Stepwise transmigration cascade of T and B cells through the perivascular channel in lymph node high endothelial venules

  1. Kibaek Choe
  2. Jieun Moon
  3. Soo Yun Lee
  4. Eunjoo Song
  5. Ju Hee Back
  6. Joo-Hye Song
  7. Young-Min Hyun
  8. Kenji Uchimura
  9. Pilhan Kim

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

High endothelial venules (HEVs) effectively recruit circulating lymphocytes from the blood to lymph nodes. HEVs have endothelial cells (ECs) and perivascular sheaths consisting of fibroblastic reticular cells (FRCs). Many studies have characterized the multiple steps of lymphocyte migration interacting with ECs at the luminal side of HEVs. However, post-luminal migration steps are not well elucidated. Herein, we performed intravital imaging to investigate post-luminal T and B cell migration, consisting of trans-EC migration, crawling in the perivascular channel (a narrow space between ECs and FRCs) and trans-FRC migration. The post-luminal migration of T cells occurred in a PNAd-dependent manner. Remarkably, we found hot spots for the trans-EC and trans-FRC migration of T and B cells. Interestingly, T and B cells preferentially shared trans-FRC migration hot spots but not trans-EC migration hot spots. Furthermore, the trans-FRC T cell migration was confined to fewer sites than trans-EC T cell migration, and trans-FRC migration of T and B cells preferentially occurred at FRCs covered by CD11c+ dendritic cells in HEVs. These results suggest that HEV ECs and FRCs with perivascular DCs delicately regulate T and B cell entry into lymph nodes.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Answers to the reviewers’ comments

    We deeply appreciate the reviewers for their thoughtful, critical and constructive comments, which have undoubtedly provided us with valuable opportunities to improve our manuscript.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Extravasation of lymphocytes from HEV in the lymph nodes is mediated by the interaction between lymphocyte L-selectin and PNAd-carrying sulfated sugars expressed by HEVs. Multiple steps of lymphocyte migration interacting with ECs at the luminal side of HEVs have been studied intensively; however, post-luminal migration steps are unclear. In this study, using intravital confocal microscopy of peripheral lymph nodes (pLNs), the authors found that GlcNAc6ST1 deficiency, required for sulfation of PNAd, delays trans-fibroblastic reticular cell (FRC) migration of lymphocytes, and hot spots of trans-HEV EC migration and trans-FRC migration. Interestingly, hot spots of trans-FRC migration are often associated with dendritic cells (DCs). Thus, the authors concluded that FRCs delicately regulate the transmigration of T and B cells across the HEV wall, which could be mediated by perivascular DCs.

    **Main comments**

    This study focused on pLNs, which are quite different from mesenteric lymph nodes (mLNs) in many ways. The authors should include mLNs in their study to make the general statement with regard to the T/B cell entry into lymph nodes. In addition, it will be more significant if this study includes challenged pLNs.

    We thank the reviewer for raising the important point. We agree that mesenteric lymph nodes are quite different from peripheral lymph node that this study focuses on. Therefore, we specified the popliteal or peripheral lymph node in the revised manuscript as follows.

    In the Abstract (page 2), “… Herein, we performed intravital imaging to investigate post-luminal T and B cell migration in popliteal lymph node, consisting of trans-EC migration, crawling in the perivascular channel (a narrow space between ECs and FRCs) and trans-FRC migration. … These results suggest that HEV ECs and FRCs with perivascular DCs delicately regulate T and B cell entry into peripheral lymph nodes.”

    In the Introduction (page 4), “Herein, we clearly visualized the multiple steps of post-luminal T and B cell migration in popliteal lymph node, including trans-EC migration, intra-PVC crawling and trans-FRC migration, using intravital confocal microscopy and fluorescent labelling of ECs and FRCs with different colours.

    In the Discussion (page 21), “… These results imply that pericyte-like FRCs, the second cellular barrier of HEVs, regulate the entry of T and B cells to maintain peripheral lymph node homeostasis more precisely and restrictively than we previously thought.”

    In addition, we discussed the difference in lymphocyte migration across HEVs between peripheral lymph node, mesenteric lymph node, and peyer’s patches in the Discussion of the revised manuscript. We also discussed inflamed lymph nodes in the Discussion as follows.

    In the Discussion (page 20), “… Although this work focused on peripheral lymph node, the other lymphoid organs have different lymphocyte homing efficiency61 due to organ-specific gene expression on HEVs62. B cells home better to mesenteric lymph nodes and peyer’s patches than peripheral lymph nodes61 by CD22-binding glycans expressed preferentially on the HEVs of mesenteric lymph nodes and peyer’s patches62.

    Inflamed peripheral lymph node become larger by recruiting more lymphocytes and even L-selectin-negative leukocytes that are excluded in the steady state63,64. Inflamed HEV ECs show different gene expression, such as downregulation of GLYCAM1 and GlcNAc6ST-160. In addition, inflamed HEV integrity may be loosen due to markedly increased leukocyte influx although the HEV FRCs can prevent bleeding by interacting with platelet CLEC-248. CD11c+ DCs are associated with inflamed HEV EC proliferation that is functionally associated with increased leukocyte entry65. The stepwise migration of lymphocyte across inflamed HEVs and their hot spots with perivascular CD11+ DCs will be interesting topic for future study.”

    The finding that GlcNAc6ST1 deficiency delays lymphocyte trans-FRC migration but not trans-HEV EC migration is surprising. However, the reason this occurs is neither shown nor discussed. Is GlcNAc6ST1 also expressed in FRCs? Or does GlcNAc6ST1 expression on HEV license lymphocytes to transmigrate across FRCs?

    This is valid point to be addressed. GlcNAc6ST-1 is predominantly involved in PNAd expression on the abluminal side rather than on the luminal side. Therefore, our results that GlcNAc6ST-1 deficiency increased the time required for trans-FRC migration but not that for trans-EC migration, could be attributable to deficiency of GlcNAc6ST-1-synthesizing L-selectin ligands in the abluminal side of HEV.

    In addition to PNAd expression in the luminal and abluminal sides of endothelial cells in HEV, PNAd expression has been observed in reticular network close to HEV as following figures. We believe that PNAds are expressed in FRCs close to HEV and can affect lymphocyte migration such as trans-FRC migration and parenchymal migration. By looking at the data (Table S1, Rodda et al., Immunity 2008), GlcNAc6ST-1 (Chst2) is expressed in T-cell-zone reticular cells while GlcNAc6ST-2 (Chst4) is absent. Therefore, it is presumable that FRC-expressed GlcNAc6ST1 may regulate trans-FRC migration in some extent.

    Figures. PNAD expression on HEVs (arrows) and reticular network (arrow heads) close to the HEVs

    We included these points in the Discussion of the revised manuscript (page 15) as follows.

    “… GlcNAc6ST-1 is predominantly involved in PNAd expression on the abluminal side rather than on the luminal side, although GlcNAc6ST-1 deficiency also modestly affects the luminal migration of lymphocytes by increasing the rolling velocity9. GlcNAc6ST-1 deficiency increased the time required for trans-FRC migration but not that for trans-EC migration. This could be attributable to deficiency of GlcNAc6ST-1-synthesizing L-selectin ligands in the abluminal side of HEV. In addition to the abluminal side of HEV endothelial cells, FRCs also express GlcNAc6ST-1, but not GlcNAc6ST-227, implying that FRC-expressed GlcNAc6ST-1 may regulate trans-FRC migration in some extent. … Thus, PNAds expressed at the endothelial junction and on the abluminal side of HEVs facilitate the efficient transmigration of lymphocytes across the HEV wall but do not slow transmigration in the perivascular region. GlcNAc6ST-1 deficiency and MECA79 antibody also decreased the parenchymal B and T cell velocities immediately after extravasation, respectively, probably because of blockade of parenchymal expression of PNAd in close proximity to HEV6,21,28.”

    Because of the adoptive transfusion experiment, the actual number of transmigrating lymphocytes in Fig. 3F is underestimated.

    We agree with the reviewer’s comment. We corrected the y-axis label in Fig. 3F from ‘average number of cells transmigrating at one site’ to ‘average number of labeled cells transmigrating at one site.’

    Whether DCs covering FRCs have a role for lymphocyte trans-migration is not shown.

    We leaved this work as future research and discussed about the potential mechanisms in the Discussion (page 17-18) that the DC may regulate lymphocyte entering by interacting FRC with LTβR or CLEC-2 signaling. We also included ‘Martinez et al Cell Rep 2019 (ref.51)’ in the discussion of the revised manuscript (page 18). In addition, we also discussed about better characterization of the CD11c+ DC in the Discussion of the revised manuscript (page 19) as follows.

    In the Discussion (page 18), “The podoplanin of FRCs also controls FRC contractility49,50 and ECM production51 by interacting with the CLEC-2 of DCs in inflamed lymph nodes. In the steady state, resident DCs in lymph nodes express CLEC-252. Thus, it is conceivable that CLEC-2+ resident DCs may control the contractility of FRCs and remodel ECM surrounding HEVs to facilitate the trans-FRC migration of T and B cells. Thus, the CLEC-2/podoplanin signalling may represent a key molecular mechanism underlying our discovery that trans-FRC migration hot spots preferentially occur at FRCs covered by CD11c+ DCs.”

    In the Discussion (page 19), “… In addition, better characterization of the CD11c+ DCs located in the hot spots of HEVs is required to differentiate them from the other CD11c+ DCs observed in the non-hot-spot regions of HEVs. Some T-cell-zone resident macrophages can also express CD11c54. Imaging of a triple-transgenic mouse with Zbtb46-cre;tdTomato and CD11b-GFP will be able to differentiate 3 types of DCs and macrophages potentially associated with the hot spots: Zbtb46+CD11b- cDC1, Zbtb46+CD11b+ cDC2, and Zbtb46-CD11b+ macrophage54,55.”

    In Fig. 1, time required for trans HEV EC migration and trans-FRC migration of T cells is shorter than that of B cells; however, this finding is not observed in Fig. 2C and E.

    Although the statistical comparison between T and B cells are not shown in Fig. 2C-F and S5., there are actually significant difference between T and B cells, which are similar results as Fig. 1 except for the dwell time in PVC. P values between T and B cells in wildtype mice are 0.0003, In the Result (page 6), “… The mean velocity of T cells (5.3 ± 1.7 μm/min) was significantly higher than that of B cells (4.1 ± 1.4 μm/min) during intra-PVC migration (Fig. 1E), while the dwell time and total path length in the PVC were not significantly different between T and B cells (Fig. 1, H and I). Similar results were obtained when both cells were imaged simultaneously, except that B cells had significant longer dwell time than T cells (Fig. 2C-F and Fig. S5). Interestingly, more than half of the T and B cells crawled from 50 μm to 350 μm inside the PVC (Fig. 1I), …”

    In the legend of Fig. 2, “… P values between T and B cells in wild-type mice were 0.0003 (C), …”

    In the legend of Fig. S5, “… P values between T and B cells in wild-type mice were 0.0240 (A), 0.3614 (B), 0.7518 (C) and 0.1337 (D). …”

    **Minor comments**

    Please provide evidence for GlcNAc6ST1 deficiency in HEV and surrounding tissues.

    Previous studies (Uchimura et al., JBC 2004, Nat. Immunol. 2005; ref9 and 10, respectively, in the manuscript) confirmed systemic deficiency of GlcNAc6ST-1 in peripheral lymph nodes of the GlcNAc6ST-1 KO mice.

    Images for delayed trans-FRC migration in GlcNAc6ST1 KO mice relative to WT are not convincing (Fig. 2G and H).

    We think the reason why the images look unconvincing is probably because it is not easy to quickly determine the images corresponding to the trans-FRC migration in the image sequence. To make the transmigration images easier to recognize, we added arrow heads indicating the transmigration site in Fig. 2G and 2H, and Fig. S4 as follows.

    Provide actual time periods required for Fig. 3F and G. Lack of isotype control IgG experiment in Fig. S3.

    We added the time periods (3 hours) in the figure legend as follows.

    “… (F) Average numbers of labeled T and B cells transmigrating at one site for 3 hours. (G) Ratio of hot spots to total transmigration sites for 3 hours. …”

    The purpose of Fig. S3 was to confirm that the anti-ER-TR7 antibody injection for labeling FRC do not alter normal T cell motility, rather than to confirm the function of ER-TR7. Therefore, we used non-injected group as control rather than control antibody injection group.

    Line 12 on page 11, "the ratio of hot spots to the total “observed” transmigration sites..." is not appropriate. The ratio must be calculated by hot spots to the total "potential" transmigration sites, although it is challenging to find total potential sites.

    We corrected the expression from ‘the total observed transmigration sites’ to ‘the total potential transmigration sites’.

    Please correct typos of angiomoduin to angiomodulin (page 16), ET-TR7 to ER-TR7 (page 17), Anti-CD3 to anti-CD3 (page 22), half the dose to half dose (page 22), the Multiple step to the multiple step (page 23).

    We thank the reviewer for finding those errors. We corrected them and performed proofreading repeatedly to correct typos and grammatic errors.

    Please provide an additional explanation of why actin-DsRed in HEVs is more strongly expressed than surrounding tissues such as FRCs in Fig. 1 although actin-DsRed should be expressed in all cell types in mice.

    We were also surprised when we found that HEV ECs expressed red fluorescence more strongly compared to surrounding tissues. Although the other cells such as FRCs and endogenous lymphocytes also express DsRed under control of a promotor gene, beta-actin, we believe that HEV ECs express more strongly, which is sufficient to image only HEV-EC by adjusting an image contrast. We revised the explanation of this point in the Methods (page 21) as follows.

    “HEV ECs of actin-DsRed mouse popliteal lymph node expressed red fluorescence much stronger than the surrounding stromal cells and endogenous lymphocytes, which was sufficient to image only HEV ECs by adjusting an image contrast (Fig. 1, A and B).”

    Reviewer #1 (Significance (Required)):

    The study focused on lymphocytes post extravasation of HEV, which is an understudied question, using intravital imaging. The in vivo imaging study was deliberately and beautifully performed, and the finding is insightful for understanding lymphocyte trafficking in lymph nodes. However, additional experimental should be performed to address some weaknesses listed in our comments.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The present study by K. Choe meticulously monitored the stepwise transmigration behavior of T cells and B cells, respectively, through the high endothelial venules of the mouse popliteal lymph node using the laser scanning confocal microscopy. In particular, the study focused on the post-luminal migration of T and B cells and reported the following. (1) Mice deficient in GlcNAc6ST-1 which is necessary for PNAd expression on the abluminal side of HEV showed significantly reduced abluminal migration of both T and B cells, (2) the footpad injection of the ER-TR7 antibody did not affect T cell transmigration across HEVs but marginally increased the parenchymal T cell velocity when compared with injection of control antibody, (3) T cells and B cells tended to share FRC migration hot spots but this was not the case with trans-EC migration hot spot, (4) the trans-FRC migration was observed at the FRCs closely associated with CD11c+ dendritic cells in HEV.

    While the present study is obviously the product of very meticulous and time-consuming work, it basically describes only a phenomenology, just reporting the lymphocyte behavior within and outside lymph node HEVs, without sufficiently analyzing the mechanistic aspect of the individual event they observed. The only antibody blocking experiments they performed to obtain mechanistic insights was by the use of commercially available monoclonal antibodies, all of which unfortunately contained a preservative, sodium azide, which potently blocks lymphocyte migration in vivo (Freitas AA & Bognacki J, Immunol 36:247, 1979). Therefore, the results of these antibody blocking experiments cannot be taken at face value.

    We thank the reviewer for raising the important point. Freitas et al used pre-treated lymphocytes with sodium azide in vitro for 1 hour while we injected the antibody into the footpad of recipient mouse 3 hours before lymphocyte injection via tail vein and imaging. Sodium azide might be highly diluted in vivo condition. In addition, Fig. S3 shows no significant difference in T cell migration in HEV between anti-ER-TR7 antibody-injected and non-injected groups although the anti-ER-TR7 antibody also contains sodium azide. We believe that the effect of sodium azide on our convincing results of the PNAd-blocking antibody compared to the control antibody (Fig. S8) may be insignificant. The potential side effect of sodium azide was mentioned in the Methods of the revised manuscript (page 22) as follows.

    “All antibodies we used contains sodium azide that has potential side effects on lymphocyte migration in lymph node57. However, Fig. S3 shows no significant difference in T cell migration in HEV between anti-ER-TR7-injected and non-injected groups.”

    Reviewer #2 (Significance (Required)):

    Real time imaging experiments were performed very carefully. However, as mentioned above, authors used sodium azide-containing antibodies for blocking experiments, and hence, these experiments cannot be interpreted properly.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This study presents a detailed investigation of T and B cell entry into lymph nodes (LN) via HEV. Substantial high quality intravital imaging is used to examine trans-EC and trans-FRC migration and define the role of PNAds in this process. The authors find that T and B cells use 'hot spots' to cross EC and FRC barriers, which supports prior similar observations by others. They also show that where T and B cells cross EC and FRC layers can differ, with regions of shared trans-FRC migration but more distinct EC crossing sites. This may relate to differences in the structure of these cellular layers, but provides novel insight into the mechanisms of cell entry into LNs via HEV. Assessment of the dependence on PNAd using antibodies or GlcNAc6ST-1 KO mice revealed perivascular and parenchymal cell behavior is also influenced by these signals. Lastly, examination of DCs that sit on the perivascular FRCs suggested that cells may prefer to cross at sites co-localized by DCs, although the reasons for this are not explored.

    This is a well performed study, with high quality imaging data and analysis. The results are convincing, with sufficient numbers of mice and adequate statistical analysis. There are a number of minor grammatical errors throughout the text, which should be easy to fix.

    We thank the reviewer for the positive evaluation. We carefully performed proofreading repeatedly to correct typos and grammatical errors.

    Reviewer #3 (Significance (Required)):

    Although 'hot spots' have been proposed by others, this detailed analysis provides new knowledge of how lymphocytes can cross the HEV and FRC barriers to enter LNs. This is an important study to advance our understanding of cell recruitment to lymph nodes. The role of perivascular and parenchymal PNAd signals observed here should also be of interest to immunologists to help define the signals required for immune cell motility in tissues.

    Reviewer #4 (Evidence, reproducibility and clarity (Required)):

    The authors have used a combination of intravital confocal imaging and transgenic models to study the migration of T and B cells through the HEVs. They move on from Moscacci et al. and Park et al., studies on lymphocyte migration. This study focuses on visualization and molecular mechanism of post-trans-EC migration, including the intra-PVC and trans-FRC migration of T and B cells in HEVs. They have been able to show how lymphocytes migrate through the HEV into the parenchyma. Using the GlcNAc6sT-1 (catalyst for sulfation of PNAds) KO model (and MECA control for PNAds blocking) they identify the role of L-selectin/PNAd for lymphocyte transmigration. The identification of hot spots of T and B cell transmigration in HEVs is novel and extremely interesting for the field however the data shown is not entirely convincing in their current form. The hot spots were defined as areas where the lymphocytes migrate through the HEV epithelial cells and pericyte (FRC) regions. These are areas where migration was greatly shared T and B cells. Using the CD11c-YFP mouse model they identified CD11c+ cells in proximity to the FRCs located at the migration hotspots which can drive further speculation regarding the mechanism by which these areas of the HEVs are more permissive.

    **Major comments**

    1. Intravital imaging of T and B cell transmigration across HEVS composed of ECs and FRCs
    • Figure 1: The authors mention that they performed similar experiments for B cells. Authors should show comparative data for T cells and B cells.
    • Panel S1B should be provided for both T and B cells in figure 1.

    We added the image sequence of B cell migration and the panels (Fig S1B of previous manuscript) showing intra-PVC segments of T or B cells in Fig. 1C of the revised manuscript as follows.

    1. T and B cells preferentially share hotspots for trans-FRC migration not EC-migration
    • Figure 4: This data is important to the storyline but as presented it is difficult to understand. Results are overstated in the text however it is difficult to see where these conclusions come from based on the figure. In Figure 4B the authors should show percentages on the Venn diagram or remove it entirely. In Figure 4C the authors should add labels to their y-axis and separate the data in order to assist with the storyline and convince of the presence of hot spots.

    We agree with the reviewer’s opinion. We removed the Venn diagram, separated the Fig. 4C into 4B and 4C, and added y-axis labels in the figures. In addition, we revised the figure legends and the text in the Results to make it easier to understand as follows.

    In the figure legend, “…(B-C) The round and diamond symbols represent predicted and observed values, respectively, for the percentage of T cell hot spots in B cell hot spots (B), for the percentage of B cell hot spots in T cell hot spots (C). …”

    In the Results (page12), “Simultaneously imaging T and B cells showed that some T and B cells transmigrated across FRCs at the same site (Fig. 4A and Movie S8). To investigate whether T and B cells share their hot spots preferentially or accidentally, we compared the percentage of T cell hot spots in total B cell hot spots (diamond symbols in Fig. 4B) with its predicted value that is the possibility of accidently sharing T and B cell hot spots (round symbols in Fig. 4B). The predicted value can be calculated as the percentage of T cell hot spots in total transmigration sites. To note, the percentage of hot spots in total sites for trans-FRC migration was higher than that for trans-EC migration (Fig. 3G and round symbols in Fig. 4B) maybe because the number of trans-FRC migration sites was less than that of trans-EC migration sites. It implies that the possibility of accidently sharing T and B cell hot spots for trans-FRC migration is higher than that for trans-EC migration. However, surprisingly, the percentage of T cell hot spots in B cell hot spots was significantly higher than its predicted value of accidently sharing hot spots for trans-FRC migration (Fig. 4B). Similarly, the percentage of B cell hot spots in T cell hot spots was also significantly higher than its predicted value for trans-FRC migration (Fig. 4C). These results imply that T and B cells preferentially share trans-FRC migration hot spots beyond the prediction for accidently sharing. However, there were no significant differences between observed and predicted values for trans-EC migration (Fig. 4B and 4C), which implies T and B cells just accidently share their trans-EC migration hot spots.”

    1. T and B cells prefer to transmigrate across FRCs covered by perivascular CD11c+ DCs
    • DCs drive changes to FRC phenotype and contractility. The interaction between CLEC-2 (on DCs and platelets) is important for driving permeability of the HEVs. The authors use the CD11c-YFP mouse model in Figure 5 (and the supporting figures) to show the proximity of the CD11c+ cells and FRCs. Data from Baratin et al., (Immunity, 2017) suggest that CD11c+ cells in the parenchyma are also T cell zone macrophages (TZMs) that were previously characterized as DCs. Macrophages have previously been shown important for perivascular transmigration of neutrophils during bacterial skin infection (Abtin et al.2014- Nat Immun). CD11c-YFP alone does not show the cells proximal to FRCs are DCs so the authors should try to stain them with CLEC-2 or use the CLEC9a-cre mouse model to better characterise these cells.

    We thank the reviewer for raising important point. We agree that the perivascular CD11c+ cells could be T-cell-zone macrophages (TZMs). Better characterization of the CD11c+ cells located in the hot spots of HEVs is required to determine if they are DCs or macrophages, and also to differentiate them from the other CD11c+ cells observed in the non-hot-spot regions of the HEVs. To differentiate DCs from TZMs, Zbtb46-GFP mouse can be used for imaging because Zbtb46-GFP are highly expressed in conventional DCs (cDCs) but not monocytes, macrophages, or other lymphoid or myeloid lineages (Satpathy et al, JEM 2012). However, endothelial cells also express Zbtb46-GFP. To visualize only DCs in HEVs, we need to make a chimeric mouse by adoptive transfer of Zbtb46-GFP bone-marrow cells into irradiated wild-type mouse. Furthermore, using a triple transgenic mouse with Zbtb46-cre;tdTomato and CD11b-GFP will be able to differentiate 3 types of DCs and TZMs potentially associated with the hot spots: Zbtb46+CD11b- cDC1 (red), Zbtb46+CD11b+ cDC2 (yellow), and Zbtb46-CD11b+ macrophage (green). However, since generation or obtaining of those transgenic mice models including CLEC9a-cre mouse will take long time, we will leave this work as future research and discussed this point in the Discussion of the revised manuscript as follows. In addition, we think that it will be difficult to differentiate the CLEC2 of perivascular DCs from that of platelets by in vivo labeling by injection of anti-CLEC2 antibody conjugated with a fluorescent dye because the CLEC2 of platelets maintains HEV integrity with interacting of FRC podoplanin (Herzog et al, Nature 2013).

    In the Discussion (page 19), “… In addition, better characterization of the CD11c+ DCs located in the hot spots of HEVs is required to differentiate them from the other CD11c+ DCs observed in the non-hot-spot regions of HEVs. Some T-cell-zone resident macrophages can also express CD11c54. Imaging of a triple-transgenic mouse with Zbtb46-cre;tdTomato and CD11b-GFP will be able to differentiate 3 types of DCs and macrophages potentially associated with the hot spots: Zbtb46+CD11b- cDC1 (red), Zbtb46+CD11b+ cDC2 (yellow), and Zbtb46-CD11b+ macrophage (green)54,55.”

    **Minor comments**

    1. Intravital imaging of T and B cell transmigration across HEVS composed of ECs and FRCs
    • The velocity differences observed could be due to location of HEV in the parenchyma. Furthermore, FRC plasticity can cause differences in secretion of chemokine gradients based on the location of cells and their niche (Rhoda et al., Immunity 2018). HEVs regulation of lymphocyte entry can be influenced by their niche (Veerman et al., Cell Reports 2019). The authors should comment on the HEV position relative to B cell areas.

    We included this point with the references (Rhoda et al, immunity 2018, ref 27; Veerman et al., Cell Rep. 2019, ref 60) in the Discussion of the revised manuscript (page 19-20) as follows.

    “Compared to T cell, B cells took a longer time to pass EC and FRC layers in HEV and had lower velocity in PVC and parenchyma just after extravasation. Furthermore, the adhesion rate of B cells to HEV EC in luminal side is lower than that of T cells5. These could be attributed to lower expression of L-selectin and CCR7 on B cells than T cells18,59. The difference in homing efficiency between T and B cells may vary depending on the HEV location due to the heterogeneous expression of chemokines and integrins on HEV EC and surrounding FRCs in peripheral lymph node27,60. The HEVs imaged in this work were located around 40-70 μm depth from the capsule where might be close to B cell follicles. B cell homing efficiency in the deeper paracortical T cell zone could be different from our data probably due to less CXCL13 that is chemoattractant for B cells highly expressed in follicles. …”

    • Images shown in Fig1A is the same as Fig S1A/B. I presume this is an error.

    Fig. 1A and Fig. S1A correspond to a 20-um-thick maximum intensity projection and single z-frame without projection, respectively. To avoid the confusion, we changed Fig.1A to the single z-frame (Fig S1A) and remove the 20-um thick maximum projection.

    • Figure S3: Data for Ab treated appears to be identical to what is shown for T cells in Fig 1. I presume this is an error and the correct control will be shown.

    We used the data of Fig. 1D-1I as the Ab-injected group in Fig. S3. We are sorry for the lack of clear explanation about this. We included the explanation in the figure legend as follows.

    In the legend of Fig. S3, “(A-E) There is no significant difference between antibody-injected group (Ab) and non-injected group (Non) in T cell migration from trans-EC migration to trans-FRC migration. Non-injected means that no substance is injected into a footpad of mouse. We used the data of Fig. 1D-1I as the antibody-injected group. …”

    1. Non-redundant role of L-selectin/PNAd interactions in post-luminal migration of T and B cells in HEV
    • Could the authors clarify the number of mice used for this analysis (same applies to figure 1)

    In the legends of Fig. 1-2, S6 and S8, there is the number of mice we used. In Fig. 1, “Four and 3 mice were used for the analysis of T and B cells, respectively.” In Fig. 2, “Four mice were analysed for each group.” In Fig. S6, “Three mice were analysed for each group.” In Fig. S8, “Five and 4 mice were analysed for the control Ab and MECA79 groups, respectively.”

    In addition, we added the number of mice in the legend of Fig. S7. In Fig. S7, “The images are representative of 4 popliteal lymph nodes of 2 mice and 2 popliteal lymph nodes of a mouse for MECA79 and control IgM antibody, respectively.”

    • Figure S6: further to percentages of T cell populations the authors should also provide the number of T cells (CD4, CD8, CM and naive) for both wildtype and KO.

    We included the analyzed cell number by FACS in Fig. S6 and revised the figure legend as follows.

    In the Fig. S6, “… (B) Analyzed cell numbers by FACS for 3 control and 3 KO mice. (C) Percentage of each type of T cells in DsRed+ T cells. No difference in the percentage of homing central memory, Naïve CD4 and CD8 T cells between wild-type and KO mice. …”

    **Methods** for the flow cytometry analysis could the details of how samples were processed (or reference) be provided.

    We added the details in the Methods (page 24) as follows.

    “Popliteal and inguinal lymph nodes were harvested and single-cell suspensions were prepared by mechanical dissociation on a cell strainer (RPMI-1640 with 10% FBS). Cell suspensions were centrifuged at 300g for 5 min. Erythrocytes in lymph nodes were lysed with ACK lysis buffer for 5 min at RT. Cell suspensions were washed and filtered through 40um filters. Non-specific staining was reduced by using Fc receptor block (anti-CD16/CD32). Cells were incubated for 30 min with varying combinations of the following fluorophore-conjugated monoclonal antibodies: anti-CD3e (clone 145-2C11, BD pharmigen), anti-CD4 (clone GK1.5, BD Pharmingen), anti-CD8 (clone 53-6.7, eBioscience), anti-CD44 (clone IM7, Biolegend) and anti-CD62L (clone MEL-14, eBioscience) antibodies (diluted at a ratio of 1:200) in FACS buffer (5% bovine serum in PBS). After several washes, cells were analyzed by FACS Canto II (BD Biosciences) and the acquired data were further evaluated by using FlowJo software (Treestar).

    **References:** The discussion covers key references in the field, but more recent studies should be included. Some examples have been suggested in the comments sections. Key references missing that can help discussion/interpretation of the data include: 1) Veerman et al 2019, Cell reports. The data in that paper shows the heterogeneity of the HEV and different regulation of genes that control lymphocyte entry. This can also be linked to the comments above regarding section 1 and 2. 2) Rhodda et al 2018, Immunity that focuses on niche-associated heterogeneity of lymph node stromal cells. The authors should also include Webster et al., 2006, JEM which describes the role of DCs in regulating vascular growth in the lymph node.

    We thank the reviewer for suggesting good references to discuss. We included the references #1 and #2 in the revised manuscript as we responded to the minor comment #1. We also cited Webster et al., JEM 2006 (as ref 65) in the Discussion of the revised manuscript (page 20) as follows.

    “Inflamed peripheral lymph node become larger by recruiting more lymphocytes and even L-selectin-negative leukocytes that are excluded in the steady state63,64. Inflamed HEV ECs show different gene expression, such as downregulation of GLYCAM1 and GlcNAc6ST-160. In addition, inflamed HEV integrity may be loosen due to markedly increased leukocyte influx although the HEV FRCs can prevent bleeding by interacting with platelet CLEC-248. CD11c+ DCs are associated with inflamed HEV EC proliferation that is functionally associated with increased leukocyte entry65. The stepwise migration of lymphocyte across inflamed HEVs and their hot spots with perivascular CD11+ DCs will be interesting topic for future study.”

    Reviewer #4 (Significance (Required)):

    This paper asks important questions and can make a significant contribution to the field if all revisions are addressed. The authors identified PNAd as an important factor for T cell migration. Further to previous studies in the field suggesting non-random transmigration sites. The authors used intra-vital confocal imaging to identify how lymphocytes cross the epithelial cells and FRCs of the HEVs to migrate to the parenchyma. The authors identify hotspots used by lymphocytes to transmigrate. Finally, the authors show that CD11c+ cells are proximal to FRCs hotspots and might have a role in driving lymphocyte transmigration.

    Audience: Lymphocyte/immune cell biology, stomal immunology, FRC and lymph node inflammation. My expertise: Stomal immunology, immunology, innate immunity

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #4

    Evidence, reproducibility and clarity

    The authors have used a combination of intravital confocal imaging and transgenic models to study the migration of T and B cells through the HEVs. They move on from Moscacci et al. and Park et al., studies on lymphocyte migration. This study focuses on visualization and molecular mechanism of post-trans-EC migration, including the intra-PVC and trans-FRC migration of T and B cells in HEVs. They have been able to show how lymphocytes migrate through the HEV into the parenchyma. Using the GlcNAc6sT-1 (catalyst for sulfation of PNAds) KO model (and MECA control for PNAds blocking) they identify the role of L-selectin/PNAd for lymphocyte transmigration. The identification of hot spots of T and B cell transmigration in HEVs is novel and extremely interesting for the field however the data shown is not entirely convincing in their current form. The hot spots were defined as areas where the lymphocytes migrate through the HEV epithelial cells and pericyte (FRC) regions. These are areas where migration was greatly shared T and B cells. Using the CD11c-YFP mouse model they identified CD11c+ cells in proximity to the FRCs located at the migration hotspots which can drive further speculation regarding the mechanism by which these areas of the HEVs are more permissive.

    Major comments:

    1. Intravital imaging of T and B cell transmigration across HEVS composed of ECs and FRCs
    • Figure 1: The authors mention that they performed similar experiments for B cells. Authors should show comparative data for T cells and B cells.
    • Panel S1B should be provided for both T and B cells in figure 1.
    1. T and B cells preferentially share hotspots for trans-FRC migration not EC- migration
    • Figure 4: This data is important to the storyline but as presented it is difficult to understand. Results are overstated in the text however it is difficult to see where these conclusions come from based on the figure. In Figure 4B the authors should show percentages on the Venn diagram or remove it entirely. In Figure 4C the authors should add labels to their y-axis and separate the data in order to assist with the storyline and convince of the presence of hot spots.
    1. T and B cells prefer to transmigrate across FRCs covered by perivascular CD11c+ DCs
    • DCs drive changes to FRC phenotype and contractility. The interaction between CLEC-2 (on DCs and platelets) is important for driving permeability of the HEVs. The authors use the CD11c-YFP mouse model in Figure 5 (and the supporting figures) to show the proximity of the CD11c+ cells and FRCs. Data from Beratin et al., (Immunity, 2017) suggest that CD11c+ cells in the parenchyma are also T cell zone macrophages (TZMs) that were previously characterised as DCs. Macrophages have previously been shown important for perivascular transmigration of neutrophils during bacterial skin infection (Abtin et al.2014- Nat Immun). CD11c-YFP alone does not show the cells proximal to FRCs are DCs so the authors should try to stain them with CLEC-2 or use the CLEC9a-cre mouse model to better characterise these cells.

    Minor comments:

    1. Intravital imaging of T and B cell transmigration across HEVS composed of ECs and FRCs
    • The velocity differences observed could be due to location of HEV in the parenchyma. Furthermore FRC plasticity can cause differences in secretion of chemokine gradients based on the location of cells and their niche (Rhoda et al., Immunity 2018).HEVs regulation of lymphocyte entry can be influenced by their niche (Veerman et al., Cell Reports 2019).The authors should comment on the HEV position relative to B cell areas.
    • Images shown in Fig1A is the same as Fig S1A/B. I presume this is an error.
    • Figure S3: Data for Ab treated appears to be identical to what is shown for T cells in Fig 1. I presume this is an error and the correct control will be shown.
    1. Non-redundant role of L-selectin/PNAd interactions in post-luminal migration of T and B cells in HEV
    • Could the authors clarify the number of mice used for this analysis (same applies to figure 1)
    • Figure S6: further to percentages of T cell populations the authors should also provide the number of T cells (CD4, CD8, CM and naive) for both wildtype and KO.

    Methods:

    for the flow cytometry analysis could the details of how samples were processed (or reference) be provided.

    References:

    The discussion covers key references in the field but more recent studies should be included. Some examples have been suggested in the comments sections.Key references missing that can help discussion/interpretation of the data include: 1) Veerman et al 2019, Cell reports. The data in that paper shows the heterogeneity of the HEV and different regulation of genes that control lymphocyte entry. This can also be linked to the comments above regarding section 1 and 2. 2) Rhodda et al 2018, Immunity that focuses on niche-associated heterogeneity of lymph node stromal cells. The authors should also include Webster et al., 2006, JEM which describes the role of DCs in regulating vascular growth in the lymph node.

    Significance

    This paper asks important questions and can make a significant contribution to the field if all revisions are addressed. The authors identified PNAd as an important factor for T cell migration. Further to previous studies in the field suggesting non-random transmigration sites. The authors used intra-vital confocal imaging to identify how lymphocytes cross the epithelial cells and FRCs of the HEVs to migrate to the parenchyma. The authors identify hotspots used by lymphocytes to transmigrate. Finally the authors show that CD11c+ cells are proximal to FRCs hotspots and might have a role in driving lymphocyte transmigration.

    Audience: Lymphocyte/immune cell biology, stomal immunology, FRC and lymph node inflammation.

    My expertise: Stomal immunology, immunology, innate immunity

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    This study presents a detailed investigation of T and B cell entry into lymph nodes (LN) via HEV. Substantial high quality intravital imaging is used to examine trans-EC and trans-FRC migration and define the role of PNAds in this process. The authors find that T and B cells use 'hot spots' to cross EC and FRC barriers, which supports prior similar observations by others. They also show that where T and B cells cross EC and FRC layers can differ, with regions of shared trans-FRC migration but more distinct EC crossing sites. This may relate to differences in the structure of these cellular layers, but provides novel insight into the mechanisms of cell entry into LNs via HEV. Assessment of the dependence on PNAd using antibodies or GlcNAc6ST-1 KO mice revealed perivascular and parenchymal cell behaviour is also influenced by these signals. Lastly, examination of DCs that sit on the perivascular FRCs suggested that cells may prefer to cross at sites co-localised by DCs, although the reasons for this are not explored.

    This is a well performed study, with high quality imaging data and analysis. The results are convincing, with sufficient numbers of mice and adequate statistical analysis. There are a number of minor grammatical errors throughout the text, which should be easy to fix.

    Significance

    Although 'hot spots' have been proposed by others, this detailed analysis provides new knowledge of how lymphocytes can cross the HEV and FRC barriers to enter LNs. This is an important study to advance our understanding of cell recruitment to lymph nodes. The role of perivascular and parenchymal PNAd signals observed here should also be of interest to immunologists to help define the signals required for immune cell motility in tissues.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The present study by K. Choe meticulously monitored the stepwise transmigration behavior of T cells and B cells, respectively, through the high endothelial venules of the mouse popliteal lymph node using the laser scanning confocal microscopy. In particular, the study focused on the post-luminal migration of T and B cells and reported the following. (1) Mice deficient in GlcNAc6ST-1 which is necessary for PNAd expression on the abluminal side of HEV showed significantly reduced abluminal migration of both T and B cells, (2) the footpad injection of the ER-TR7 antibody did not affect T cell transmigration across HEVs but marginally increased the parenchymal T cell velocity when compared with injection of control antibody, (3) T cells and B cells tended to share FRC migration hot spots but this was not the case with trans-EC migration hot spot, (4) the trans-FRC migration was observed at the FRCs closely associated with CD11c+ dendritic cells in HEV.

    While the present study is obviously the product of very meticulous and time-consuming work, it basically describes only a phenomenology, just reporting the lymphocyte behavior within and outside lymph node HEVs, without sufficiently analyzing the mechanistic aspect of the individual event they observed. The only antibody blocking experiments they performed to obtain mechanistic insights was by the use of commercially available monoclonal antibodies, all of which unfortunately contained a preservative, sodium azide, which potently blocks lymphocyte migration in vivo (Freitas AA & Bognacki J, Immunol 36:247, 1979). Therefore, the results of these antibody blocking experiments cannot be taken at face value.

    Significance

    Real time imaging experiments were performed very carefully. However, as mentioned above, authors used sodium azide-containing antibodies for blocking experiments, and hence, these experiments cannot be interpreted properly.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Extravasation of lymphocytes from HEV in the lymph nodes is mediated by the interaction between lymphocyte L-selectin and PNAd-carrying sulfated sugars expressed by HEVs. Multiple steps of lymphocyte migration interacting with ECs at the luminal side of HEVs have been studied intensively; however, post-luminal migration steps are unclear. In this study, using intravital confocal microscopy of peripheral lymph nodes (pLNs), the authors found that GlcNAc6ST1 deficiency, required for sulfation of PNAd, delays trans-fibroblastic reticular cell (FRC) migration of lymphocytes, and hot spots of trans-HEV EC migration and trans-FRC migration. Interestingly, hot spots of trans-FRC migration are often associated with dendritic cells (DCs). Thus, the authors concluded that FRCs delicately regulate the transmigration of T and B cells across the HEV wall, which could be mediated by perivascular DCs.

    Main comments:

    1. This study focused on pLNs, which are quite different from mesenteric lymph nodes (mLNs) in many ways. The authors should include mLNs in their study to make the general statement with regard to the T/B cell entry into lymph nodes. In addition, it will be more significant if this study includes challenged pLNs.
    2. The finding that GlcNAc6ST1 deficiency delays lymphocyte trans-FRC migration but not trans-HEV EC migration is surprising. However, the reason this occurs is neither shown nor discussed. Is GlcNAc6ST1 also expressed in FRCs? Or does GlcNAc6ST1 expression on HEV license lymphocytes to transmigrate across FRCs?
    3. Because of the adoptive transfusion experiment, the actual number of transmigrating lymphocytes in Fig. 3F is underestimated.
    4. Whether DCs covering FRCs have a role for lymphocyte trans-migration is not shown.
    5. In Fig. 1, time required for trans HEV EC migration and trans-FRC migration of T cells is shorter than that of B cells; however, this finding is not observed in Fig. 2C and E.

    Minor comments:

    1. Please provide evidence for GlcNAc6ST1 deficiency in HEV and surrounding tissues.
    2. Images for delayed trans-FRC migration in GlcNAc6ST1 KO mice relative to WT are not convincing (Fig. 2G and H).
    3. Provide actual time periods required for Fig. 3F and G. Lack of isotype control IgG experiment in Fig. S3.
    4. Line 12 on page 11, "the ratio of hot spots to the total;observed' transmigration sites..." is not appropriate. The ratio must be calculated by hot spots to the total "potential" transmigration sites, although it is challenging to find total potential sites.
    5. Please correct typos of angiomoduin to angiomodulin (page 16), ET-TR7 to ER-TR7 (page 17), Anti-CD3 to anti-CD3 (page 22), half the dose to half dose (page 22), the Multiple step to the multiple step (page 23).
    6. Please provide an additional explanation of why actin-DsRed in HEVs is more strongly expressed than surrounding tissues such as FRCs in Fig. 1 although actin-DsRed should be expressed in all cell types in mice.

    Significance

    The study focused on lymphocytes post extravasation of HEV, which is an understudied question, using intravital imaging. The in vivo imaging study was deliberately and beautifully performed, and the finding is insightful for understanding lymphocyte trafficking in lymph nodes. However, additional experimental should be performed to address some weaknesses listed in our comments.

  6. Version published to 10.1101/2020.12.20.423079 on bioRxiv