros / counterparts (Fig. 3). This suggests that, in Ikaros null thymocytes, the threshold of positive selection is lowered such that CD4 T cells expressing TCRs with suboptimal avidity for MHC/ FIGURE 2. Increased percentage of functional CD4 SP thymocytes in Ikaros null mice. Thymocytes were isolated from 3- to 4-wk-old Ikaros null (Ik / ) and wild-type (Ik / ) mice and cultured in plates coated with PBS or anti-cd3 plus anti-cd28. Cells were then stained with anti- CD4-allophycocyanin, anti-cd8 -FITC, and anti-cd40-l-pe. Cells were gated as described in Fig. 1. A, Representative histograms of CD40-L upregulation on Ik / and Ik / CD4 SP cells stimulated (anti-cd3 /CD28, black line) and unstimulated (PBS, shaded) ex vivo. B, Scatter plot representing the corrected percentages of CD4 CD40-L, taking into account the decreased thymic cellularity and increased percentage of CD4 SP thymocytes in Ik / mice. Each point represents an individual mouse. The percentage of functional CD4 SP thymocytes was determined using the following formula: ((no. CD4 SP cells)(% CD4 CD40-L )]/(total cell no.). The mean SEM of the CD40-L CD4 SP percent is Ik / (n 4) %, Ik / (n 5) %, and Ik / (n 8) %, respectively. Up-regulation of CD40-L on Ik / CD4 SP cells is statistically different from Ik / and Ik / at a p 0.05, as determined using a two-tailed t test.

5 The Journal of Immunology 4473 FIGURE 3. CD5 surface expression is decreased on the surface of CD4 SP thymocytes in Ikaros null mice. Thymocytes were isolated from 3- to 4-wk-old Ikaros null ( / ), Ikaros heterozygous ( / ), and wild-type ( / ) mice and stained with anti-cd4-allophycocyanin, anti-cd8 -PE, and anti-cd5-fitc. Cells were gated as described in Fig. 1. Shown is an experiment representative of four independent experiments. Mean SEM of the CD5 mean fluorescence intensity (MFI) on CD4 SP thymocytes for all experiments are Ik / (n 7) , Ik / (n 15) , and Ik / (n 11) , respectively. The CD5 mean fluorescence intensity on Ik / CD4 SP cells is statistically different from Ik / at a p 0.09 and Ik / at p 0.06, as determined using a two-tailed t test. self-peptide that would normally die of neglect are now allowed to mature. In the absence of Ikaros, thymic CD4-like thymocytes are observed that express an MHC class I-restricted TCR Next, analysis of the role of Ikaros in selection of the CD8 lineage was investigated by using Ikaros null Rag1 / mice backcrossed onto the H-Y and F5 TCR Tg backgrounds. The TCR expressed in F5 Tg mice is a MHC class I-restricted TCR specific for an influenza nucleoprotein peptide in the context of H-2D b (25). The H-Y TCR recognizes a male-specific Ag in the context of H-2D b, allowing the study of negative selection using H-Y Tg male mice and positive selection using H-Y Tg female mice (26). Because the Rag1 gene product is necessary for TCR rearrangement, thymocytes can express only the F5 or the H-Y TCR. Data presented here are from female mice on the H-Y Rag1 / background. Mice on the F5 Rag1 / background show a similar phenotype (Ref. 27 and data not shown). Female H-Y, Ikaros null Rag1 / display no significant increase in the proportion of CD8 SP thymocytes compared with their Ikaros wild-type counterparts (Fig. 4A). However, female H-Y, Ikaros null Rag1 / thymi contain a significant population of CD4 SP thymocytes, suggesting that Ikaros null CD4 SP thymocytes can develop that inappropriately express a MHC class I-restricted TCR. This had been previously shown in F5, Ikaros null Rag1 / mice, although no studies were performed to analyze the functionality of these CD4 SP thymocytes (27). CD4 SP thymocytes were not observed in Ikaros / Rag1 / mice on either the H-Y or the F5 TCR Tg background, suggesting that 50% of Ikaros activity is sufficient to prevent their appearance (data not shown). To ascertain whether the CD4 SP thymocytes observed in the F5 and H-Y, Ikaros null Rag1 / thymi had transited through positive selection, we analyzed expression of the maturation markers TCR, CD5, and CD69. In contrast to the CD4 SP cells in the Ikaros null and DO11.10, Ikaros null Rag1 / thymi, the CD4 SP FIGURE 4. CD4-like SP cells arise in Ikaros null mice solely expressing a MHC class I-restricted TCR in the absence of an increased percentage of CD8 SP thymocytes. Thymocytes were isolated from 3- to 4-wk-old female HY /Rag / Ikaros null (Ik / ) mice and their Ikaros wild-type (Ik / ) counterparts. Cells were stained with anti-cd4-allophycocyanin, anti-cd8 -PE, and anti-tcr -FITC or anti-cd69-fitc. Cells were gated as described in Fig. 1. A, The percentage of live CD8 SP thymocytes is compared for Ik / and Ik / mice. Mean SEM of the percentage of CD8 SP thymocytes for all experiments is Ik / (n 9) % and Ik / (n 11) %, respectively. There is no significant difference between Ik / and Ik / mice, as determined using a two-tailed t test. B, Representative dot plots indicate CD4 vs CD8 profiles of live cells and numbers in quadrants are percentage of live cells. Boxes within quadrants indicate the gating used in the histograms comparing expression of TCR and CD69 on DP (shaded), CD8 SP (thick line), and CD4 SP (thin line) thymocytes in female HY /Rag / Ik / (left) and Ik / (right) mice. C, Cells were cultured in plates coated with PBS or anti-cd3 plus anti- CD28. Cells were then stained with anti-cd4-allophycocyanin, anti- CD8 -FITC, and anti-cd40-l-pe. Representative histograms of CD40-L staining on female HY /Rag / Ik / (right) CD4 SP thymocytes stimulated (anti-cd3/cd28, black line) and unstimulated (PBS, shaded) ex vivo are shown. Female HY /Rag / Ik / mice do not have CD4 SP cells and therefore CD40-L expression could not be examined.

6 4474 IKAROS NULL MICE DISPLAY DEFECTS IN T CELL SELECTION thymocytes that arise on the F5 and the female H-Y TCR Tg backgrounds do not up-regulate CD69 or TCR relative to their DP counterparts (Fig. 4B and data not shown). Next, to assay their functionality, thymocytes from F5 and female H-Y, Ikaros null Rag1 / mice were activated with platebound anti-cd3 plus anti-cd28, and CD40-L up-regulation was analyzed. Neither the CD4 SP thymocytes from the F5, Ikaros null Rag1 / nor those from the female H-Y, Ikaros null Rag1 / thymi were able to up-regulate CD40-L in response to TCR stimulation (Fig. 4C and data not shown). Taken together, these data provide evidence that these CD4 SP thymocytes are neither functional nor mature. FIGURE 5. Male HY /Rag / Ikaros null mice have impaired negative selection to conventional Ag. A, Thymocytes from 3- to 4-wk-old male and female HY /Rag / Ikaros null (Ik / ), Ikaros heterozygous (Ik / ) mice, and Ikaros wild-type (Ik / ) mice were isolated and stained with trypan blue to determine numbers of live thymocytes. The mean SEM of thymic cellularity ( 10 5 ) for female HY /Rag / mice is Ik / (n 9) , Ik / (n 10) , Ik / (n 9) and for male HY /Rag / mice is: Ik / (n 6) , Ik / (n 3) , and Ik / (n 6) , respectively. Male Ik / and Ik / were significantly different from their female counterparts at p 0.05 and 0.06, respectively, as determined by a two-tailed t test. Male and female Ik / mice were not significantly different. B, Thymocytes from 3- to 4-wk-old male and female HY /Rag / Ikaros null (Ik / ) and Ikaros wild-type (Ik / ) mice were stained with anti-cd4-allophycocyanin, anti- CD8 -PE, and anti-cd5-fitc. Representative dot plots of CD4 vs CD8 profiles of live cells are shown. The percentage of live cells in each quadrant is indicated. C, A representative histogram overlay of CD5 expression on total thymocytes in HY /Rag / mice on the following backgrounds: female Ik / (shaded), male Ik / (black line), male Ik / (dotted line). Numbers on plot indicate CD5 mean fluorescence intensity. In the male Ik / thymus, the percentages of cells that fall into the CD5 high vs CD5 low populations are equivalent for DP, CD4 SP, and CD8 SP subsets (data not shown). D, Thymocytes from 3- to 4-wk-old Ikaros null HY /Rag / mice were isolated and cultured in plates coated with PBS or anti-cd3 plus anti-cd28. Cells were then stained with anti-cd4-allophycocyanin, anti- CD8 -FITC, and anti-cd40-l-pe. Cells were gated as described in Fig. 1. Shown is a representative histogram of CD40-L up-regulation on CD4 SP Ikaros null thymocytes display impaired negative selection in response to conventional Ag Since decreasing Ikaros levels leads to decreased thresholds of activation in mature T cells and, as has been suggested in this report, decreased TCR signaling thresholds for positive selection, the expectation was that Ikaros null thymocytes would demonstrate enhanced negative selection. To test this prediction, thymic populations from male H-Y, Ikaros null Rag1 / mice were compared with those from their Ikaros wild-type counterparts. The H-Y TCR recognizes a male-specific Ag, making every thymocyte in the male H-Y Tg Rag1 / mouse a target for negative selection. In contrast to predictions, thymi from male H-Y, Ikaros null Rag1 / mice demonstrate signs of impaired negative selection. First, there is no decrease in thymic cellularity in male compared with female H-Y, Ikaros null Rag1 / thymi as is observed in their Ikaros wild-type counterparts (Fig. 5A). The decreased thymic cellularity observed in both the male and female H-Y, Ikaros null Rag1 / mice as compared with their Ikaros wild-type counterparts is similar to that described in non-tg Ikaros null mice and is hypothesized to be the consequence of decreased thymic progenitor input as has been described previously (27). Second, since negative selection occurs in the DP population, thymi from male H-Y, Ikaros wild-type Rag1 / mice contain primarily double-negative (CD4 CD8 ) cells. However, thymi from H-Y, Ikaros null Rag1 / mice contain DP, CD4 SP, and CD8 SP cells (Fig. 5B). All of these subsets are expressing high levels of TCR (data not shown). This strongly suggests that not only are Ikaros null DP thymocytes immune from negative selection, but they are able to continue in their development to the CD8 SP and, paradoxically, the CD4 SP stages. One explanation for the impaired negative selection observed in Ikaros null thymi is that the thymocytes are not receiving the high levels of TCR signal necessary to induce negative selection. To investigate this possibility, we compared the levels of CD5 expression on the surface of H-Y, Ikaros null Rag1 / thymocytes from female and male mice. Male H-Y, Ikaros null Rag1 / thymocytes expressed significantly higher levels of CD5 than their female counterparts whose thymocytes are solely receiving positive selection signals (Fig. 5C). This suggests that although these thymocytes are receiving a stronger TCR signal, as would be expected since their TCRs are being engaged by MHC/agonist peptide as opposed to MHC/self-peptide, they are not undergoing negative selection. thymocytes stimulated (anti-cd3 /CD28, black line) and unstimulated (PBS, shaded). E, Splenocytes from 3- to 4-wk-old male HY /Rag / Ikaros null (Ik / ) and Ikaros wild-type (Ik / ) mice were stained with anti-cd4-allophycocyanin and anti-cd8 -PE. Representative dot plots of CD4 vs CD8 profiles of live cells are shown. The percentage of live SP T cells is indicated in each quadrant.

7 The Journal of Immunology In male H-Y, Ikaros null Rag1 / thymi, functional CD4 SP thymocytes develop which express a MHC class I-restricted TCR As shown above, male as well as female H-Y, Ikaros null Rag1 / thymi inappropriately contain CD4 SP cells. To determine whether the CD4 SP thymocytes observed in male H-Y, Ikaros null Rag1 / thymuses are functional T cells, we analyzed CD40-L up-regulation after activation with plate-bound anti- CD3 plus anti-cd28. Dramatically, CD40-L up-regulation was observed on a subset of the CD4 T cells that develop in the male H-Y, Ikaros null Rag1 / thymi, providing evidence that, in the absence of Ikaros activity, functional, mature helper T cells can develop that inappropriately express class I-restricted TCRs (Fig. 5D). Significantly, a small percentage of mature CD4 T cells are also observed in the spleens of male H-Y, Ikaros null Rag1 / mice ( %, n 5; Fig. 5E). The splenic CD4 T cells express high levels of TCR and can up-regulate CD40-L in response to TCR activation ( %, n 5; data not shown). This is in sharp contrast to the results obtained with the CD4 T cells that develop in the female H-Y, Ikaros null Rag1 / thymi, which are not functional as discussed above. The difference between these two models is that the female mice solely express the positively selecting ligand, whereas the male mice also express the high-affinity agonist ligand. Therefore, these data suggest that functional Ikaros null CD4 SP thymocytes can develop that inappropriately express a MHC class I-restricted TCR if the selecting ligand is of high affinity. Ikaros null thymocytes display impaired negative selection in response to endogenous superantigen To further study the role of Ikaros in negative selection, the efficiency of thymocyte deletion induced by endogenous retroviral superantigens was analyzed in Ikaros null mice with polyclonal T cell repertoires. Superantigens, which are expressed by endogenous mouse mammary tumor viruses (Mtv), cause deletion of CD4 thymocytes that express reactive V TCRs through their ability to interact directly with MHC class II outside the conventional Ag binding site (28). BALB/c mice are positive for Mtv-6, Mtv-8, and 4475 Mtv-9 (29). As a result, CD4 thymocytes expressing V 3 (Mtv-6), V 11 (Mtv-8 and Mtv-9), and V 12 (Mtv-8 and Mtv-9) are deleted in this strain. Therefore, Ikaros / mice (C57BL/6 Sv129) were backcrossed onto the BALB/c background and then intercrossed to generate Ikaros null mice. TCRs containing V 8.1/8.2 would not be targets for superantigen-mediated negative selection, and, therefore, analysis of thymocytes expressing these TCRs was used as a control. Strikingly, a significantly higher percentage of CD4 SP thymocytes expressing TCRs containing V 11 and V 12 were observed in a subset of Ikaros null mice as compared with their Ikaros wild-type counterparts (Fig. 6). This suggests that negative selection of thymocytes expressing V 11 and V 12, both of which rely upon expression of superantigen from Mtv-8 and Mtv-9, is reduced in the absence of Ikaros (Fig. 6). In contrast, negative selection of thymocytes expressing V 3, which relies upon expression of superantigen from Mtv-6, was not affected. These results were also obtained in mice deficient for B7-1 and B7-2, two costimulatory molecules expressed on the surface of thymic dendritic cells (30). This could suggest, therefore, that the defect in negative selection in Ikaros null mice is related to the inability of their thymocytes to receive the necessary costimulatory signals for negative selection. Ikaros null thymocytes display unimpaired apoptosis in ex vivo death assays To eliminate the possibility that Ikaros null thymocytes have an intrinsic inability to undergo apoptosis, thymocytes were plated in the presence of plate-bound anti-cd3 plus anti-cd28 and assessed for apoptosis by CD4/CD8 down-regulation as well as annexin V staining. By both criteria, Ikaros null thymocytes show similar levels of apoptosis as compared with their Ikaros wild-type counterparts (Fig. 7). This result suggests that Ikaros null thymocytes have no intrinsic block in apoptotic pathways. Discussion Although the cell surface receptors and cytoplasmic signaling pathways have been well described for T cell selection events, not FIGURE 6. Ikaros null mice have impaired superantigen-mediated deletion of T cells bearing V 11- and V 12-containing TCRs. The Ikaros mutation was backcrossed for five generations onto the BALB/c genetic background. Thymocytes were isolated from 3- to 4-wk-old Ikaros null (Ik / ), Ikaros heterozygous (Ik / ), and wild-type (Ik / ) mice and stained with anti-cd4-allophycocyanin, anti-cd8 -PE, and either anti-v 3-FITC, anti-v 8.1/8.2- FITC, anti-v 11-FITC, or anti-v 12-FITC. The percentage of CD4 SP cells expressing V 8.1/8.2 (A, control), V 11 (B), V 12 (C), and V 3 (D) are shown. E, Averages and SEM for the percentage of CD4 SP cells expressing each TCR chain in A D are listed in the table. The values are the mean SEM of 9 20 animals for each genotype. Statistical significance was determined with the Wilcoxon test; a represents p 0.05 compared with Ik /, b is p 0.05 compared with Ik /, and c is p compared with Ik /.

8 4476 IKAROS NULL MICE DISPLAY DEFECTS IN T CELL SELECTION FIGURE 7. Ikaros null thymocytes do not display impaired apoptosis in an ex vivo-negative selection assay. Thymocytes were isolated from 3- to 4-wk-old Ikaros null (Ik / ), Ikaros heterozygous (Ik / ), and wild-type (Ik / ) mice. They were cultured in plates coated with PBS or anti-cd3 plus anti-cd28. Cells were then stained with anti-cd4-allophycocyanin, anti-cd8 -FITC, and Annexin V PE. A, Percentage of DP thymocytes demonstrating dulling of CD4 and CD8 expression was determined by gating on the DP bright and DP dull populations and calculating the percentage using the formula: (DP dull )/(DP dull DP bright ). The mean percent SEM of DP dulling is Ik / (n 3) %, Ik / (n 7) %, and Ik / (n 6) %, respectively. Ik / is significantly different from Ik / and Ik / at a p 0.05, as determined by a two-tailed t test. B, The percentage of DP apoptotic cells was determined by gating on the DP total populations and determining the percentage in that population that are annexin V. The mean percent SEM of annexin V DP cells is Ik / (n 3) %, Ik / (n 7) %, Ik / (n 6) %, respectively. Ik / is significantly different from Ik / at a p 0.05, as determined by a two-tailed t test. much is known about the nuclear factors involved in these developmental processes. In this report, we have provided evidence that Ikaros null mice display defects in negative selection as well as CD4 vs CD8 lineage decisions during positive selection, suggesting that the nuclear factor, Ikaros, plays a central role in both of these processes. The role of Ikaros in positive selection is most clearly observed in the development of the CD4 T cell lineage. We hypothesize that either Ikaros has a CD4 lineage-specific function or that there is a lowest level of signaling that must be received for transition from the DP to the SP stage, and Ikaros plays no role in setting this level. Rather, it may be that levels of Ikaros activity play a role in interpreting signaling levels that lead to CD4 (higher levels of signal) vs CD8 (lower levels of signal) lineage decisions. A recently published study demonstrated that decreased levels of Ikaros activity might result in variegated expression of the CD8 gene (31). In this report, it was suggested that the increase in the percentage of CD4 SP thymocytes observed in the thymi of Ikaros null mice is the result of abnormal down-regulation of CD8 on immature DP thymocytes and not the result of increased maturation into the CD4 lineage. However, these studies were not performed using Ikaros null mice. We have demonstrated that the increased percentage of CD4 SP cells in the Ikaros null and FIGURE 8. Model for the role of Ikaros in thymic selection events. A, Wild-type thymocytes require a minimum level of TCR signal to progress from the DP to the SP stage during positive selection. This signal ensures that CD4 and CD8 T cells develop with MHC class II- and MHC class I-restricted TCRs, respectively. The threshold of signaling that gives rise to CD4 T cells is hypothesized to be higher than that which gives rise to CD8 T cells. Once positive selection has occurred, TCRs are screened for their affinity for self-ag. This process requires the input of thymic APCs such as dendritic cells. Thymocytes expressing TCRs that show too high an affinity for MHC/self-Ag are negatively selected and undergo apoptosis. B, In the absence of Ikaros, positive selection occurs with lower signal strength. This is most apparent within the CD4 T cell compartment. In addition, Ikaros null thymocytes do not undergo negative selection. Instead, strong TCR signals can promote development of CD4 T cells that inappropriately express a MHC class I-restricted TCR. This defect in negative selection may be due to lack of normal numbers of dendritic cells in the Ikaros null thymi (Model 1) or to lack of up-regulation of a costimulatory molecule on the surface of Ikaros null thymocytes (Model 2) that is required for interaction with APCs.

9 The Journal of Immunology DO11.10, Ikaros null Rag1 / TCR Tg thymi represent an increased percentage of mature and functional CD4 SP thymocytes. Ikaros null SP thymocytes show up-regulation of TCR, CD5, and CD69, markers associated with transit through positive selection. In addition, there is an increased percentage of Ikaros null CD4 SP thymocytes, as compared with their Ikaros wild-type counterparts, which have the ability to up-regulate CD40-L in response to a TCR signal, a characteristic response of a functional Th cell. Moreover, if variegated expression of CD8 was solely responsible for the higher percentage of CD4 SP thymocytes, it would be expected that CD4 SP thymocytes would appear in Ikaros null thymi where T cell differentiation is blocked at the DP stage. However, this is not the case. In a previous report, it was shown that only a very low percentage of CD4 T cells ( 4%) is observed in the thymi of Ikaros null TCR / mice in which T cell differentiation is blocked at the DP stage (27). Therefore, variegated CD8 expression could only account for a limited increase in the percentage of CD4 SP thymocytes and not the significant increase that is observed in Ikaros null thymi. We provided evidence, through analysis of CD5 surface expression, that differentiation to the CD4 SP stage can occur in Ikaros null thymocytes with lower levels of TCR signal. Although a direct role for Ikaros in regulating CD5 expression is formally possible, the decreased surface expression of CD5 on Ikaros null CD4 thymocytes is more likely a reflection of signaling intensity since Ikaros deficiency has no effect on CD5 expression on the surface of DP and CD8 SP thymocytes. Therefore, these data support the hypothesis that the increased percentage of CD4 SP thymocytes observed in Ikaros null mice is the result of decreased thresholds of signaling for positive selection of this lineage. Dramatically, lack of Ikaros can also result in differentiation of functional and mature CD4 thymocytes inappropriately expressing a MHC class I TCR as observed in the male H-Y, Ikaros null Rag1 / thymi. This abnormal selection appears to be induced only in mice where the selecting ligand is of a high enough affinity that it would normally induce negative selection. Under conditions in which the agonist peptide is absent and the selecting peptide is one that induces positive selection, as in the F5 and female H-Y TCR Tg model systems, lack of Ikaros results in the inappropriate appearance of CD4 SP thymocytes, but these cells are neither mature nor functional. Abnormally early expression of a mature TCR at the double-negative stage, which occurs in TCR Tg mice, may potentiate the appearance of these abnormal CD4-like T cells in the Ikaros null F5 and female H-Y model systems (25, 32). Whereas lack of Ikaros activity facilitates positive selection toward the CD4 lineage, it impairs negative selection, both in response to conventional Ag and to endogenous superantigen. Expression of CD5 is higher on the surface of male H-Y, Ikaros null Rag1 / thymocytes than on their female counterparts, suggesting that TCR signaling pathways are intact and able to deliver a strong signal, yet they fail to undergo negative selection. Instead, they continue on in their developmental program to the SP stage, as if they had received signals for positive selection. Stronger TCR signals have been correlated with development into the CD4 lineage (3, 4). This suggests that, in the absence of Ikaros, a strong signal, as provided by agonist ligand/self-mhc, is being interpreted as a signal for development into the CD4 lineage, since negative selection is impaired. The defect in negative selection in Ikaros null thymocytes is not attributable to an intrinsic defect in apoptotic pathways, since they can die ex vivo in response to plate-bound anti-cd3 plus anti- CD28. In fact, if anything, apoptosis is increased in Ikaros null thymocyte populations compared with their wild-type counterparts. Yet, despite this, Ikaros null thymocytes are not undergoing 4477 negative selection in vivo. One explanation for this discrepancy is that Ikaros null thymocytes may not be receiving the costimulatory signal(s) in vivo that is necessary for negative selection to occur. As previously shown, Ikaros null thymi contain 15-fold fewer thymic dendritic cells than their Ikaros wild-type counterparts (33). This reduction is significantly greater than the reduction seen in thymocyte numbers, which is 3-fold. Therefore, in Ikaros null thymi, the thymocyte:apc ratio is considerably higher than that in Ikaros wild-type thymi. It is possible that there are insufficient APCs to provide the costimulatory signals required for negative selection to occur. Future studies will address whether increasing numbers of thymic dendritic cells can restore normal negative selection in Ikaros null thymi. Alternatively, Ikaros null thymocytes may be unable to up-regulate the costimulatory molecules needed to interact effectively with thymic dendritic cells or other thymic APCs. Both of these possibilities are supported by our data demonstrating that Ikaros null thymocytes can be efficiently deleted by only a subset of endogenous superantigens. Whereas a subset of Ikaros null mice demonstrate a decreased ability to delete thymocytes expressing V 11 and V 12 containing TCRs (as mediated by Mtv-8 and Mtv-9), deletion of thymocytes expressing V 3 containing TCRs (mediated by Mtv-6) occurs normally. It has been suggested that deletion of thymocytes expressing V 11 and V 12 containing TCRs is dependent upon the CD28/CTLA-4 costimulatory pathways, but that deletion of thymocytes expressing V 3 containing TCRs occurs independently of costimulatory signals (30). Therefore, this would suggest that Ikaros null thymocytes are impaired in their ability to undergo negative selection if cell death is dependent upon initiation and/or translation of costimulatory pathways. If Ikaros null mice have defective negative selection, autoimmunity should develop over time in mice with a polyclonal T cell repertoire. However, investigation for evidence of autoimmunity has proven difficult in the Ikaros null mice since they develop fatal leukemia so rapidly. In addition, they do not develop B cells, making it impossible to look for autoantibodies, the most widely used test for autoimmunity. Nevertheless, investigation of negative selection using the H-Y TCR Tg system combined with studies of thymocyte deletion by endogenous superantigen clearly point to severe defects in negative selection in the absence of Ikaros. In conclusion, we have provided evidence that Ikaros null mice have defects in both negative and positive selection, including CD4 vs CD8 lineage decisions, in the thymus (Fig. 8). To our knowledge, the Ikaros null mice are the only model system defective for the expression of a nuclear factor to demonstrate this dual phenotype. Therefore, Ikaros is the first identified nuclear factor that impacts both of these important developmental events, although its role in negative selection may not be thymocyte autonomous. In future studies, we will focus on defining the molecular role of Ikaros in regulating TCR signaling thresholds during T cell development. In addition, we will use the Ikaros null model as a unique system with which to characterize the role of costimulation in negative selection. Acknowledgments We gratefully acknowledge Dr. Katia Georgopoulos (Massachusetts General Hospital/Harvard Medical School) for the Ikaros null mice. We thank Erin Griffiths, Rachelle Lorenz, and Angela Minniti for excellent animal care and genotyping and Ciara Shaver for help with statistical analyses. We also thank Drs. Melissa Brown, Geoff Kansas, Neil Clipstone, and Paul Stein for critical reading of this manuscript. References 1. Starr, T. K., S. C. Jameson, and K. A. Hogquist Positive and negative selection of T cells. Annu. Rev. Immunol. 21:139.

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