Intestinal fibroblastic reticular cell niches control innate lymphoid cell homeostasis and function

Innate lymphoid cells (ILCs) govern immune cell homeostasis in the intestine and protect the host against microbial pathogens. Various cell-intrinsic pathways have been identified that determine ILC development and differentiation. However, the cellular components that regulate ILC sustenance and function in the intestinal lamina propria are less known. Using single-cell transcriptomic analysis of lamina propria fibroblasts, we identify fibroblastic reticular cells (FRCs) that underpin cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Genetic ablation of lymphotoxin-β receptor expression in Ccl19-expressing FRCs blocks the maturation of CPs into mature ILFs. Interactome analysis shows the major niche factors and processes underlying FRC-ILC crosstalk. In vivo validation confirms that a sustained lymphotoxin-driven feedforward loop of FRC activation including IL-7 generation is critical for the maintenance of functional ILC populations. In sum, our study indicates critical fibroblastic niches within the intestinal lamina propria that control ILC homeostasis and functionality and thereby secure protective gut immunity.

Based on our scRNA-seq data from adult intestinal fibroblasts, we have performed a differentiation trajectory analysis using the Slingshot algorithm. A distinct trajectory could be identified starting from CD81 + trophocytes to PDGFRα lo fibroblasts and subsequently bifurcating into two differentiation populations, i.e. SILT FRC and muscularis mucosae myocytes ( Fig. 1R for the attention of the reviewer). In contrast, the trajectory analysis indicates that three separate cell clusters (PDG-FRα hi telocytes, Thy1 + fibroblasts and mural cells) are not connected to the trophocyte-to-SILT FRC trajectory (Fig. R1). This result suggests that SILT FRCs may originate from CD81 + trophocytes in the adult small intestine. However, due to a lack of a suitable fate-mapping model, this pathway cannot be further validated.
Relation between FRCs in CPs, imILFs and mILFs: Indeed, cell numbers are too low to elaborate potential differences. The Ccl19-EYFP model would be suitable to enrich for SILT FRCs. We are planning to investigate in future studies the activation and differentiation of SILT FRCs under conditions of acute and chronic inflammation. Ccl19+ FRC (Fig S7). Given the scRNAseq data generated in this study, could the authors evaluate the importance of some of the other interactions which may sustain FRC differentiation and ILC recruitment prior to LTBR engagement? KIT-KITL interaction seems like a good candidate and has been shown to be involved in Peyer's patches ' formation (Chappaz, JI 2010). It has been shown previously that SILT development depends on the presence of Rorc-expressing lymphoid tissue inducer (LTi) cells and that the interaction with stromal cells depends on LTβR signalling (Tsuji et al., 2008, Immunity, PMID 18656387). Hence, LTi cells (= embryonic ILC3) initiate SILT development through the provision of LTβR ligands. In the adult, LTβR ligands are provided mainly by SILT B cells, which are important for ILF maturation (Hamada et al., 2002, J Immunol, PMID 11751946). We found that ILF maturation was not affected in SILTs of Ccl19-Cre Il7 fl/fl mice despite the reduction in ILC3 numbers. It is therefore conceivable -and in line with previous data -that both ILC3 and B cells provide LTβR ligands to promote ILF maturation.

Figure R1. Differentiation trajectory analysis of adult intestinal fibroblasts. Slingshot pseudotime analysis of scRNA-seq data from adult intestinal fibroblasts. Left UMAP indicates different cell clusters. Right UMAP indicates predicted lineages using pseudotime inference of adult intestinal fibroblast differentation. Lta by ILCs compared to B cells for example? LTa from B cells have been shown to be sufficient to drive ILF maturation. While IL-7 is very important for recruitment of ILCs, loss of ILCs in SILTs does not prevent the maturation of CP and imILF into mILFs when Il7 is lost in
We thank the reviewer for the suggestion to assess whether the KIT-KITL signaling affects ILC-FRC interaction. We found that ablation of Kitl in Ccl19-Cre + SILT FRCs did not alter ILC or lymphocyte numbers in the small intestine (Fig. R2a). Likewise, the development of Peyer's patches (Fig. R2b) or SILT structures (Fig. R2c) remained unaffected. This data suggest that KITL signaling in SILT FRCs is not critical for the process of SILT formation and maturation.

The findings relative to Citrobacter infection have to be analysed and interpreted in the context
of the loss of Ltbr in all FRCs not just SILT FRCs. In particular, the formation and function of Peyer's patches and mLNs will be affected and will probably impact the immune response to Citrobacter rodentis.
We appreciate the comment on the role of Peyer's patches and mesenteric lymph nodes during Citrobacter infection. Indeed, genetic Ltbr ablation in Ccl19-Cre + cells mice leads to impaired Peyer's patch development (Prados et al., 2020, Nat Immunol., PMID 33707780). However, as shown in Extended Data Figure 6, Peyer's patch development was not affected when Ltbr expression in adult Ccl19-iEYFP Ltbr fl/fl mice was prevented through Doxycycline withdrawal. Likewise, development of mesenteric lymph nodes was not affected (Fig. R3a). Hence, adult Ccl19-iEYFP Ltbr fl/fl mice (Dox 8 wk off) still possess Peyer's patches and mesenteric lymph nodes, but exhibit a loss of mILFs ( Figure 4 of the manuscript). Moreover, B and T cell zone segregation remained unaltered (Fig. R3a) and we did not detect significant differences in the hematopoietic cell composition and ILC numbers from Peyer's patches or mesenteric lymph nodes in adult Ccl19-iEYFP Ltbr fl/fl mice after withdrawal of the Doxycycline treatment for 8 weeks (Fig. R3b). Taken together, these results provide further evidence that it is the LTR signaling in SILT FRCs of adult mice that affects the local immune response against Citrobacter rodentium.

Figure R3. Transgene activity and hematopoietic cell numbers in Ccl19-iEYFP Ltbr fl/fl mice after withdrawal the doxycycline treatment for 8 weeks. a, Representative confocal microscopy images of mLN and PP structures stained with indicated markers. Scale bar, 300 µm and 50 µm (mLN), 150 µm and 50 µm (PP). b, Cell numbers of CD45+ cells, B cells and T cell subsets (left panels) and ILC subsets (right panels) from adult Ccl19-iEYFP Ltbr fl/fl mice and co-housed littermate controls after withdrawal of the doxycycline treatment for 8 weeks. (b) n=9 and 12 from 3 independent experiments, geometric mean ± SD. Statistical analyses were performed using and nonparametric Mann-Whitney test.
Minor comments:

-Could you clarify how cells were sorted for the scRNAseq analysis?
The sorting markers have been added in the legend of Figure 1a.
-It would be informative to have a full list of DEGs for SILT FRCs.
A full list of differentially expressed marker gene is now provided in the Extended Data Table 1 and mentioned in the revised manuscript.
-What is the level of expression of Ltbr across SI fibroblast populations?
The Ltbr gene is expressed by all SI fibroblasts but slightly enriched in SILT FRC (Fig. R4). Yes, the opacity indicates the p values derived from the Cellphone analysis. (Taylor et al. 2004, PMID: 15585839). This paper needs to be cited and discussed. We have taken into consideration the comments of Reviewer 2 concerning previously published studies that have used globally gene-deficient animals for the characterization of FRC-ILC interaction. As highlighted in the discussion, cell type-specific and timely regulated ablation of specific genes does not merely "refine" previously published work, but pinpoints specific cellular and molecular interactions that are of biological importance. We thank the Reviewer for the productive comments. We agree that SILT FRCs may indeed act on ILC precursor cells (ILCp). However, the current animal models or markers, such as Id2 or Zbtb16 are limited in their ability to label ILC progenitors in the bone marrow. In addition, these reporter genes broadly target ILCs and even different lymphocyte subsets in other organs (Xu et al., 2019, Immunity, PMID 30926235). Therefore, we feel that it is currently not feasible to properly identify and locate ILC progenitors in the adult small intestine.

Overall, this is a technically well-performed study, provides convincing results and a clear message. However, my excitement is limited because this study doesn't provide novel insights but instead redemonstrates well established findings in a technically refined manner. This refers to the role of LTb with regards to FRC functionality in general and specifically for CP and ILFs
The Ccl19-EYFP model highlights FRCs in SILTs as shown in Figure 1b- To address the question whether ILC3 are preferentially affected during inflammation, we infected Ccl19-Cre Ltbr fl/fl mice and littermate controls with Citrobacter rodentium and evaluated lymphocyte and ILC changes on day 6 after infection. This analysis did not reveal significant differences between Ccl19-Cre Ltbr fl/fl mice and cohoused littermate controls after infection (Fig. R5a). However, total CD127 + ILC and ILC3 numbers were significantly reduced in Ccl19-Cre Ltbr fl/fl mice after bacterial infection, whereas ILC1 and ILC2 numbers showed only a slight reduction under conditions of FRC-specific Ltbr deficiency (Fig. R5b). Taken together, these results indicate that the cellular niche formed by SILT FRCs is particularly important for ILC3.
Ccl19-Cre + FRCs in the SI lamina propria are located in SILTs, as highlighted in the manuscript and shown here again for the attention of the Reviewer in Figure R6.

The impact of CCL19+ FRCs in lymph nodes and Peyer's patches could be significant and is not discussed or tested here. As such, the specificity of the approach for intestinal FRCs (and intestinal ILCs) is not demonstrated. In a related point, the actual contribution of intestinal ILCs (and not other intestinal lymphoid subsets) to the colitis phenotype is not demonstrated and would strengthen the author's most important contention, that CCL19+ FRCs specifically support ILCs.
To address this question, we have evaluated the structural integrity and hematopoietic cell composition in the PPs and mLN from Ccl19-iEYFP Ltbr fl/fl mice (Dox 8 wk off). Please see our response to Reviewer 1 point 3.

The authors find that inducible loss of IL-7 in CCL19+ cells lead to fewer of all intestinal ILCs, including fewer Rorgt+ cells in ILFs (but not CPs). However, these mice are not well explored. What is phenotype after colitis? Does this impact intestinal (comparing to SLO) Tcell, innate-like T-cell, and ILC composition and function at rest or after inflammation?
To address this point, we have further characterized T cell subset composition, including conventional CD4 and CD8 T cells, Th17 cells and Treg cells, as well as innate T cell subsets such as γδ T cells and CD1d-tetramer + iNKT cells. Conditional ablation of Il7 gene in SILT FRCs did not affect T cell subset composition in the lamina propria of Ccl19-Cre Il7 fl/fl mice compared to cohoused littermate controls (new Extended Data Fig. 7e-g). The infection with Citrobacter rodentium in these mice revealed shortened colon length and increased bacterial concentration in the faeces and colon (new Extended Data Fig. 7h-i). These data further underpin the conclusion that SILT FRCs provide critical niche factors that support ILC sustenance and function. -Line 56 -see above, tissue ILCs can come from tissue resident mature ILC proliferation, ILCp maturation (in tissue or from circulation), or trafficked mature ILCs from circulation. The relative contribution of each of these pathways is not definitively established, although proliferation of tissue-resident mature ILC pools is certainly a major one… -I do not believe the authors ever define for the reader the definitions and/or differences between SILTs, CPs, immature ILFs, and mature ILFs. Brief description in intro is warranted.
In Figure 1 and supplemental, the authors describe a cluster as vascular smooth muscle cells. However, these cells seem to be more consistent with 'pericytes', often defined by the exact markers shown by the authors…perhaps a more inclusive term would be 'mural' cells to avoid the nebulous distinction between pericytes and vascular smooth muscle cells using scRNAseq.
We have amended the text and the figures according to the reviewer's comments in the revised version. We have thoroughly analyzed the transgene activity in the small intestine. The Ccl19-EYFP transgene almost exclusively detectable in FRC of Peyer's patches (Prados et al., 2021, Nat Immunol., PMID 33707780) and SILTs ( Fig. R6 and Fig. 1b-c). Likewise, CLU expression is highly expressed FRCs of Peyer's patches (Prados et al., 2021, Nat Immunol., PMID 33707780) and SILTs ( Fig. R6 and Extended Fig. 1e). Indeed, the shift of cell clusters in presented UMAPs represents differential gene expression between subsets/subclusters. Ltbr-deficient FRCs underpinning CP and immature ILF exhibit the expression of conventional SILT FRC markers such as Cxcl16 and Kitl, but show lower expression of some chemokine and cytokine genes (Fig. 2e). The Ltbr-dependent shift in gene expression/phenotype founds the basis for the impediment of ILF maturation. To address this question, we have analyzed the composition of innate T cells including γδ T cells and iNKT cells in Ccl19-Cre Ltbr fl/fl mice and co-housed littermate controls. In sum, we conditional ablation of the Ltbr gene in SILT FRCs did not affect T cell subset composition in the lamina propria (Fig. R7). Since only few T cells can be detected in SILTs, it is conceivable that an altered SILT maturation has no or only very limited effect on the immune functions T cell subsets in the small intestine. We have evaluated the B and T cell number from Ccl19-iEYFP Ltbr fl/fl mice and co-housed littermate controls after withdrawl of Doxycycline for 8 weeks (Dox off 8 wk). There could not detect no significant differences in either B or T cell numbers (Fig. R8). To address this question, we have infected Ccl19-Cre Ltbr fl/fl mice and cohoused littermate controls with L3 larvae of Heligmosomoides polygyrus helminths. In line with the fact that neither ILC2 nor eosinophils were altered in the deficiency of LTβR signalling in SILT FRC ( Fig. 3c and Extended Fig. 4d), there was no significant difference in the granuloma formation or worm burden in the small intestine on day 8 after helminth infection ( Fig. R9a-b). In contrast, conditional ablation of Ltbr gene in SILT FRCs resulted in the reduction of ILC3 cell numbers in naive mice ( Fig.  3c and Fig. 4d) and after Citrobacter rodentium infection (Fig. R5b). Taken together, these results support the notion that the interaction between SILT FRCs and ILC3 mainly promotes intestinal immune responses against bacterial infection. Certainly, further studies are required to fully elaborate the extent of SILT FRC-ILC2 interaction in type 2 immunity against parasite infection.