Author response: CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance

Field: Neuroscience (Life Sciences)

Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Central tolerance ensures autoreactive T cells are eliminated or diverted to the regulatory T cell lineage, thus preventing autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their T-cell receptors (TCRs) for autoreactivity against the diverse self-antigens displayed by antigen-presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, synchronized positive selection assays, two-photon time-lapse microscopy, and quantification of TCR-signaled apoptotic thymocytes, demonstrate that CCR4 and CCR7 promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets in mice. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and is required for medullary localization and negative selection of mature thymocytes. In addition, CCR4 and CCR7 differentially enforce self-tolerance, with CCR4 enforcing tolerance to self-antigens presented by activated APCs, which express CCR4 ligands. Our findings show that CCR7 expression is not synonymous with medullary localization and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of negative selection, respectively, via interactions with distinct APC subsets. Editor's evaluation This important paper reveals the key steps associated with intrathymic central tolerance. Using elegant live imaging approaches, the authors provide convincing evidence in support of an updated model for how positive-selected thymocytes are called into the thymus medulla to interact with distinct antigen-presenting cells. The work makes an important contribution to the field by identifying previously unappreciated complexities related to cellular movement during T cell generation. https://doi.org/10.7554/eLife.80443.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Autoimmune diseases occur when immune cells mistakenly identify the body's own tissues as 'foreign' and attack them. To reduce the risk of this happening, the body has multiple ways of removing self-reactive immune cells, including T cells. One such way, known as central tolerance, occurs in the thymus – the organ where T cells develop. In the center of the thymus – the medulla – specialized cells display fragments of the majority of proteins expressed by healthy cells throughout the body. Developing T cells enter the medulla, where they scan these specialized cells to determine if they recognize the presented protein fragments. If an immature T cell recognizes and binds to these 'self-antigens' too strongly, it is either destroyed, or it develops into a regulatory cell, capable of actively suppressing T cell responses to that self-antigen. This ensures that T cells won't attack healthy cells in the body that make those self-antigens, and therefore, it is important that T cells enter the medulla and carry out this scanning process efficiently. T cells are recruited to the medulla from the outer region of the thymus by chemical signals called chemokines. These signals are recognized by chemokine receptors on T cells, which are expressed at different times during T cell development. Previous work has shown that one of these receptors, called CCR7, guides T cells to the medulla. Although it was thought that CCR7 was solely responsible for this migration, prior work suggests another receptor, CCR4, may also contribute to T cell migration into the medulla and central tolerance. To determine whether CCR7 and CCR4 play the same or different roles in central tolerance, Li, Tipan et al. used a combination of experimental methods, including live imaging of the thymus, to study T cell development in mice. The experiments revealed that CCR4 is expressed first, and this receptor alone guides immature T cells into the medulla and ensures that they are the first to be checked for self-reactivity. In contrast, CCR7 is expressed by more mature developing T cells two to three days later, ensuring they also accumulate within the medulla and become tolerant to self-antigens. Both receptors are required for protection from autoimmunity, with results suggesting that CCR4 and CCR7 promote tolerance against different tissues. Taken together, the findings provide new information about the distinct requirement for CCR4 and CCR7 in guiding immature T cells into the medulla and ensuring central tolerance to diverse tissues. One outstanding question is whether defects in T cells entering the medulla earlier or later alter tolerance to distinct self-antigens and lead to different autoimmune diseases. Future work will also investigate whether these observations hold true in humans, potentially leading to therapies for autoimmune diseases. Introduction Self-tolerance of the T cell repertoire is established in thymus through the process of central tolerance, which encompasses both negative selection and regulatory T cell (Treg) induction (Klein et al., 2014). To avoid autoimmunity, developing T cells must be broadly tolerized not only to ubiquitously expressed self-antigens, but also to proteins expressed by distinct differentiated cell types. The thymic medulla is a specialized environment in which central tolerance to such diverse self-antigens is enforced. After positive selection in the cortex, thymocytes migrate into the medulla, where they interact with medullary antigen-presenting cells (APCs), including thymic dendritic cells (DCs) and medullary thymic epithelial cells (mTECs). Collectively, these APCs enforce self-tolerance by presenting self-peptides from the majority of the proteome on MHC complexes, such that thymocytes expressing autoreactive T cell receptors undergo clonal deletion or diversion to the Treg lineage (Ehrlich, 2016; Klein et al., 2014; Klein et al., 2019; Lancaster et al., 2019). AIRE+ mTECs express >80% of the proteome, including Aire-dependent tissue restricted antigens (TRAs) that are otherwise expressed in only a few peripheral tissues (Abramson and Anderson, 2017; Anderson and Su, 2016; Mathis and Benoist, 2007; Meredith et al., 2015). DCs also present numerous self-antigens, including those acquired from mTECs, from circulation, or from peripheral tissues and trafficked into the thymus (Atibalentja et al., 2009; Bonasio et al., 2006; Koble and Kyewski, 2009). The importance of inducing thymocyte tolerance to medullary self-antigens is evidenced by multi-organ autoimmunity that ensues in Aire-deficient mice and APECED patients (Anderson et al., 2002; Finnish-German APECED Consortium, 1997; Nagamine et al., 1997), in whom thymic expression of TRAs is greatly diminished. Failure to express even a single medullary TRA can result in impaired tolerance and subsequent T cell-mediated autoimmune pathology (DeVoss et al., 2006). Notably, individual TRAs are expressed by only 1–3% of Aire+ mTECs (Brennecke et al., 2015; Meredith et al., 2015; Sansom et al., 2014), resulting in a sparse mosaic of self-antigen display throughout the medulla. Because thymocytes reside in the medulla for only 4–5 days (McCaughtry et al., 2007), tight spatiotemporal regulation is required to ensure that post-positive selection thymocytes encounter the full spectrum of medullary self-antigens required to enforce self-tolerance. To facilitate medullary entry after positive selection, thymocytes upregulate chemokine receptors that promote their directional migration toward medullary biased chemokine gradients (Bleul and Boehm, 2000; Hu et al., 2015a; Lancaster et al., 2018; Petrie and Zúñiga-Pflücker, 2007). Notably, CCR7 has been shown to play a critical role in directing chemotaxis of post-positive selection thymocytes toward the medulla, where CCR7 ligands are expressed, enhancing thymocyte accumulation within the medulla, enforcing negative selection to TRAs, and averting autoimmunity (Ehrlich et al., 2009; Kozai et al., 2017; Kurobe et al., 2006; Nitta et al., 2009; Ueno et al., 2004). Given the significance of CCR7 in promoting thymocyte medullary entry, CCR7 expression is widely considered to be synonymous with thymocyte medullary localization. Based largely on this definition, recent studies indicate that despite the well-established role of the medulla in inducing central tolerance, ~75% of negative selection occurs in CCR7− 'cortical' thymocytes, while only 25% occurs in CCR7+ 'medullary' cells (Breed et al., 2019; Daley et al., 2013; Hu et al., 2016). It has also been suggested that cortical negative selection may eliminate thymocytes reactive to ubiquitous self-antigens while medullary deletion tolerizes TRA-responsive cells. Our previous research implicates chemokine receptors other than CCR7 in promoting thymocyte medullary localization (Ehrlich et al., 2009). We found that CCR4, which is upregulated by post-positive selection thymocytes, also promotes medullary entry and negative selection (Hu et al., 2015b). These findings raise the question of whether CCR4 and CCR7 play distinct or redundant roles in promoting thymocyte medullary entry and central tolerance. In this paper, we use a combination of approaches, including chemotaxis assays, two-photon live-cell microscopy, and synchronized positive selection assays, to distinguish the contributions of CCR4 versus CCR7 to thymocyte medullary entry and negative selection. We find that CCR4 is upregulated by thymocytes as early as a few hours after positive selection, while CCR7 is expressed days later by mature post-positive selection cells. CCR4 expression promotes chemotaxis and medullary entry of early post-positive selection thymocytes, which do not yet express CCR7. Initial CCR7 expression results in only moderate thymocyte chemotaxis toward CCR7 ligands and modest accumulation in the medulla, such that these thymocytes migrate in both the cortex and medulla. These findings indicate that CCR7 expression is not a definitive marker of medullary versus cortical localization. CCR7 is, however, required for robust accumulation of mature (CD4SP) thymocytes in the medulla. Notably, consistent with their differential activity in distinct thymocyte subsets, CCR4 and CCR7 are required in early and late phases of polyclonal negative selection, respectively. While CCR7 is known to promote tolerance to TRAs (Kozai et al., 2017; Nitta et al., 2009), we present evidence that CCR4 promotes tolerance to self-antigens presented by activated APCs. Collectively, our study establishes non-redundant roles for CCR4 and CCR7 in governing localization of post-positive selection thymocyte subsets and central tolerance. Results CCR4 is expressed by immature post-positive selection thymocyte subsets, while CCR7 expression is restricted to more mature thymocyte subsets We first investigated expression of CCR4 and CCR7 by distinct thymocyte subsets using cell-surface markers that delineate developmental stages after positive selection (Figure 1A; Sinclair et al., 2013; Xing et al., 2016). To validate the developmental trajectory of these thymocyte subsets, we analyzed their GFP expression levels in Rag2p-GFP mice, in which GFP expression is driven by the Rag2 promoter. In this model, expression of GFP distinguishes newly generated thymocytes (GFP+) from recirculating T cells (GFP−), and declining GFP expression reflects time elapsed after positive selection, when the Rag2 promoter becomes inactive (Boursalian et al., 2004). In pre-positive selection CD4+CD8+ double-positive (DP) CD3−CD69− thymocytes, the Rag2 promoter is active, resulting in maximal GFP expression (Figure 1B). After receiving a TCR signal, thymocytes upregulate CD3 and CD69 (Fu et al., 2009), generating early post-positive selection DP CD3loCD69+ cells; the brief time elapsed after positive selection is indicated by continued high GFP expression by this subset (Figure 1B). To further evaluate if DP CD3loCD69+ thymocytes represent post-positive selection thymocytes, as opposed to cells undergoing strong TCR signaling driving clonal deletion, we evaluated thymocyte subsets from Nr4a1GFP mice, in which GFP levels reflect the strength of TCR signaling (Figure 1—figure supplement 1; Moran et al., 2011). Elevation of GFP expression by DP CD3loCD69+ cells, relative to DP CD3−CD69− cells, confirmed this subset was TCR signaled. Thymocytes rescued from negative selection express Nr4a1GFP at levels comparable to thymic Tregs, which express self-reactive TCRs (Stritesky et al., 2013). GFP levels were substantially lower in DP CD3loCD69+ cells than in CD25+ CD4SPs, which are mostly Tregs (Figure 1—figure supplement 1A). Together, these data are consistent with DP CD3loCD69+ cells representing mainly post-positive selection DPs, and not strongly self-reactive thymocytes undergoing negative selection. Sequential maturation through CD4+ single-positive semi-mature cells (CD4SP SM) and then CD4SP or CD8SP mature 1 (M1) and mature 2 (M2) subsets was confirmed by progressive reductions in GFP levels for both the CD4 and CD8 lineages (Figure 1B; Xing et al., 2016). A distinct population of DP CD3+CD69+ cells expressed lower levels of GFP than CD4SP SM cells (Figure 1B), placing it temporally between CD4SP SM and CD8SP M1 cells (Figure 1B). Thus, we considered whether DP CD3+CD69+ cells represent MHC-I-restricted thymocytes in the process of differentiating into CD8SP cells through co-receptor reversal downstream of the CD4SP SM stage (Brugnera et al., 2000). Consistent with this possibility, DP CD3+CD69+ cells were significantly reduced in B2m−/− mice, in which positive selection of CD8SP thymocytes is abrogated, as is selection of innate iNKT cells and CD8αα+ IELps (Bendelac et al., 1994; Ruscher et al., 2017). Furthermore, DP CD3+CD69+ cells were enriched in mice with a deletion spanning the MHC-II genes H2-Aa, H2-Eb1, and H2-Eb2 (MHCII−/−) in which only MHC-I-restricted thymocytes are positively selected (Figure 1—figure supplement 1B, C). Together, these data demonstrate that DP CD3+CD69+ cells are MHC-I restricted and have persisted for an intermediate time between CD4SP SM and CD8SP M1 cells after positive selection, consistent with a co-receptor reversing DP subset. Figure 1 with 2 supplements see all Download asset Open asset CCR4 and CCR7 are expressed by immature and mature subsets of post-positive selection thymocytes, respectively. (A) Flow cytometry gating scheme to delineate thymocyte subsets in Rag2p-GFP mice. Post-positive selection DP (CD3loCD69+ and CD3+CD69+), CD4SP (SM, M1, and M2), and CD8SP (M1 and M2) subsets are annotated. (B) Relative Rag2p-GFP Geometric Mean Fluorescent Intensity (GMFI) of each subset normalized to pre-positive selection DP CD3−CD69− cells. Individual data points represent average GFP GMFI of the indicated subset from each mouse. (C) Representative flow cytometry plots showing CCR4 and CCR7 expression by post-positive selection thymocyte subsets. (D) Quantification of the percentage of cells of the indicated subset that express CCR4, CCR7, or both chemokine receptors. For (B) and (D), data were compiled from four independent experiments (mean ± standard error of the mean [SEM]; N = 10 mice). Having delineated the temporal sequence of thymocyte development post-positive selection, we quantified expression of CCR4 and CCR7 by each subset. CCR4 is upregulated by the majority of early post-positive selection DP CD3loCD69+ thymocytes, which do not yet express CCR7 (Figure 1C, D, Figure 1—figure supplement 2A, B). CCR7 is subsequently expressed at high levels by ~40% CD4SP SM cells, and CCR4 expression persists on this subset (Figure 1C, D). In comparison to Ccr7−/− cells, low-level expression of CCR7 can be detected on additional Ccr7+/+ CD4SP SM cells, consistent with initial upregulation of CCR7 by this subset (Figure 1—figure supplement 2A, B). Expression of CCR4 and CCR7 by MHC-I-restricted DP CD3+CD69+ cells is similar to that of CD4SP SM cells, with slightly more CCR7 and less CCR4 (Figure 1C, D, Figure 1—figure supplement 2A, B). As thymocytes mature through CD4SP and CD8SP M1 and M2 stages, CCR4 is progressively downregulated. CCR7 expression peaks in both CD4SP and CD8SP M1 subsets and is diminished in M2 cells (Figure 1C, D, Figure 1—figure supplement 2A, B). Because cell-surface expression of CCR7 on mature CD4+ T cells can be downregulated due to ligand-induced internalization of the chemokine receptor (Britschgi et al., 2008), we tested whether receptor internalization could have obscured detection of CCR7 or CCR4 on ex vivo thymocyte subsets. Thymocytes were incubated at 37°C for up to 2 hr, to allow re-expression of internalized receptors, before immunostaining with anti-CCR7 and anti-CCR4 antibodies. Increased expression of cell-surface CCR7 or CCR4 was not detected with increasing incubation time (Figure 1—figure supplement 2C, D). These findings confirm that CCR4 expression is an early indicator of positive selection at the DP stage, while CCR7 expression is upregulated later by more mature CD4SP and CD8SP subsets. Early post-positive selection thymocytes undergo chemotaxis toward CCR4 ligands, while more mature subsets respond to CCR7 ligands We next determined whether expression of CCR4 and CCR7 corresponds to thymocyte chemotactic responses to their respective ligands. DP CD3loCD69+ cells, which express CCR4 but not CCR7, underwent chemotaxis in response to the CCR4 ligand CCL22, but not the CCR7 ligands CCL19 or CCL21 (Figure 2A–D, Figure 2—figure supplement 1A). CCL22-induced chemotaxis of all subsequent post-positive selection thymocytes, except the most mature M2 thymocyte subsets (Figure 2B, Figure 2—figure supplement 1A), largely in keeping with CCR4 expression patterns. Although CD4SP SM and DP CD3+CD69+ subsets expressed CCR7 (Figure 1C), CCR7-mediated chemotaxis of these subsets did not reach statistical significance when compared to all thymocyte subsets (Figure 2C, D). However, when considered separately, moderate CCR7-mediated chemotaxis of these subsets was revealed, although DP CD3loCD69+ cells still did not undergo CCR7-mediated chemotaxis (Figure 2—figure supplement 1A). Consistent with expression of CCR7 by more mature thymocyte subsets, the CCR7 ligands CCL19 and CCL21 induced highly efficient chemotaxis of mature CD4SP and CD8SP M1 and M2 subsets (Figure 2C, D). Figure 2 with 2 supplements see all Download asset Open asset CCR4 and CCR7 promote chemotaxis of immature and mature post-positive selection thymocyte subsets, respectively. Transwell assays were used to quantify chemotaxis of thymocyte subsets to the indicated concentrations of the CCR4 ligands CCL17 (A) and CCL22 (B) and the CCR7 ligands CCL19 (C) and CCL21 (D). Migration index was calculated as the frequency of input cells of each subset that migrated toward the chemokine relative to the frequency of input cells that migrated in the absence of chemokine (Blank). Data were compiled from three independent experiments with triplicate wells per assay. Two-way ANOVA with Dunnett's multiple comparisons correction was used for statistical analysis (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). A recent study indicated that expression of the chemokine receptor CXCR4 must be extinguished before post-positive selection thymocytes can leave the cortex, where CXCL12 is expressed by cortical thymic epithelial cells, to migrate into the medulla (Kadakia et al., 2019). Thus, we sought to place CXCR4 expression and function into context with CCR4 and CCR7. CXCR4 expression decreased after positive selection, but all post-positive selection subsets continued to express CXCR4 (Figure 3A, B), and all except for CD4SP SM and DP CD3+CD69+ subsets underwent chemotaxis in response to CXCL12 (Figure 3C). Although the earliest post-positive selection thymocyte subsets exhibited reduced CXCR4 responsiveness, perhaps allowing them to exit the cortex, the ability to respond to CXCR4 signals does not preclude medullary localization, as evidenced by CXCR4-mediated chemotaxis of medullary CD4SP and CD8SP M2 cells. Taken together, chemotaxis assays show that early post-positive selection DP CD3loCD69+ thymocytes are responsive to CCR4 but not CCR7 ligands, with low responsiveness to CXCR4 ligands. At the next stage, CD4SP SM cells also undergo chemotaxis toward CCR4 ligands, and begin to migrate at a low level toward CCR7 ligands, but not to CXCR4 ligands. As thymocytes mature to the CD4SP and CD8SP M1 and M2 stages, they progressively gain the ability to respond robustly to CCR7 ligands, regain CXCR4 responsiveness, and lose responsiveness to CCR4 ligands (Figure 3D). Figure 3 Download asset Open asset Persistent CXCR4 expression was detected on post-positive selection thymocytes, and CXCR4 activity declines only in intermediate subsets. (A) Representative flow cytometry plots showing CXCR4 surface expression by the indicated thymocyte subsets, compared to isotype control stains. (B) Quantification of relative GMFI of CXCR4 for the indicated thymocyte subsets (mean ± SEM; N = 10). (C) Transwell assays were used to quantify chemotaxis of thymocyte subsets to the indicated concentrations of CXCL12. Migration index was calculated as the frequency of input cells of each subset that migrated toward the chemokine relative to the frequency of input cells that migrated in the absence of chemokine (Blank). Data were compiled from three independent experiments with triplicate wells per assay. Two-way ANOVA with Dunnett's multiple comparisons correction was used for statistical analysis (mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001). (D) Graphical summary showing relative expression and chemotactic activity of CXCR4, CCR4, and CCR7 on post-positive selection thymocyte subsets based on data in Figures 1—3. Initial expression of CCR7 by CD4SP SM thymocytes does not result in strong chemotaxis to CCR7 ligands (Figure 2D), and expression of CXCR4 by this subset does not enable detectable chemotaxis toward CXCL12 (Figure 3C), highlighting a temporal disconnect between chemokine receptor expression and function on maturing post-positive selection thymocytes. We considered the possibility that CD4SP SM cells could be intrinsically unable to respond to chemokine receptor signaling. However, these cells responded efficiently to CCR4 ligands (Figure 2B), and they underwent chemotaxis to the CCR9 ligand CCL25 at levels comparable to DP CD3loCD69+ cells (Figure 2—figure supplement 2A), consistent with a previous study showing CCL25 induces migration of most thymocyte subsets (Campbell et al., 1999). Thus, regulation of thymocyte responsiveness to chemokine receptors is more complex than altered cell-surface expression levels. CCL21a establishes a chemokine gradient that drives accumulation of CD4SP thymocytes in the medulla (Kozai et al., 2017; Ueno et al., 2002; Ueno et al., 2004). Because we found that functional CCR4 is expressed by early post-positive selection DP thymocytes, we examined whether CCL22 could establish a similar chemokine gradient to recruit these CCR4-responsive cells into the medulla. Immunofluorescence analysis revealed that CCL22 is expressed predominantly in the medulla (Figure 2—figure supplement 2B), consistent with a potential role in inducing medullary entry of early post-positive selection thymocytes. The timing of CCR4 upregulation following positive selection correlates with thymocyte medullary entry Given that the majority of early post-positive selection DP thymocytes respond to CCR4 but not CCR7 ligands, we sought to determine how rapidly CCR4 versus CCR7 are upregulated following initiation of positive selection and to assess whether expression of either receptor correlates with the timing of medullary entry. To accomplish these goals, we used a synchronized positive selection thymic slice assay, coupled with flow cytometry or two-photon (Figure mice as a of pre-positive selection DP thymocytes that did not yet express CCR4 or CCR7 due to of positive selection in the absence of MHC-II expression (Figure thymocytes were prior to on positively or or (MHCII−/−) live thymic At time expression of CCR4 and CCR7 by thymocytes in the thymic was by flow or medullary entry was quantified by two-photon live-cell (Figure Figure with 1 supplement see all Download asset Open asset upregulation of CCR4 following positive selection of thymocytes correlates with medullary entry. (A) of synchronized thymic slice positive selection assays to determine the timing of CCR4 and CCR7 expression by flow cytometry and medullary entry by two-photon (B) Representative flow cytometry plots showing CCR4 and CCR7 expression of thymocytes to thymic and analyzed after incubation in or as indicated (C) of and thymocytes in thymic of the indicated at the indicated time Data were compiled from three experiments with triplicate analysis with multiple comparisons correction was used for statistical analysis (mean ± SEM; *p < 0.05, **p < (D) Representative of two-photon imaging data showing a thymic slice thymocytes at and after thymocytes were to the and cortical were as indicated by the Quantification of medullary of thymocytes at indicated time points after incubation on thymic Individual data points represent relative medullary input cell from each compared to the average of medullary from all Data were compiled from four ANOVA with multiple comparison correction was used for statistical analysis (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). DP cells upregulated CCR4 as early as 3 after positively thymic (Figure The frequency of cells continued to on selection was required for initiation of CCR4 as by the to upregulate CCR4 on CCR7 was not upregulated within of positive selection (Figure thymocytes were into positively thymic and by two-photon microscopy, they were to the cortex, consistent with our previous findings that pre-positive selection thymocytes the medulla 1; et al., 2009). after positive selection, thymocytes the medulla, as evidenced by a in medullary (Figure As CCR7 is not yet expressed by these cells (Figure CCR4 drives medullary entry of early post-positive selection thymocytes. 1 Download asset This be in place does support may still the for Download as Download as Download as live imaging of input thymocytes in a thymic slice after to positively thymic as shown in Figure were acquired for at time through a of and are 2 Download asset This be in place does support may still the for Download as Download as Download as live imaging of input thymocytes in a thymic slice after to positively thymic as shown in Figure were acquired for at time through a of and are To test if the timing of CCR4 and CCR7 upregulation and medullary entry are comparable for MHC-I-restricted thymocytes, we out similar experiments using pre-positive selection thymocytes that in the of Although positive selection was largely in these about of the DP cells expressed CCR4 at (Figure supplement B). CCR4 was upregulated as early as 3 after positive selection in thymic but not in and (Figure supplement B). CCR7 was not expressed during the first after initiation of positive