Essential role of submandibular lymph node dendritic cells in protective sublingual immunotherapy against murine allergy

While sublingual immunotherapy (SLIT) is known as an allergen-specific treatment for type-1 allergies, how it controls allergic pathogenesis remains unclear. Here, we show the prerequisite role of conventional dendritic cells in submandibular lymph nodes (ManLNs) in the effectiveness of SLIT for the treatment of allergic disorders in mice. Deficiency of conventional dendritic cells or CD4+Foxp3+ regulatory T (Treg) cells abrogates the protective effect of SLIT against allergic disorders. Furthermore, sublingual antigenic application primarily induces antigen-specific CD4+Foxp3+ Treg cells in draining ManLNs, in which it is severely impaired in the absence of cDCs. In ManLNs, migratory CD11b+ cDCs are superior to other conventional dendritic cell subsets for the generation of antigen-specific CD4+Foxp3+ Treg cells, which is reflected by their dominancy in the tolerogenic features to favor this program. Thus, ManLNs are privileged sites in triggering mucosal tolerance mediating protect effect of SLIT on allergic disorders that requires a tolerogenesis of migratory CD11b+ conventional dendritic cells.


Overall impressions
The authors showed SLIT had a potential to protect the development of Ag-specific allergic disorders by using mouse model. It is interesting to examine the immunological mechanisms in the experimental prophylactic SLIT. Especially, the author clearly showed the induction of Ag-specific Tregs after sublingual Ag application, and the roles and characteristics of cDC subset in the induction of Tregs. These results may be a reference to understand the mechanism of conventional therapeutic administration of SLIT as well.
Specific comments 1) I think it is more appropriate to add the phrase "prophylactic" or "protective" before "sublingual immunotherapy" in the title.
3) The authors described the aim is to clarify the contribution of cDCs to the effectiveness of SLIT protecting against Th2-mediated allergic air way responses in P6.L3-4 and concluded the absence of cDCs impairs protective effect of SLIT on the development of these Th2-mediated allergic pathogenesis in P6L16-17 and P7L8-9 by two results that the number of eosinophils in BALF cells and systemic anaphylaxis were not recovered with SLIT in CD11c-DTR/EGFR Tg mice with DT. However, I think the contributions of cDCs were not fully shown to the effectiveness of SLIT protecting against Th2-mediated allergic responses in Fig 1. What were the results regarding Th2 responses such as IgG1, IgE, airway function, or Ag-specific Th2 cell responses in CD11c-DTR/EGFR Tg mice?
4) The author showed the generation of ST2+CD4+pathogenic T cells were suppressed in the asthmatic mice and mice with food allergy with prophylactic SLIT in Fig2 a-c and sFig2 d-f. And in conclusion, the author described migratory CD11b+cDCs are involved in the generation of Agspecific CD4+Foxp3+ Tregs that inhibit the pathogenic Th2-mediated allergic immune response P13L4-6. However, the contribution of pathogenic ST2+ T cells on the pathogenesis of allergic disorders in the model mouse, the relationship between pathogenic T cells and Ag-specific CD4+Foxp3+ Tregs or cDCs, or the molecular and immunological mechanisms were not examined in this study. To make the conclusion, I think the author should show some mechanism.
5) The author clearly showed that sublingually administrated OVA-Ag was captured and retained in migratory cDCs in ManLNs in Fig3d Reviewer #2: Remarks to the Author: The manuscript by Miyanaga, et. al. reports that migratory CD11b+ cDCs in ManLNs provide a milieu of Treg-driven mucosal tolerance inhibiting allergic Th2-responses that mediates the efficacy of SLIT on acute and chronic type I allergic disorders. The research topic is very important. The experiments conducted are detailed and thorough and the mechanistic findings are novel and intriguing. Despite the enthusiasm, a few concerns need to be addressed by the authors, before the manuscript can be considered for publication. 1. In Fig. 1, the authors need to detect the Th2 cytokine expression (such as IL4 or IL13) by ELISA to see if deficiency of cDCs really attenuates the protective effect of SLIT on OVA induced Th2mediated allergic airway inflammation. 2. In Fig. 2i, the authors need to include the scale bar for the IFA figures. 3. In the figure legend of Fig. 1j, there are mistakes for the methacholine doses (j; 3.125-50 mg/ml). The authors need to revise it. 4. In the figure legend of Supplementary Fig.2, the authors need to change the capital A-F into small.

Response to the reviewer #1
We carefully revised the manuscript according to the reviewers' comments and Specific comments 1) I think it is more appropriate to add the phrase "prophylactic" or "protective" before "sublingual immunotherapy" in the title.
-As suggested, we added "protective" before "sublingual immunotherapy" in the title in the revision.

2) What mouse strain were used in Fig1 e-h, j-l? Balbc/WT?
-We used Balb/c WT mice. As suggested, we added "in Balb/c WT mice" in the Figure   1 legend in the revision. -As suggested, we showed that CD4 + CD25 + T reg cells, but not CD4 + CD25 -T cells obtained from mice received sublingual antigenic application exhibited a more inhibitory effect on Ag-specific proliferation of KJ1-26 + CD4 + ST2 + CD44 + T cells than CD4 + CD25 + T reg cells from naïve mice in Supplementary Fig. 7e-g in the revision. -Regarding Fig. 4i, the adaptive transfer with CD4 + CD25 + T reg cells obtained from mice received sublingual antigenic application exhibited a more potent protection against the development of systemic anaphylaxis than CD4 + CD25 + T reg cells from naive mice. We added the statistics symbol ( †) between CD25 + from SLIT mice and CD25 + from naive mice in Fig. 4i in the revision.

Response to the reviewer #2
We -Possibly due to the B6-backgroud, we could not detect serum production of IL-4 and IL-13 in CD11c-DTR/EGFP mice that had received immunization with OVA protein plus alum adjuvant. On the other hand, the sublingual administration with OVA protein