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

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1. 

   Version published to 10.7554/elife.80443 on eLife

   Jun 2, 2023
2. 

   eLife

   May 23, 2023

   Author Response

   Reviewer #1 (Public Review)

   Using in vitro assays that take advantage of thymic slices, with or without the ability to present pMHC antigens, the authors define an early period in which CCR4 expression is induced, which induces their migration to the medulla and likely encounter with cDC2 and other APCs. Notably, the timing for CCR4 expression precedes that of CCR7 and illustrates the potential role for this early expression to initiate the movement of post-positive selection thymocytes to the medulla. The evidence for supporting a role for CCR4, as well as CCR7, in sequential tolerance induction is provided using multiple approaches, and although the observed changes amount to small percent changes, the significance is clear and likely biologically relevant over the lifespan of a developing T cell repertoire. …

   Author Response

   Reviewer #1 (Public Review)

   Using in vitro assays that take advantage of thymic slices, with or without the ability to present pMHC antigens, the authors define an early period in which CCR4 expression is induced, which induces their migration to the medulla and likely encounter with cDC2 and other APCs. Notably, the timing for CCR4 expression precedes that of CCR7 and illustrates the potential role for this early expression to initiate the movement of post-positive selection thymocytes to the medulla. The evidence for supporting a role for CCR4, as well as CCR7, in sequential tolerance induction is provided using multiple approaches, and although the observed changes amount to small percent changes, the significance is clear and likely biologically relevant over the lifespan of a developing T cell repertoire. Overall, the model provides a holistic view of how tolerance to self-antigens is likely induced during T cell development, which makes this work highly topical and influential to the field.

   We thank the reviewer for their comments and for highlighting the significance of identifying distinct roles for CCR4 and CCR7 in promoting medullary localization and inducing self-tolerance of thymocytes at different stages of T-cell development.

   Reviewer #2: (Public Review )

   This manuscript describes that CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance. Overall, the data are presented clearly. The distinct roles of CCR4 and CCR7 at different phases of thymocyte deletion (shown in Figure 6C) are novel and important. However, the conclusion that expression profiles of CCR4 and CCR7 are different during DP to SP thymocyte development was documented previously. More importantly, the data presented in this manuscript do not support the conclusion that CCR7 is uncoupled from medullary entry. Moreover, it is unclear how the short-term thymus slice culture experiments reflect thymocyte migration from the cortex to the medulla.

   We thank the reviewer for pointing out the significance of our finding that CCR4 and CCR7 regulate different phases of thymocyte deletion. We agree that prior reports, including our own (Cowan et al. 2014, Hu et al., 2015) have shown that CCR4 and CCR7 are expressed by different post-positive selection thymocytes. However, the expression data we present here provides a higher resolution perspective on the specific thymocyte subsets that express these two receptors, as well as the different timing with which the receptors are expressed after positive selection. These data, coupled with chemotaxis assays of the granular thymocyte subsets responding to CCR4 versus CCR7 ligands, and 2-photon imaging data showing that CCR4 and CCR7 are required for medullary accumulation of distinct thymocyte subsets, are critical for delineating the unexpectedly distinct roles of these two chemokine receptors in promoting medullary entry and central tolerance.

   The reviewer raises an important question about our conclusion that CCR7 is “uncoupled” from medullary entry. We think there was likely a misunderstanding of our intended meaning, as we did not mean to imply that CCR7 does not promote medullary entry of thymocyte subsets; we have modified the wording of the abstract to replace “uncoupled” to clarify. As we detail in the Introduction, the role of CCR7 in directing chemotaxis of single-positive thymocytes towards the medulla and inducing their medullary accumulation is well established (Ehrlich et al., 2009; Kurobe et al., 2006; Kwan & Killeen, 2004; Nitta et al., 2009; Ueno et al., 2004). Instead, our data demonstrate that 1) the most immediate post-positive selection thymocyte subset (DP CD3loCD69+) does not require CCR7 for medullary entry, and 2) the next stage of post-positive selection thymocytes (CD4SP SM) express CCR7, but CCR7 recruits these cells only modestly into medulla. In contrast, CCR7 promotes robust medullary accumulation of more mature thymocyte subsets (CD4SP M1+M2), in keeping with the well-known role of CCR7 in promoting thymocyte medullary localization. We think these findings are highly significant for the field because currently, there is a widely held assumption that post-positive selection thymocytes that do not express CCR7 are located in the cortex, while those that express CCR7 are located in the medulla. Our data show that neither of these assumptions is true: CCR4 drives medullary accumulation of cells that do not yet express CCR7, and the earliest post-positive selection cells that express CCR7 continue to migrate in both the cortex and medulla. These findings form the basis of our statement that CCR7 expression is “not synonymous with” medullary localization. The finding that thymocytes do not robustly accumulate in the medulla in a CCR7-dependent manner until more the mature SP stages has important implications for central tolerance, as localization of thymocytes in the cortex versus medulla will impact which APCs and self-antigens they encounter when testing their TCRs for self-reactivity.

   The reviewer also raised concerns about whether short-term thymus slice cultures reflect physiological thymocyte migration. Short-term live thymic slice cultures have been widely used to investigate the development, localization, migration, and positive and negative selection of thymocytes, as they have been shown to faithfully reflect these in vivo processes, including confirming the role of CCR7 in inducing chemotaxis of mature thymocytes from the cortex into the medulla (Au-Yeung et al., 2014; Dzhagalov et al., 2013; Ehrlich et al., 2009; Lancaster et al., 2019; Melichar et al., 2013; Ross et al., 2014). However, we acknowledge that thymic slices are not equivalent to intact thymuses and have now discussed limitations of this system in our revised Discussion.

   Comment 1: Differential profiles in the expression of chemokine receptors, including CCR4, CCR7, and CXCR4, during DP to SP thymocyte development were well documented. Previous papers reported an early and transient expression of CCR4, a subsequent and persistent expression of CCR7, and an inverse reduction of CXCR4 (Campbell, et al., 1999, Cowan, et al., 2014, and Kadakia, et al. 2019). The data shown in Figures 1, 2, and 3 are repetitive to previously published data.

   The expression profile of CCR4, CCR7 and CXCR4 on thymocytes has been documented previously in the studies cited above and in our prior publication (Hu et al., 2015). Campbell et al. (Campbell, Haraldsen, et al., 1999) investigated chemotactic effects of chemokines, but did not directly address expression of chemokine receptors by thymocyte subsets. Cowan et al. (Cowan et al., 2014) examined the expression of CCR4 versus CCR7 on DP and CD4SP thymocytes. However, our data provide a more detailed analysis of expression of these distinct chemokine receptors by subsets of DP, CD4SP, and CD8SP thymocyte subsets along the trajectory of differentiation after positive selection, using a gating scheme inspired by a study published after the above-cited papers (Breed et al., 2019). Our more nuanced evaluation of CCR4 versus CCR7 expression sets the stage for finding that they play distinct roles in promoting medullary entry and central tolerance of early- versus late-stage post-positive selection thymocytes. Without examining CCR4 and CCR7 expression patterns by distinct thymocyte subsets in detail, we would not have made the unexpected observation that although CCR7 is expressed at high levels by many CD4SP SM thymocytes, it does not induce strong chemotaxis or medullary accumulation of this subset, relative to its role in more mature SP thymocyte subsets. This finding has important implications for which APCs thymocytes encounter as they are tested for self-reactivity to enforce central tolerance. As we were working on these studies, Kadakia et al. reported that extinguishing CXCR4 expression was important for enabling medullary entry (Kadakia et al., 2019). Thus, we thought it was important to place CXCR4 in the context of CCR4 and CCR7 expression on thymocyte subsets in our study, and in doing so found another example of asynchronous chemokine receptor expression and function, further indicating that expression of a chemokine receptor alone is not a reliable marker of functional activity or thymocyte localization, as cells migrate dynamically between the cortex and medulla.

   Through more extensive gating and simultaneous investigation of chemokine receptor expression and function, our data have provided new insights into how thymocytes respond to chemokine cues at different time points during their post-positive selection development. Moreover, our refined gating scheme (Figure 1) can be used to distinguish thymocyte subsets at different development stages without relying on chemokine receptor expression, thus providing an unbiased way of investigating chemokine receptor expression at different developmental stages.

   Comment 2: The manuscript describes the lack of CCR7 at early stages during DP to SP thymocyte development (Figure 1-3). However, CCR7 expression is detected insensitively in this study. Unlike CCR4 detection with a wide fluorescence range between 0 and 2x104 on the horizontal axis, CCR7 detection has a narrow range between 0 and 2x103 on the vertical axis (Figure 1C, 1D, 4B, 4C, 6B, S2, S3), so that flow cytometric CCR7 detection in this study is 10-times less sensitive than CCR4 detection. It is therefore likely that the "CCR7-negative" cells described in this manuscript actually include "CCR7-low/intermediate" thymocytes described previously (for example, Figure S5A in Van Laethem, et al. Cell 2013 and Figure 6 in Kadakia, et al. J Exp Med 2019).

   We provide new data to address the possibility that we were failing to detect low levels of CCR7 expression on early post-positive selection DPs (CD3loCD69+). We agree that CCR7 immunostaining of mouse cells is known to be more challenging than immunostaining of other chemokine receptors, including CCR4 and CXCR4. CCR7 immunostaining needs to be carried out at 37°C, which we did throughout our studies. We provide new data comparing CCR7 expression by Ccr7+/+ versus Ccr7-/- thymocyte subsets (Figure 1—figure supplement 2A-B), which confirm that CCR7 is not expressed at detectable levels by CD3loCD69+ DP cells above the background seen in CCR7-deficient cells. As thymocytes transition to theCD4SP SM stage, low/intermediate to high expression of CCR7 can be detected (Figure 1—figure supplement 2A). To further test whether we were failing to detect low levels of CCR7 by post-positive selection DPs, we incubated thymocytes at 37°C for up to 2 hours prior to immunostaining for CCR4 and CCR7, as a prior study indicated in vitro culture would enable increased cell surface expression of CCR7 by alleviating ligand-mediated CCR7 internalization (Britschgi et al., 2008). However, we did not observe increased CCR7 (or CCR4) expression by any thymocyte subset incubated at 37°C (Figure 1—figure supplement 2C-D). Lack of expression of CCR7 by CD3loCD69+ DP cells is consistent with their failure to undergo chemotaxis to CCR7 ligands in vitro, and initial expression of CCR7 by CD4SP SM is consistent with their chemotaxis towards CCR7 ligands in vitro (now show in greater detail in Figure 2—figure supplement 1), albeit at a much lower migration index than subsequent thymocyte subsets.

   Comment 3: Low levels of CCR7 expression could be functionally evaluated by the chemotactic assay as shown in Figure 2. However, the data in Figure 2 are unequally interpreted for CCR4 and CCR7; CCR4 assays are sensitive where a migration index at less than 1.5 is described as positive (Figure 2A and 2B), whereas CCR7 assays are dismissal to such a small migration index and are only judged positive when the migration index exceeds 10 or 20 (Figure 2C and 2D). CCR7 chemotaxis assays should be carried out more sensitively, to equivalently evaluate the chemotactic function of CCR4 and CCR7 during thymocyte development.

   We thank the reviewer for his insight about the possibility that we could have overlooked CCR7-mediated chemotaxis at lower migration indexes. When data from the chemotaxis assays were evaluated separately for each thymocyte subset, CCR7-mediated chemotaxis of CD4SP SM and subsequent DP CD3+CD69+ co-receptor reversing thymocytes could be detected. However, DP CD3loCD69+ thymocytes still did not undergo CCR7-meidated chemotaxis, but were responsive to the CCR4 ligand CCL22 (Figure 2—figure supplement 1).

   We did not detect CCR7-mediated chemotaxis of CD4SP SM and DP CD3+CD69+ subsets in our previous analysis because their lower-level chemotactic index relative to mature thymocytes did not reach statistical significance when chemotaxis of all subsets were compared simultaneously (Figure 2D). We note that the magnitude of difference in the responsiveness of CD4SP SM cells compared to mature CD4SP and CD8SP M1 & M2 thymocytes (Figure 2D) is likely physiologically important as CCR7 deficiency results in severely reduced medullary accumulation of CD4SP M1+M2 cells, but only a very mild reduction in medullary accumulation of CD4SP SM cells, which is only detected with our new paired analyses in Figure 5C. We feel these new analyses provide important new insights and thank the reviewer for this suggestion.

   Comment 4: Together, this manuscript suffers from the poor sensitivity for CCR7 detection both in flow cytometric analysis and chemotactic functional analysis. Conclusions that CCR7 is absent at early stages of DP to SP thymocyte development and that CCR7 is uncoupled from medullary entry are the overinterpretation of those results with the poor sensitivity for CCR7. The oversimplified scheme in Figure 3D is misleading.

   We agree that the scheme in Figure 3D, as previously constructed, did not ideally display the difference in scale between thymocyte responses to CCR7 ligands versus CCR4 and CXCR4 ligands (as detected in vitro). Thus, we have now modified the schematic to include the mild response to CCR7 ligands that we observed in CD4SP SM thymocytes (comment 3) and to emphasize the higher chemotactic response of mature thymocytes to CCR7 ligands than of DPs and CD4SP SM to CCR4 ligands. Likewise, we have modified the manuscript to clarify the importance of CCR7 expression in the medullary entry and accumulation of mature thymocyte subsets.

   We respectfully disagree that the sensitivity of CCR7 detection was poor in our flow cytometry and chemotactic analyses. Our CCR7 stains identified a range of CCR7 expression levels, from no expression by pre- and post-positive positive selection DP cells to high expression by CD4SP M1 cells, and we now provide new data confirming our ability to detect CCR7 expression (Figure 1—figure supplement 2), as described in response to Comment 3. Our chemotaxis assays detected CCR7 responses over a range of migration indexes from ~ 2 up to 100, showing our sensitive ability to detect CCR7-mediated chemotaxis in vitro (Figure 2 and Figure 2—figure supplement 1). In live thymic slices, we were also able to capture a range of biologic activities of CCR7, from mediating modest medullary accumulation of CD4SP SM cells to robust medullary accumulation of CD4SP M1+M2 cells (Figure 5A-C). Importantly, our results demonstrate that CCR7 is not the only chemokine receptor responsible for medullary entry and accumulation of thymocytes. Complex spatiotemporal regulation of thymocytes at distinct stages of development is achieved through tight orchestration of expression and signaling through multiple chemokine receptors, including CCR4, as shown by our data. However, our study does not negate an important role for CCR7 in mediating medullary entry of thymocytes, which we have clarified in the text.

   Comment 5: The short-term thymus slice culture experiments should be described more carefully in terms of selection events during DP to SP thymocyte development, which takes at least 2 days for CD4 lineage T cells and approximately 4 days for CD8 lineage T cells (Saini, et al. Sci Signal 2010 and Kimura, et al. Nat Immunol 2016). The slice culture experiments in this manuscript examined cellular localization within 12 hours and chemokine receptor expression within 24 hours (Figures 4, 5) even for the development of CD8 lineage T cells (Figure S2), which are too short to examine entire events during DP to SP thymocyte development and are designed to only detect early phase events of thymocyte selection.

   Experiments in Figures 4 and 5 were indeed designed to capture behaviors of thymocytes relatively early after introduction onto thymic slices. Figure 4 (and Figure 4—figure supplement 1) shows that the timing of CCR4 versus CCR7 expression after positive selection is dramatically different: CCR4 is expressed within hours of positive selection, concomitant with medullary entry, while CCR7 expression takes several days in the slices (sufficient time for CD8SP development, Figure 4—figure supplement 1). Figure 5 shows that medullary accumulation of CD4SP M1+M2 cells occurs robustly in the medulla of thymic slices within a couple of hours after introduction into the slices, and this localization is CCR7 dependent, while CCR4 induces more mild medullary accumulation of post-positive selection DPs. As indicated by the reviewer, it has been shown that it takes days for DP thymocytes to develop into mature CD4SP and CD8SP cells (Kimura et al., 2016; Lutes et al., 2021; Saini et al., 2010), as recapitulated in the thymus slice system (Figure 4—figure supplement 1) (Lutes et al., 2021). The relatively short time frame of our time-course experiments (up to 12 hours after addition of pre-positive selection thymocytes to positively selecting thymic slices) allowed us to detect expression of CCR4 within a few hours after positive selection and to determine that this timing correlated with medullary entry. Thus, the 12-hour time-course was important for temporal resolution of chemokine receptor expression and medullary localization after initial stages of positive selection.

   Comment 6: It is unclear what the medullary density alteration measured in the thymus slice culture experiments represents. Although the manuscript describes that the increase in the medullary density reflects the entry of cortical thymocytes to the medulla (Figure 4E and S2E), this medullary density can be affected by other mechanisms, including different survival of the cells seeded on the top of different thymus microenvironments. Thymocytes seeded on the medulla may be more resistant to cell death than thymocytes seeded in the cortex, for example, because of the rich supply of cytokines by the medullary cells. So, the detected alterations in the medullary density may be affected by the differential survival of thymocytes seeded in the cortex and the medulla. Also, the medullary density is measured only within a short period of up to 12 hours. The use of MHC-II-negative slices and CCR4- or CCR7-deficient thymocytes in the thymus slice cultures may verify whether the detected alteration in the medullary density is dependent on TCR-initiated and chemokine-dependent cortex-to-medulla migration.

   We thank the reviewer for pointing out these possibilities. The purpose of the positive selection timing experiment (Figure 4) was to establish the early correlation between receiving a positive selection signal, upregulating CCR4, and migrating into the medulla. In this system, cells only enter only the cortex in the first hour after migration in the slice, consistent with prior studies of localization of pre-positive selection thymocytes to the cortex (Ehrlich et al., 2009; Ross et al., 2014); subsequently, they move into the medulla. Because CCR7 is widely accepted to be essential for medullary entry, we feel it is important to demonstrate the disconnect between the timing of medullary entry and CCR7 expression in multiple ways. The timing experiment design utilized MHCII-/- and β2m-/- slices to show that positive selection was necessary for expression of CCR4. To test whether CCR4 or CCR7 were required for medullary entry of early post-positive selection DPs, we evaluated medullary accumulation of this subset from WT, Ccr4-/-, Ccr7-/-, and Ccr4-/-Cc7-/- mice. This experiment provided a more robust means of determining the extent to which CCR4 deficiency impacted medullary localization of a large cohort of cells that had passed positive selection (Figure 5), and again showed that the post-positive selection thymocytes, which express CCR4 but not CCR7, accumulate in the medulla in a CCR4-dependent manner. We note that in Figure 5, we show that all Ccr4-/-Ccr7-/- thymocyte subsets imaged have medullary:cortical density ratios of ~1, indicating an even distribution across cortex and medulla, which is highly consistent with an essential role for these two chemokine receptors in cooperating to mediate medullary accumulation of different stages of developing T cells.

   The reviewer makes an interesting point that survival cues could differ in the cortex versus medulla. However, if thymocytes lacking one or both chemokine receptors had impaired survival because they didn’t enter a region of the thymus efficiently to receive survival cues, we would expect to detect increased apoptosis in Ccr4-/-, Ccr7-/-and Ccr4-/-Cc7-/- thymocytes. However, we found that chemokine receptor deficiencies resulted in diminished apoptosis of different thymocyte subsets (Figure 6). This finding is more consistent with reduced negative selection of these subsets due to reduced clonal deletion. We nonetheless discuss this possibility in our revised manuscript, as it important to consider that chemokine-mediated migration of thymocytes into different microenvironments could alter their access cytokines and other pro-survival cues.

   Reviewer #3 (Public Review)

   In this manuscript, Li et al. examine how the expression of the chemokine receptor CCR4 impacts the movement of thymocytes within the thymus. It is currently known that the chemokine receptor CCR7 is important for developing thymocytes to migrate from the cortical region into the medullary region and CCR7 expression is therefore often used to define medullary localization. This is important because key developmental outcomes, like enforcing tolerance to self-antigens amongst others, occur in the medullary environment. The authors demonstrate that the chemokine receptor CCR4 is induced on thymocytes prior to expression of CCR7 and thymocytes exhibit responsiveness to CCR4 ligands earlier in development. Using elegant live confocal microscopy experiments, the authors demonstrate that CCR4 expression is important for the entry and accumulation of specific thymocyte subsets while CCR7 expression is needed for the accumulation of more mature thymocyte subsets. The use of cells deficient in both CCR4 and CCR7 and competitive migration/accumulation experiments provide strong support for this conclusion. The elimination of CCR4 expression results in decreases in apoptosis of thymocyte subsets that have been signalled through their antigen receptor and are responsive to CCR4 ligands. As expected, more mature thymocyte subsets show decreased apoptosis when CCR7 is absent. Distinct antigen-presenting cells in the thymus express CCR4 ligands supporting a model where CCR4 expressing thymocytes can interact with thymic antigen-presenting cells for induction of apoptosis. The absence of CCR4 results in an increase in peripheral T cells that can respond to self-antigens presented by LPS-activated antigen-presenting cells providing further support for the model. Collectively, the manuscript convincingly demonstrates a previously unappreciated role for CCR4 in directing a subset of thymocytes to the medulla.

   We thank the reviewer for appreciating the novelty of the finding that CCR4 directs distinct subsets of thymocytes into the medulla relative to CCR7, as supported by multiple lines of evidence.

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3. 

   eLife

   May 22, 2023

   eLife assessment

   This paper will be of broad interest to immunologists that study T cell responses and formation of the peripheral T cell compartment. Using elegant live imaging approaches, the authors provide convincing evidence in support of a revised model for how positive-selected thymocytes are called to 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.

   Read the original source
4. 

   eLife

   May 22, 2023

   Reviewer #1 (Public Review):

   Using in vitro assays that take advantage of thymic slices, with or without the ability to present pMHC antigens, the authors define an early period in which CCR4 expression is induced, which induces their migration to the medulla and likely encounter with cDC2 and other APCs. Notably, the timing for CCR4 expression precedes that of CCR7 and illustrates the potential role for this early expression to initiate the movement of post-positive selection thymocytes to the medulla. The evidence for supporting a role for CCR4, as well as CCR7, in sequential tolerance induction is provided using multiple approaches, and although the observed changes amount to small percent changes, the significance is clear and likely biologically relevant over the lifespan of a developing T cell repertoire. Overall, the model …

   Reviewer #1 (Public Review):

   Using in vitro assays that take advantage of thymic slices, with or without the ability to present pMHC antigens, the authors define an early period in which CCR4 expression is induced, which induces their migration to the medulla and likely encounter with cDC2 and other APCs. Notably, the timing for CCR4 expression precedes that of CCR7 and illustrates the potential role for this early expression to initiate the movement of post-positive selection thymocytes to the medulla. The evidence for supporting a role for CCR4, as well as CCR7, in sequential tolerance induction is provided using multiple approaches, and although the observed changes amount to small percent changes, the significance is clear and likely biologically relevant over the lifespan of a developing T cell repertoire. Overall, the model provides a holistic view of how tolerance to self-antigens is likely induced during T cell development, which makes this work highly topical and influential to the field.

   Read the original source
5. 

   eLife

   May 22, 2023

   Reviewer #2 (Public Review):

   This manuscript describes that CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance. Overall, the data are presented clearly. The distinct roles of CCR4 and CCR7 at different phases of thymocyte deletion (shown in Figure 6C) are novel and important. However, the conclusion that expression profiles of CCR4 and CCR7 are different during DP to SP thymocyte development was documented previously. More importantly, the data presented in this manuscript do not support the conclusion that CCR7 is uncoupled from medullary entry. Moreover, it is unclear how the short-term thymus slice culture experiments reflect thymocyte migration from the cortex to the medulla.

   1. Differential profiles in the expression of chemokine receptors, including CCR4, CCR7, and CXCR4, …

   Reviewer #2 (Public Review):

   This manuscript describes that CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance. Overall, the data are presented clearly. The distinct roles of CCR4 and CCR7 at different phases of thymocyte deletion (shown in Figure 6C) are novel and important. However, the conclusion that expression profiles of CCR4 and CCR7 are different during DP to SP thymocyte development was documented previously. More importantly, the data presented in this manuscript do not support the conclusion that CCR7 is uncoupled from medullary entry. Moreover, it is unclear how the short-term thymus slice culture experiments reflect thymocyte migration from the cortex to the medulla.

   1. Differential profiles in the expression of chemokine receptors, including CCR4, CCR7, and CXCR4, during DP to SP thymocyte development were well documented. Previous papers reported an early and transient expression of CCR4, a subsequent and persistent expression of CCR7, and an inverse reduction of CXCR4 (Campbell, et al., 1999, Cowan, et al., 2014, and Kadakia, et al. 2019). The data shown in Figures 1, 2, and 3 are repetitive to previously published data.

   2. The manuscript describes the lack of CCR7 at early stages during DP to SP thymocyte development (Figure 1-3). However, CCR7 expression is detected insensitively in this study. Unlike CCR4 detection with a wide fluorescence range between 0 and 2x10*4 on the horizontal axis, CCR7 detection has a narrow range between 0 and 2x10*3 on the vertical axis (Figure 1C, 1D, 4B, 4C, 6B, S2, S3), so that flow cytometric CCR7 detection in this study is 10-times less sensitive than CCR4 detection. It is therefore likely that the "CCR7-negative" cells described in this manuscript actually include "CCR7-low/intermediate" thymocytes described previously (for example, Figure S5A in Van Laethem, et al. Cell 2013 and Figure 6 in Kadakia, et al. J Exp Med 2019).

   3. Low levels of CCR7 expression could be functionally evaluated by the chemotactic assay as shown in Figure 2. However, the data in Figure 2 are unequally interpreted for CCR4 and CCR7; CCR4 assays are sensitive where a migration index at less than 1.5 is described as positive (Figure 2A and 2B), whereas CCR7 assays are dismissal to such a small migration index and are only judged positive when the migration index exceeds 10 or 20 (Figure 2C and 2D). CCR7 chemotaxis assays should be carried out more sensitively, to equivalently evaluate the chemotactic function of CCR4 and CCR7 during thymocyte development.

   4. Together, this manuscript suffers from the poor sensitivity for CCR7 detection both in flow cytometric analysis and chemotactic functional analysis. Conclusions that CCR7 is absent at early stages of DP to SP thymocyte development and that CCR7 is uncoupled from medullary entry are the overinterpretation of those results with the poor sensitivity for CCR7. The oversimplified scheme in Figure 3D is misleading.

   5. The short-term thymus slice culture experiments should be described more carefully in terms of selection events during DP to SP thymocyte development, which takes at least 2 days for CD4 lineage T cells and approximately 4 days for CD8 lineage T cells (Saini, et al. Sci Signal 2010 and Kimura, et al. Nat Immunol 2016). The slice culture experiments in this manuscript examined cellular localization within 12 hours and chemokine receptor expression within 24 hours (Figures 4, 5) even for the development of CD8 lineage T cells (Figure S2), which are too short to examine entire events during DP to SP thymocyte development and are designed to only detect early phase events of thymocyte selection.

   6. It is unclear what the medullary density alteration measured in the thymus slice culture experiments represents. Although the manuscript describes that the increase in the medullary density reflects the entry of cortical thymocytes to the medulla (Figure 4E and S2E), this medullary density can be affected by other mechanisms, including different survival of the cells seeded on the top of different thymus microenvironments. Thymocytes seeded on the medulla may be more resistant to cell death than thymocytes seeded in the cortex, for example, because of the rich supply of cytokines by the medullary cells. So, the detected alterations in the medullary density may be affected by the differential survival of thymocytes seeded in the cortex and the medulla. Also, the medullary density is measured only within a short period of up to 12 hours. The use of MHC-II-negative slices and CCR4- or CCR7-deficient thymocytes in the thymus slice cultures may verify whether the detected alteration in the medullary density is dependent on TCR-initiated and chemokine-dependent cortex-to-medulla migration.

   Read the original source
6. 

   eLife

   May 22, 2023

   Reviewer #3 (Public Review):

   In this manuscript, Li et al. examine how the expression of the chemokine receptor CCR4 impacts the movement of thymocytes within the thymus. It is currently known that the chemokine receptor CCR7 is important for developing thymocytes to migrate from the cortical region into the medullary region and CCR7 expression is therefore often used to define medullary localization. This is important because key developmental outcomes, like enforcing tolerance to self-antigens amongst others, occur in the medullary environment. The authors demonstrate that the chemokine receptor CCR4 is induced on thymocytes prior to expression of CCR7 and thymocytes exhibit responsiveness to CCR4 ligands earlier in development. Using elegant live confocal microscopy experiments, the authors demonstrate that CCR4 expression is …

   Reviewer #3 (Public Review):

   In this manuscript, Li et al. examine how the expression of the chemokine receptor CCR4 impacts the movement of thymocytes within the thymus. It is currently known that the chemokine receptor CCR7 is important for developing thymocytes to migrate from the cortical region into the medullary region and CCR7 expression is therefore often used to define medullary localization. This is important because key developmental outcomes, like enforcing tolerance to self-antigens amongst others, occur in the medullary environment. The authors demonstrate that the chemokine receptor CCR4 is induced on thymocytes prior to expression of CCR7 and thymocytes exhibit responsiveness to CCR4 ligands earlier in development. Using elegant live confocal microscopy experiments, the authors demonstrate that CCR4 expression is important for the entry and accumulation of specific thymocyte subsets while CCR7 expression is needed for the accumulation of more mature thymocyte subsets. The use of cells deficient in both CCR4 and CCR7 and competitive migration/accumulation experiments provide strong support for this conclusion. The elimination of CCR4 expression results in decreases in apoptosis of thymocyte subsets that have been signalled through their antigen receptor and are responsive to CCR4 ligands. As expected, more mature thymocyte subsets show decreased apoptosis when CCR7 is absent. Distinct antigen-presenting cells in the thymus express CCR4 ligands supporting a model where CCR4 expressing thymocytes can interact with thymic antigen-presenting cells for induction of apoptosis. The absence of CCR4 results in an increase in peripheral T cells that can respond to self-antigens presented by LPS-activated antigen-presenting cells providing further support for the model. Collectively, the manuscript convincingly demonstrates a previously unappreciated role for CCR4 in directing a subset of thymocytes to the medulla.

   Strengths:

   Relevant model systems and elegant experimental techniques are used throughout the manuscript. The experiments are extensively replicated resulting in robust and convincing data sets. These findings represent an important conceptual advance in our understanding of the processes and cellular regulation of T cell development in the thymus.

   Weaknesses:

   Evidence demonstrating a direct interaction between CCR4 expressing thymocytes and CCR4-ligand expressing antigen-presenting cells is lacking. Furthermore, increased self-reactivity in the absence of CCR4 is measured using mature peripheral CD4 T cells, but altered self-reactivity of thymocytes is not evaluated similarly.

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7. 

   Version published to 10.1101/2022.04.03.486911v2 on bioRxiv

   May 13, 2023
8. 

   Version published to 10.1101/2022.04.03.486911v1 on bioRxiv

   Apr 4, 2022