Fate mapping analysis reveals a novel murine dermal migratory Langerhans-like cell population
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Evaluation Summary:
The present study uses innovative approaches to further our knowledge of skin immunobiology. The corresponding results explain the expression of langerin on two fractions of dermal DC (CD103+ and CD103-) observed several years ago. The demonstration that epidermal LC do not contribute to LN populations in the steady state is completely unexpected and raises important questions about the in vivo function of the LC-like subset unveiled in the present study.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Dendritic cells residing in the skin represent a large family of antigen-presenting cells, ranging from long-lived Langerhans cells (LC) in the epidermis to various distinct classical dendritic cell subsets in the dermis. Through genetic fate mapping analysis and single-cell RNA-sequencing, we have identified a novel separate population of LC-independent CD207 + CD326 + LC like cells in the dermis that homed at a slow rate to the lymph nodes (LNs). These LC like cells are long-lived and radio-resistant but, unlike LCs, they are gradually replenished by bone marrow-derived precursors under steady state. LC like cells together with cDC1s are the main migratory CD207 + CD326 + cell fractions present in the LN and not, as currently assumed, LCs, which are barely detectable, if at all. Cutaneous tolerance to haptens depends on LC like cells, whereas LCs suppress effector CD8 + T-cell functions and inflammation locally in the skin during contact hypersensitivity. These findings bring new insights into the dynamism of cutaneous dendritic cells and their function opening novel avenues in the development of treatments to cure inflammatory skin disorders.
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Reviewer #3 (Public Review):
In this manuscript, Sheng et al. have demonstrated that Langerhans cells (LCs) do not exit the skin both under steady-state conditions and after skin sensitization, using newly generated DC-SIGN DT mice and others. In addition, through a combined use of genetic fate mapping and novel inducible LC ablating mouse models, they show that the originally described lymph node LC fraction is actually an independent LClike cell population that originates from the dermis, not from the epidermis. Moreover, these LClike cells, which are replaced over time by bone marrow-derived counterparts, are characterized by their slow turnover rate and trafficking to the LN. This study contains novel and important findings. This reviewer has several comments on the manuscript.
Major comments:
The functional roles of LClike cells …
Reviewer #3 (Public Review):
In this manuscript, Sheng et al. have demonstrated that Langerhans cells (LCs) do not exit the skin both under steady-state conditions and after skin sensitization, using newly generated DC-SIGN DT mice and others. In addition, through a combined use of genetic fate mapping and novel inducible LC ablating mouse models, they show that the originally described lymph node LC fraction is actually an independent LClike cell population that originates from the dermis, not from the epidermis. Moreover, these LClike cells, which are replaced over time by bone marrow-derived counterparts, are characterized by their slow turnover rate and trafficking to the LN. This study contains novel and important findings. This reviewer has several comments on the manuscript.
Major comments:
The functional roles of LClike cells remains unclear, especially in the relationship with conventional LCs. Thus far it has been considered that LCs play essential roles in OVA-induced atopic dermatitis like models. But it remain unclear whether conventional LC s or LC like cells play important roles.
The authors stated that a fraction of CD11bhiF4/80hi cells co-expressed CD326 and CD207 are detected in the dermis (Fig. 1b, upper panel), which is likely to be derived from the epidermis. But it remains unclear whether this subset is just a contamination through the separation process or a truly migratory one from the epidermis. The authors can demonstrate clear localization of LCs in the epidermis, LClike cells, and LCs in the dermis.
Related to the above question, the authors claim that LCs can emigrate from the epidermis to the dermis. Given that LCs can emigrate from the epidermis to the dermis by transmigrating through the basement membrane, why LCs cannot migrate into the lymphatic vessels. LCs are known to express CCR7 highly that is important for migrating into the lymph nodes from the skin. What is the functional (APC, migration, etc) difference between LCs and LClike cells?
I agree that the novel subset exists as CD207+CD326+ LClike cells in the dermis, which is different from conventional LC. But the term, "LC-independent" CD207+CD326+ LClike cells, which the authors used often through the manuscript, is a bit confusing, because it is not totally clear whether LClike cells are completely independent of LCs or not. It would be informative if the authors can demonstrate whether LClike cells contain bierbeck granules (this is also a hallmark of LCs) or not, since bierbeck granule-positive cells were detected in the LN (https://pubmed.ncbi.nlm.nih.gov/4758275).
The authors can discuss the relationship between LClike cells and short lived LCs that were previously described (https://pubmed.ncbi.nlm.nih.gov/23159228).
Where do LClike cells locate by FACS plots analysis using CD11b and CD103?
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Reviewer #2 (Public Review):
The authors used several approaches to define a discrete population of Langerhans cell-like (LC-like) dendritic cells (DC) in the dermis of mice. By flow cytometry, these cells expressed langerin/CD207 and EpCAM/CD236 and were found in the CD103-CD11b- fraction of dermal cells. It was also shown that LC-like cells, rather than LC, are the main contributors to CD103- langerin+ cells in the lymph node. By single cell RNAseq of dermal cells they clustered with Langerhans cells but lacked expression of DC-SIGN/CD209. Fate-mapping with Kitmercremer/Rosa26loxPSTOPloxPeYFP showed a similar origin to other dermal DC, with no yolk sac signal as observed in LC. Bone marrow chimeras, however, indicated a much slower turnover compared with other dermal DC. Independence from LC and other DCSIGN+ DC was also demonstrated …
Reviewer #2 (Public Review):
The authors used several approaches to define a discrete population of Langerhans cell-like (LC-like) dendritic cells (DC) in the dermis of mice. By flow cytometry, these cells expressed langerin/CD207 and EpCAM/CD236 and were found in the CD103-CD11b- fraction of dermal cells. It was also shown that LC-like cells, rather than LC, are the main contributors to CD103- langerin+ cells in the lymph node. By single cell RNAseq of dermal cells they clustered with Langerhans cells but lacked expression of DC-SIGN/CD209. Fate-mapping with Kitmercremer/Rosa26loxPSTOPloxPeYFP showed a similar origin to other dermal DC, with no yolk sac signal as observed in LC. Bone marrow chimeras, however, indicated a much slower turnover compared with other dermal DC. Independence from LC and other DCSIGN+ DC was also demonstrated by unchanged kinetics during continuous ablation of DCSIGN+ fractions in a DTR model.
The results explain the expression of langerin on two fractions of dermal DC observed several years ago (CD103+ and CD103-). The demonstration that epidermal LC do not contribute to LN populations in the steady state is completely unexpected and raises an important question of the in vivo function of the LC-like subset. LC-like cells appear to be related to LC but a more in-depth analysis of their gene expression differences compared with LC would be interesting. Also, their potential relationships with other DC (cDC1 or cDC2), was not defined. Langerin expression by human cDC2 is well described and possibly correlated with these observations in mice. Finally, the turnover of LC-like cells was slow, yet they contributed as many langerin+ cells to the resting lymph node as cDC1, which turnover quickly. Proliferation in situ might explain this observation but there were no data on this.
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Reviewer #1 (Public Review):
In the days of the COVID-19 pandemic vaccines, mechanisms of vaccine administration are important and of broad interest. Vaccines are most often given into the skin. Antigen-presenting cells of the skin are responsible for eliciting the immune response in draining lymph nodes. Langerhans cells, the dendritic cell variant of the epidermis, are one of these cutaneous antigen presenting cells that are believed to do this job. They migrate from the skin, the site of antigen/vaccine uptake to the draining lymph nodes, where lymphocytes are located and where the immune reaction will be initiated. With their sophisticated experiments, the authors challenge this view. They use leading edge methodology (mouse models) that strongly suggest that there may be yet another subset of skin antigen presenting cells, that is …
Reviewer #1 (Public Review):
In the days of the COVID-19 pandemic vaccines, mechanisms of vaccine administration are important and of broad interest. Vaccines are most often given into the skin. Antigen-presenting cells of the skin are responsible for eliciting the immune response in draining lymph nodes. Langerhans cells, the dendritic cell variant of the epidermis, are one of these cutaneous antigen presenting cells that are believed to do this job. They migrate from the skin, the site of antigen/vaccine uptake to the draining lymph nodes, where lymphocytes are located and where the immune reaction will be initiated. With their sophisticated experiments, the authors challenge this view. They use leading edge methodology (mouse models) that strongly suggest that there may be yet another subset of skin antigen presenting cells, that is responsible for carrying antigen from skin to lymph — at least in the steady-state skin. This population resides in the dermis (the connective tissue part of skin), as opposed to the classical Langerhans cells, which sit in the epidermis. This may be relevant to the maintenance of immunologic tolerance to innocuous substances in the absence of an overt inflammation. The data suggest that Langerhans cells may not play the crucial role they were thought to play. This is certainly a conceptual advance that — like always in science, especially when experimental systems are complex, as they are here — needs to be underpinned by future studies. In the long run, it will be very interesting (but much more difficult to study) to see whether this also holds true for human skin.
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Evaluation Summary:
The present study uses innovative approaches to further our knowledge of skin immunobiology. The corresponding results explain the expression of langerin on two fractions of dermal DC (CD103+ and CD103-) observed several years ago. The demonstration that epidermal LC do not contribute to LN populations in the steady state is completely unexpected and raises important questions about the in vivo function of the LC-like subset unveiled in the present study.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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