RORγt-dependent antigen-presenting cells direct regulatory T cell-mediated tolerance to food antigen

The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microbes with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents1-4. While RORγt+ antigen-presenting cells (RORγt-APCs) were shown to program gut microbiota-specific pTregs5-7, understanding of their characteristics remains incomplete, and the APC subset responsible for food tolerance has remained elusive. Here, we demonstrate that RORγt-APCs are similarly required for differentiation of food antigen-specific pTregs and establishment of oral tolerance. The ability of these cells to direct both food and microbiota-specific pTreg cell differentiation is contingent on expression of RORγt and on a unique cis-regulatory element within the Rorc gene locus (Rorc(t) +7kb). Absent this +7kb element, there was a notable increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTregs, leading to compromised tolerance in a mouse asthma model. By employing single-cell analyses across these models, as well as freshly resected mesenteric lymph nodes from a human organ donor, we identified a rare subset of evolutionarily conserved APCs that are dependent on RORγt, uniquely express the Prdm16 transcription factor, and are endowed with essential mediators for inducing pTreg cell differentiation. Our findings suggest that a better understanding of how RORγt-APCs develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance.

Notably, targeting by RORγt-cre of conditional alleles for MHCII, α v β 8 integrin, and CCR7 in these APCs resulted in loss of pTregs, indicating that the APCs express RORγt during ontogeny, but it remained uncertain whether RORγt is itself required for these cells to develop or perform their function.
We previously showed that inactivation of the same target genes in CD11c-cre mice also resulted in loss of pTreg-inducing APC function.
To investigate a potential role for RORγt in these APCs, we therefore inactivated Rorc(t) in CD11c-cre mice and determined the fate of T cells specific for the large intestine pathobiont Helicobacter hepaticus .CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which this version posted July 25, 2024.; https://doi.org/10.1101/2024.07.23.604803 doi: bioRxiv preprint (Hh).Naïve Hh-specific CD4 + T cells from Hh7-2 TCR transgenic mice 15 were transferred into Hh-colonized mice.Two weeks post-transfer, the large intestine lamina propria (LILP) of control mice displayed a predominance of pTregs expressing both RORγt and FOXP3 (Fig. 1a and Extended Data Fig. 1a).In contrast, in the LILP of Cd11c cre Rorc(t) fl/gfp (Rorc(t) ΔCD11c ) mice, the differentiation of adoptively transferred Hh-specific pTregs was abrogated.Instead, these mice exhibited an increase in RORγt-and T-bet-expressing Hh-specific T cells (Fig. 1a and Extended Data Fig. 1a), indicative of a shift towards a pro-inflammatory profile.Similarly, among endogenous T cells in these mutant mice there were fewer RORγt + pTregs and substantially more inflammatory T helper 17 (Th17)/Th1 cells (Extended Data Fig. 1b).Consistent with findings in Rorc(t) ΔCD11c mice, Hh-specific T cells in Rorc(t) gfp/gfp mice failed to differentiate into pTregs, and instead adopted a Th1 phenotype, expressing T-bet (Fig. 1a and Extended Data Fig. 1a,b).These results indicate that RORγt expression in CD11c lineage APCs is required for them to direct gut microbiota-specific pTreg cell differentiation.

Lineage-specific Rorc(t) cis elements
RORγt is a transcription factor whose expression is largely confined to diverse lymphoid lineage cells, in which it contributes to distinct phenotypic programs [16][17][18][19][20][21] .Because different cis-regulatory elements (CREs) within a gene locus can govern its cell-specific expression, as best exemplified by the erythroid-specific enhancer in Bcl11a 22 , a therapeutic target for sickle cell disease, we hypothesized that distinct CREs within the Rorc locus may selectively modulate expression in the RORγt + lineages, including the pTreg-inducing APCs.While prior research delineated several CREs involved in RORγt expression in Th17 cells and ILC3, Rorc regulatory regions in other cell types that express the transcription factor remain less characterized [23][24][25] .To identify cell type-specific regulatory sequences, we conducted bulk ATAC-seq analyses in several RORγt-expressing cell types, including CD4 + CD8 + thymocytes, in vitro differentiated Th17 cells, and small intestine lamina propria (SILP)-derived Th17 cells, Tγδ17 cells, and presumptive type 3 innate lymphoid cells (ILC3).These studies revealed distinct patterns of chromatin accessibility within the Rorc locus across the different cell types (Fig. 1b).
Notably, regions situated +6kb and +7kb from the Rorc(t) transcription start site exhibited pronounced accessibility in in vitro differentiated Th17 cells and intestinal ILC3, respectively (Fig. 1b).Additionally, the +11kb element exhibited open chromatin configuration in all RORγt + cell types, with the notable exception of in vitro polarized Th17 cells (Fig. 1b), consistent with our previous findings 24 .Further studies using dual reporter BAC transgenic mice specifically lacking a +3kb element (Tg (Δ+3kb Rorc(t)-mCherry);Rorc(t) +/gfp ) indicated that Rorc(t) +3kb is a pivotal enhancer in Th17 and Tγδ17 cells in vivo, as well as in vitro differentiated Th17 cells, but not in ILC3 (Extended Data Fig. 2a,b).
To further explore the functional importance of the Rorc(t) +6 kb and +7kb elements, we engineered mice with deletions of these sequences.Rorc(t) +6kb -/-(Δ+6kb) mice had significant reduction in both the proportion of Th17 cells in the SILP and level of RORγt expression within the remaining cells, but no change in ILC3 and Tγδ17 cells (Extended Data Fig. 2c,d).Notably, when naïve CD4 T cells from these knockout mice were subjected to Th17 differentiation conditions, they failed to upregulate RORγt (Extended Data Fig. 2f), confirming the regulatory importance of the +6kb region.In contrast, analysis of Rorc(t) +7kb -/-(Δ+7kb) mice showed significantly reduced RORγt + SILP ILC3 and Tγδ17 populations, with reduced RORγt expression in the remaining cells, but no effect in Th17 cells (Extended Data Fig. 2c,e).Naïve CD4 + T cells isolated from Δ+7kb mice displayed normal RORγt expression upon in vitro Th17 cell differentiation (Extended Data Fig. 2f), consistent with a role of this element only in innate-type lymphocytes.
The ILC3 subsets in the SILP and LILP of mutant mice were skewed towards Nkp46-expressing NCR + ILC3, with reduction in CCR6-expressing LTi-like ILC3 (Extended Data Fig. 3a,b).Notably, the number of Peyer's patches remained unchanged (Extended Data Fig. 3c), indicating that RORγt-dependent lymphoid tissue inducer (LTi) cell functionality was not compromised.When the Δ+7kb mice were challenged with the enteric pathogen Citrobacter rodentium, there was no difference in bacterial titers and weight loss compared to wild-type controls, nor was there any defect in IL-22 production (Extended Data Fig. 3d-f), which is essential for bacterial clearance 26 .These results suggested that mature ILC3 in the LILP of Δ+7kb mice remain functional, despite the reduction or loss of RORγt, which is required early for ILC3 development and for repression of T-bet, which, in turn, promotes expression of Nkp46 and transition to the ILC1 phenotype.This finding is consistent with previous studies showing that while RORγt is crucial for restraining transcriptional networks associated with type 1 immunity, it is not essential for robust IL-22 production among mature ILC3 27,28 .

Tolerogenic APC-specific Rorc(t) CRE
Despite the apparent maintenance of ILC3 function in the Δ+7kb mice, there was severe disruption of adoptively transferred Hh-specific pTreg cell differentiation in the LILP and expansion of inflammatory Th17/Th1 cells (Fig. 1c and Extended Data Fig. 3g).There was also a reduction of endogenous RORγt + pTregs and increase of Th17/Th1 cells in the Hh-colonized mice (Extended Data Fig. 3h).Thus, the Rorc(t) +7kb CRE is required for RORγt-APCs to promote microbiota-specific pTreg cell differentiation even though known ILC3/LTi cell functions remain intact, which raise the possibility that such APCs belong to a different cell lineage than the ILCs.
Because RORγt is expressed in multiple immune system cell types, the T cell phenotype observed in the Δ+7kb mice could stem from direct or indirect effects.To clarify which cell types are affected by the deletion of the CRE, we established competitive bone marrow (BM) chimeric mice by co-transplanting CD45.1/2 wild-type BM along with either CD45.2 wild-type control or CD45.2Δ+7kb BM into irradiated CD45.1 wild-type recipients (Extended Data Fig. 4a).We conducted a detailed analysis of donor chimerism across various intestinal immune cell subsets, including RORγt + pTreg, ILC3, Th17, and Tγδ17 cells, normalizing these measurements to splenic B cells as an internal control.As expected, CCR6 + RORγt + ILC3 derived from the CD45.2 mutant donor were underrepresented, while Nkp46 + RORγt + ILC3 from the same donor were increased.However, RORγt + pTreg, Th17 and Tγδ17 cells repopulated to similar extents in the mixed chimeras (Extended Data Fig. 4b,c).Moreover, we investigated RORγt expression within RORγt + cell populations, discovering that Rorc(t) +7kb intrinsically regulates RORγt expression in ILC3 and Tγδ17, but not in RORγt + pTreg and Th17 cells (Extended Data Fig. 4d-f).Thus, the Rorc(t) +7kb mutation does not intrinsically affect T cell differentiation, which is consistent with the observed T cell phenotype being attributed to deficient RORγt-APC function.

RORγt-APCs required for oral tolerance
When the Δ+7kb mice were examined in the absence of H. hepaticus colonization, we observed the expected reduction, compared to wild-type mice, of RORγt + pTregs within both SILP and LILP (Extended Data Fig. 4g).Intriguingly, this decrease did not coincide with an increase in Th17 cell proportions, but was associated with a substantial elevation of intestinal Th2 cell levels (Extended Data Fig. 4g,h).This observation in the mutant mice suggested that tolerogenic APCs induce pTregs specific not only for microbiota, but also for food antigens with potential to induce allergy-related Th2 cells.To test this hypothesis, we transferred naïve ovalbumin (OVA)-specific CD4 + T cells from OT-II TCR transgenic mice 29 into both control and Δ+7kb mice and administered OVA either via gavage or in drinking water (Fig. 2a).Interestingly, OVA-specific OT-II pTregs in wild-type mice consisted of both RORγt -and RORγt + phenotypes (Fig. 2b,c).At five days post-transfer, there were few OT-II pTregs in the mesenteric lymph nodes (mLN) of Δ+7kb mice, and, instead, the OVA-specific T cells displayed Th2 and T follicular helper (Tfh) phenotypes, with no notable changes in Th17 and Th1 profiles (Fig. 2b and Extended Data Fig. 5a).By the twelfth day post-transfer, the reduction in OT-II pTregs persisted in both SILP and LILP of mutant mice, coinciding with variable increases across all OT-II Th cell subsets (Fig. 2c and Extended Data Fig. 5b,c).These results highlight the critical role of RORγt-APCs in promoting food antigen-specific pTreg cell differentiation.A dysfunction of these APCs correlates with intensified effector Th cell responses, although the specific Th cell subset favored depends on the local tissue environment.
Peripheral pTreg cells are pivotal in the induction and maintenance of oral tolerance, a critical mechanism that suppresses diverse immune responses not only in the gastrointestinal tract but also systemically 30,31 .We therefore aimed to explore whether RORγt-dependent APCs are essential for directing food antigen-specific pTregs to mediate oral tolerance.We used an allergic lung response model, in which oral administration of antigen prior to sensitization results in pTreg-mediated inhibition of the inflammatory process.Mice were not pre-treated or were administered OVA intragastrically before being primed with OVA in alum and subsequently exposed to intranasal OVA challenge (Fig. 3a).
Wild-type mice that had previously been fed OVA exhibited significant resistance to allergic lung inflammation, demonstrated by lower lung inflammation score, diminished eosinophil numbers in the bronchoalveolar lavage fluid (BALF) and lungs, reduced lung Th2 cells, and decreased levels of serum OVA-specific IgE and IgG1 compared to non-tolerized controls (Fig. 3b-d and Extended Data Fig. 6a-f).
However, the same pre-feeding strategy failed to induce oral tolerance in Δ+7kb mice.These knockout mice showed similar increases in lung inflammation score, eosinophils, Th2 cells, and OVA-specific IgE and IgG1 production as non-tolerized Δ+7kb mice (Fig. 3b-d and Extended Data Fig. 6a-f).We then focused on OVA-specific T cell responses in these mice, using OVA:I-A b tetramers to identify those cells.In tolerized wild-type mice, tetramer-positive T cells were significantly fewer compared to non-tolerized animals, and most cells were GATA3 + FOXP3 + Treg cells, with limited RORγt expression (Fig. 3e and Extended Data Fig. 6g,h).In contrast, in both tolerized and non-tolerized Δ+7kb mice there was loss of OVA:I-A b tetramer-binding pTregs in the lung, accompanied by an increase in tetramer-positive Th2, Th17, and Th1 cells, with a predominant increase in Th2 cells (Fig. 3e and Extended Data Fig. 6h,i), consistent with loss of tolerance.These findings indicate that RORγt-APCs are crucial for the development of oral tolerance, highlighting their essential role in regulating immune responses to dietary antigens and preventing allergic responses.

RORγt regulates Prdm16-expressing APCs
To better define the RORγt-dependent APCs required for intestinal pTreg cell differentiation, we performed single cell RNA sequencing (sc-RNA-seq) of MHCII-expressing cells from mLN of control and mutant mice, including both Δ+7kb and Rorc(t) ΔCD11c models.Cells harvested from Δ+7kb mutants and littermate controls at 3 weeks age yielded a total of 21,504 high-quality transcriptomes, comprising 11,150 and 10,354 cells from mutant and control animals, respectively (Fig. 4a,b).We analyzed these data with an unsupervised computation (Methods).
Populations of DCs and ILCs and distinct clusters of B cell and macrophage lineages were readily annotated (Fig. 4d, Extended Data Fig. 7a).Juxtaposed ILCs were partitioned based on canonical expression of Ncr1/Tbx21 (Type 1), Il1rl1/Il4 (Type 2), or Cxcr6/Ccr6/Nrp1 (Type 3), versus a contiguous population of natural killer cells positive for Eomes and Gzma.cDC1 occupied a distinct cluster positive for Xcr1 and Clec9a.While subsets of cDC2 (all positive for Sirpa) were contiguous, they could be separated based on expression of Clec4a4/Cd4/Dtx1 (cDC2A) versus Cd209a/Cx3cr1/Mgl2 (cDC2B), with all Tbx21 expression contained within the former, as previously reported 32 .We observed two sizable populations of Ccr7 + migratory DCs that were denoted Mig_DC_1 and Mig_DC_2, both consistent with a signature known to include Socs2, Fas, and Cxcl16 5,33 .
On querying for Rorc positive cells (Fig. 4c and Extended Data Fig. 7b), beyond expected ILC3 there were two additional distinct clusters (arrows, Fig. 4b,c).One population expressed genes consistent with the described TC I subset 6 , including Aire, Trp63, Tnfrsf11b, Nrxn1, Nrn1, and Ncam1 (Fig. 4d and Extended Data Fig. 7c).Beyond the TC I gene signature, this cluster also harbored a signature closely associated with the fibroblastic reticular cell (FRC) subset found in mucosal lymph nodes 34 , including Madcam1, Mfge8, Twist1, Vcam1, and Nid1.Both non-ILC3 Rorc + clusters expressed CD45, as well as MHCII at levels comparable to DC subsets.Therefore, the TC I cells are not bona fide stromal cells nor medullary thymic epithelial cells (mTEC), but they harbor prominent FRC and mTEC attributes.
The second Rorc + cluster displayed exclusively high expression of Prdm16, previously shown to be expressed across the TC subsets 6 (Extended Data Fig. 7d).The Prdm16 + cluster was notably higher in Itgb8, Aire, Cd40, and Ccr7 and lower in Cd80 and Cd86 expression when compared to TC I (Fig 4d).
After annotating, we deconvolved each cluster according to its origin from Δ+7kb mutant or control animals.While the contribution from each condition varied minimally around the numeric split of total high-quality transcriptomes captured (Fig. 4e, dotted line), we observed a striking 60% loss of the Prdm16 + cluster from the expected Δ+7kb contribution.A violin plot examining Rorc illustrates a clear decrease of that gene's expression in the mutant-origin ILC3 population (Extended Data Fig. 7e), yet the overall cluster of ILC3 from mutant mice remained otherwise numerically intact.As there was also no apparent difference in the TC I cluster distribution when comparing control versus Δ+7kb mutant mice, the results suggested that the tolerogenic APCs are contained in the Prdm16 + population.
Comparison of mLN single cell transcriptomes from Rorc(t) ΔCD11c (8,474 cells) and littermate control mice (11,245 cells) yielded results congruent with those observed in the Δ+7kb model (Extended Data Fig. 8a and Extended Data Fig. 9a,b).In these mutants, however, there was complete loss of the Prdm16_Pos cluster when we deconvolved biological conditions (Extended Data Fig. 8b), despite a 43% overall contribution of total transcriptomes from Rorc(t) ΔCD11c mice (Fig. 4f, dotted line).Together, the results are most consistent with a requirement for RORγt expression during development of the Prdm16 + APCs and suggest that these cells have tolerogenic function in response to both microbiota and food antigens.
To determine whether the RORγt-APCs identified in mice are also present in humans, we performed sc-RNA-seq on HLA-DR-enriched cells from freshly isolated mLNs of a 22-year-old trauma patient (Fig. 5a).Following the same unsupervised computation as for the murine experiments, we clustered the resulting single-cell dataset (Fig. 5b).A population of ILC3 was demarcated by expression of IL7R, KIT, and NRP1 (Fig. 5c and Extended Fig. 10a).This was juxtaposed by an ILC1 cluster expressing NCR1 and TBX21, as well as NK cells expressing EOMES, all consistent with prior literature 35 .A cluster positive for XCR1 and CLEC9A was readily distinguished as cDC1.As compared to murine cDC2, there is less known about human cDC2 that allows for confident subset assignment.Nevertheless, we could identify a SIRPA + population overlapping with CD1C expression, as would be expected for human cDC2 36 .We annotated a plasmacytoid DC cluster adjacent to this, as these cells were negative for CD1C while positive for IL3RA (CD123) and included rare cells positive for TLR7 and TLR9 expression 37 .
On querying for RORC in this human dataset, we observed non-ILC3 expression in a single set of cells clustering at the extremity of the cDC2 annotation (arrow, Fig. 5b,c).Strikingly, this same population exclusively displayed a high PRDM16 signal.Unlike the murine sc-RNA-seq analysis, this population was not spontaneously demarcated by an unsupervised workflow, so we manually sub-clustered and similarly annotated it as Prdm16_Pos.This human cluster was also positive for ZBTB46, CD40, CCR7, and ITGAX, although AIRE was not detected in it or any other HLA-DR + mLN population.
Because our results appeared to coincide across datasets, we sought to more closely inspect the non-ILC3 mouse and human cell populations expressing Rorc (or RORC) for their top differentially upregulated genes as compared to all other cell types within each species' mLN.This revealed an overlap of 29 genes that were expressed by these cells in both species (Fig. 5d,e).Examining this shared gene list on a volcano plot measuring differential expression within mouse mLN again highlighted Prdm16 as the most statistically significant gene for this cluster, with a ~1,000-fold change in gene expression compared to all other cell types.Our list also notably included the neural adhesion genes Nrxn1 (also upregulated in TC I) and Plxna4, as well as the transcription factor Runx3, which has been described to be required for expression of RORγt in ILC3 38 .Differential expression in the human dataset similarly revealed PRDM16 to be near top-ranked in terms of statistical significance and fold change gene expression, indicating that this transcription factor is evolutionarily conserved in its co-expression with RORC in a presumptive tolerogenic APC.

Discussion
In maintaining intestinal homeostasis, the gut immune system is tasked with tolerating a complex array of dietary and microbial antigens, largely through the action of peripheral pTregs.This study expands upon the role of RORγt-APCs from their established function in driving microbiota-specific pTreg cell differentiation to facilitating oral tolerance to food antigens.Oral tolerance can be enhanced by oral immunotherapy for food allergies 39,40 and has been demonstrated effective in animal models to control antigen-specific autoimmune diseases 41,42 .The potential role of RORγt-APCs in modulating autoimmune diseases and transplantation tolerance remains to be elucidated.Our findings indicate that the competency of RORγt-APCs in establishing mucosal tolerance crucially relies on the transcription factor RORγt and a specific CRE within the Rorc locus.Because the subset of these cells that also express PRDM16 corresponds to cells found in human mLN 6,43 and is selectively depleted in the absence of RORγt, we propose that this is the most likely candidate tolerogenic APC.The transcriptional regulatory network in which RORγt participates to influence development and function of these APCs remains to be elucidated.A comprehensive understanding of the key components will likely reveal human genetic variants that can predispose to inflammatory and allergic conditions.An understanding of the ontogeny of the tolerogenic APCs may also provide critical insights into inflammatory diseases and potential therapeutic avenues.Crucially, these tolerogenic APCs possess a remarkable ability to induce pTregs despite their low abundance in the intestinal secondary lymphoid organs.Unraveling the mechanisms behind this potent immunomodulatory effect is essential and will likely require elucidation of the spatial distribution of the APCs and temporal dynamics of differentiation of T cell subsets specific for antigens encountered in the alimentary tract.The unique ability of RORγt-APCs to induce antigen-specific pTreg cells suggests that these APCs could serve as valuable therapeutic targets in multiple immune-related diseases.and Th1 (FOXP3 -RORγt -T-bet + ) cells in the LILP of Hh-colonized control (Rorc(t) fl/gfp , Rorc(t) +/gfp , and Cd11c cre Rorc(t) +/gfp ; n = 7) , Rorc(t) ΔCD11c (Cd11c cre Rorc(t) fl/gfp ; n = 5) and Rorc(t) gfp/gfp (n = 4) mice at 14 days after adoptive transfer of naïve Hh7-2tg CD4 + T cells.The lower panels of flow cytometry plots are gated on FOXP3 -cells.b, Bulk ATAC-seq data showing accessible regions in the Rorc locus of several RORγt-expressing cell types, including CD4 + CD8 + thymocytes, in vitro differentiated Th17 cells, and SILP-derived Th17 (TCRβ + CD4 + IL23R-GFP + ) cells, Tγδ17 (TCRγδ + IL23R-GFP + ) cells and ILC3 (Lin -IL-7R + Klrb1b + NK1.1 -).c, Phenotype of Hh-specific T cells in the LILP of Hh-colonized control (Rorc(t) +7kb +/+ ; n = 6) and Δ+7kb (Rorc(t) +7kb -/-; n = 4) mice at 14 days after adoptive transfer of naïve Hh7-2tg CD4 + T cells.The lower panels of flow cytometry plots are gated on FOXP3 -cells.Data in a are pooled from two independent experiments.Data in c are representative of two independent experiments.Data are means ± s.e.m.; ns, not significant; statistics were calculated by unpaired two-sided t-test.Anti-mouse CD16/32 (Bio X Cell) was used to block Fc receptors.Live/dead fixable blue (ThermoFisher) was used to exclude dead cells.I-A b OVA 328-337 tetramers (HAAHAEINEA) were provided by the NIH Tetramer Core Facility.
For labeling OVA-specific T cells, cells were incubated with tetramers for 60 min at 37 °C prior to surface staining.For intracellular staining, cells were stained for surface markers, followed by fixation and permeabilization before intracellular staining according to the manufacturer's protocol (FOXP3 staining buffer set from Thermo Fisher).For cytokine analysis, cells were stimulated ex vivo for 3 h with IL-23 (10 ng/mL; R&D systems) and GolgiStop (BD Biosciences) in complete RPMI-1640 culture medium (RPMI-1640 with 10% FBS, 1% GlutaMAX, 1% penicillin-streptomycin, 10 mM HEPES, and 1 mM sodium pyruvate).Flow cytometric analysis was performed on an LSR II (BD Biosciences) or a Cytek Aurora (Cytek Biosciences) and analyzed using FlowJo software (Tree Star).

Isolation of lymphocytes
For isolation of cells from lymph nodes and spleens, tissues were mechanically disrupted with the plunger of a 1-ml syringe and passed through 70-μm cell strainers.Bone marrow cells were harvested by flushing out the marrow from cleaned bones using a syringe containing RPMI-1640 wash medium (RPMI-1640 with 3% FBS, 1% GlutaMAX, 1% penicillin-streptomycin, 10 mM HEPES, and 1 mM sodium pyruvate).Red blood cells were lysed with ACK buffer (Thermo Fisher).Cells in bronchoalveolar lavage fluids (BALF) were isolated by flushing the lung with two washes of 0.75 ml PBS via a catheter inserted into a cut made in the trachea.
Lung tissues were cut into small pieces and digested in RPMI-1640 wash medium containing 0.5 mg/ml collagenase D (Sigma) and 0.5 mg/ml DNase I (Sigma) at 37 °C for 45 min with shaking.After removal of Peyer's patches and cecal patches, the intestines were opened longitudinally, cut into 0.5 cm pieces, and washed in PBS twice.Intestines were then incubated with shaking in HBSS medium (without Ca2 + and Mg2 + ) containing 3% FBS, 1 mM DTT, 5 mM EDTA, and 10 mM HEPES at 37 °C for 20 min twice.After washing with HBSS medium (without Ca2 + and Mg2 + ) containing 3% FBS and 10 mM HEPES, the tissues were then digested in RPMI-1640 wash medium containing 1 mg/ml collagenase D (Sigma), 0.25 mg/ml DNase I (Sigma), and 0.1 U/ml Dispase (Worthington) at 37 °C for 35 min (small intestines) or 55 min (large intestines) with shaking.To isolate leukocytes from the lungs and intestines, the digested tissues were homogenized and passed through 70-μm cell strainers.Mononuclear cells were then collected from the interphase of an 80% and 40% Percoll gradient after a spin at 2,000 rpm for 20 min.
Mice were followed for the next 14 days to measure body weight change.Fecal pellets were collected and used to measure C. rodentium burden with serial dilutions on MacConkey agar plates.

H. hepaticus culture and oral infection
H. hepaticus was provided by J. Fox (MIT).H. hepaticus was cultured and administered as previously described 15 .Frozen stock aliquots of H. hepaticus were stored in Brucella broth with 20% glycerol and frozen at −80 °C.The bacteria were grown on blood agar plates (TSA with 5% sheep blood, Thermo Fisher).Inoculated plates were placed into a hypoxia chamber (Billups-Rothenberg), and anaerobic gas mixture consisting of 80% nitrogen, 10% hydrogen and 10% carbon dioxide (Airgas) was added to create a micro-aerobic atmosphere, in which the oxygen concentration was 3-5%.The micro-aerobic jars containing bacterial plates were left at 37 °C for 4 days before animal inoculation.For oral infection, H. hepaticus was resuspended in Brucella broth by application of a pre-moistened sterile cotton swab applicator tip to the colony surface.0.2 ml bacterial suspension was administered to each mouse by oral gavage.Mice were inoculated for a second dose after 4 days.
Adoptive transfer of OT-Ⅱ CD4 + T cells was performed as previously described 31 , with minor modifications.Lymph nodes from donor OT-Ⅱ TCR transgenic mice were collected and mechanically dissociated.Naïve OT-Ⅱ CD4 + T cells were sorted as CD4 + TCRβ + CD44 low/-CD62L + CD25 − Vα2 + Vβ5.1/5.2 + (OT-Ⅱ), on the Aria II (BD Biosciences).Cells were resuspended in PBS on ice and 100,000 cells were then transferred into congenic recipient mice by retro-orbital injection.Recipient mice received OVA by oral gavage (50 mg; A5378; Sigma) for 4 consecutive days, followed by drinking water containing OVA (2.5 mg/ml) for an additional 7 days after transfer.Cells from mLN were analyzed 5 days after transfer and cells from intestines were analyzed 12 analysis that retained 50 dimensions, and clustering with the Leiden algorithm set to resolution = 1.0.This matched together cells with shared biological states across mutant and control animals, ensuring the final clusters were not driven by effects within either condition.The Rorc(t) ΔCD11c murine sc-RNA-seq dataset was appended to the Δ+7kb dataset, also clustered in an unsupervised manner, and then analyzed for the same populations as before.The human sc-RNA-seq dataset also underwent a predominantly unsupervised pipeline, but the Prdm16_Pos population was subsequently manually sub-clustered from a juxtaposed cDC2 population.

Statistical analysis
Unpaired two-sided t-test and paired two-sided t-test were performed to compare the results using GraphPad Prism, Version 10 (GraphPad Software).No samples were excluded from analysis.We treated less than 0.05 of P value as significant differences.*P < 0.05, **P < 0.01, and ***P < 0.001.Details regarding number of replicates and representative data can be found in figure legends.

Fig. 4 |
Fig. 4 | RORγt regulates development of Prdm16-expressing APCs within mLN.a, Input scheme for both murine sc-RNA-seq experiments: mLN harvest from mutant and control animals, followed by indicated sorting.b,c, UMAP of 21,504 resultant transcriptomes from an experiment combining Δ+7kb mutants (n = 7) and controls (n = 7), clustering data together (b).Feature plot of Rorc expression (c).Black arrows label two non-ILC3 clusters positive for Rorc.d, Dot plot of indicated clusters from b examining expression of genes previously ascribed to proposed RORγt-APC subsets.e, Stacked bar plots comparing proportion of each cluster in b, as derived from Δ+7kb mutant versus control animals.Dotted line at 51.2% indicates total contribution from Δ+7kb mutants.Red arrow indicates 60% loss within Prdm16_Pos cluster of expected Δ+7kb contribution.f, Stacked bar plots analogous to experiment b-e, but now comparing proportion of cell clusters as derived from Rorc(t) ΔCD11c mutants versus controls (n = 4 mice, each condition).Dotted line at 43% indicates total contribution from Rorc(t) ΔCD11c mutants.Red arrow indicates complete loss within Prdm16_Pos cluster of expected mutant contribution.

Fig. 5 |
Fig. 5 | PRDM16-expressing APCs are conserved in human mLN.a, Input scheme for human sc-RNA-seq, with four mLN resected and enriched for APCs.b, UMAP of 12,928 resultant human mLN transcriptomes.c, Dot plot of indicated clusters from b examining expression of genes previously ascribed to RORγt-APC subsets.d,e, Volcano plots of differentially up-regulated genes in the murine (d) or human (e) Prdm16_Pos cluster compared to the rest of each species' mLN cell types.A list of 29 genes shared by both species is annotated in each.Red dots indicate differential gene expression meeting P > 10 -6 and Log 2 fold change > 1.