Blimp-1 and c-Maf regulate immune gene networks to protect against distinct pathways of pathobiont-induced colitis

Intestinal immune responses to microbes are controlled by the cytokine IL-10 to avoid immune pathology. Here, we use single-cell RNA sequencing of colon lamina propria leukocytes (LPLs) along with RNA-seq and ATAC-seq of purified CD4+ T cells to show that the transcription factors Blimp-1 (encoded by Prdm1) and c-Maf co-dominantly regulate Il10 while negatively regulating proinflammatory cytokines in effector T cells. Double-deficient Prdm1fl/flMaffl/flCd4Cre mice infected with Helicobacter hepaticus developed severe colitis with an increase in TH1/NK/ILC1 effector genes in LPLs, while Prdm1fl/flCd4Cre and Maffl/flCd4Cre mice exhibited moderate pathology and a less-marked type 1 effector response. LPLs from infected Maffl/flCd4Cre mice had increased type 17 responses with increased Il17a and Il22 expression and an increase in granulocytes and myeloid cell numbers, resulting in increased T cell–myeloid–neutrophil interactions. Genes over-expressed in human inflammatory bowel disease showed differential expression in LPLs from infected mice in the absence of Prdm1 or Maf, revealing potential mechanisms of human disease.

Intestinal immune responses to microbes are controlled by the cytokine IL-10 to avoid immune pathology.Here, we use single-cell RNA sequencing of colon lamina propria leukocytes (LPLs) along with RNA-seq and ATAC-seq of purified CD4 + T cells to show that the transcription factors Blimp-1 (encoded by Prdm1) and c-Maf co-dominantly regulate Il10 while negatively regulating proinflammatory cytokines in effector T cells.Double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice infected with Helicobacter hepaticus developed severe colitis with an increase in T H 1/NK/ILC1 effector genes in LPLs, while Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice exhibited moderate pathology and a less-marked type 1 effector response.LPLs from infected Maf fl/fl Cd4 Cre mice had increased type 17 responses with increased Il17a and Il22 expression and an increase in granulocytes and myeloid cell numbers, resulting in increased T cell-myeloid-neutrophil interactions.Genes over-expressed in human inflammatory bowel disease showed differential expression in LPLs from infected mice in the absence of Prdm1 or Maf, revealing potential mechanisms of human disease.
The immune response has evolved to protect the host against infection; however, mechanisms such as the cytokine IL-10 are in place to regulate immune responses to pathogens and pathobionts to prevent untoward inflammation and host damage [1][2][3][4][5] .Mice deficient in IL-10 (Il10 −/− ) can develop colitis 6 , although less evidently in specific-pathogen-free-reared Il10 −/− mice or germ-free mice 7 , triggered by pathobionts such as Helicobacter hepaticus (H.hepaticus) 8 .T cell-derived IL-10 dominantly controls intestinal responses with T cell-specific IL-10 mutant mice developing colitis to a similar level as Il10 −/− mice 9 .Rare loss-of-function mutations in Il10, Il10ra or Il10rb genes result in inflammatory bowel disease (IBD) in childhood, although it is unclear whether infection by pathobionts contributes to these pathologies 10 .Genome-wide association studies have identified more than 230 loci linked to human IBD, including those associated with proinflammatory cytokines and transcription factors upstream of immune effector molecules 11 , such as Blimp-1, encoded by Prdm1 (ref.12).
Both common and cell-specific transcriptional mechanisms regulate Il10 and proinflammatory gene expression in T cells to ensure a Resource https://doi.org/10.1038/s41590-024-01814-zwith H. hepaticus, these T cell-specific transcription-factor-deficient mice developed colitis with varying degrees of pathology, with an overall trend of double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice developing the most severe disease, and the Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice each developing mild to moderate colitis, respectively compared to Prdm1 fl/fl Maf fl/fl control mice (hereafter referred to as control mice), which showed no inflammation or colitis (Fig. 1a-c and Methods).Total LPLs increased in all three knockouts compared to controls (Fig. 1d), and CD4 + T cell numbers were increased in the infected single Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre and most significantly increased in the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice compared to controls (Fig. 1e).
Given that Blimp-1 and c-Maf induce Il10 gene expression 14,15 while negatively regulating a large network of proinflammatory cytokines 15 , we determined whether the effects of T cell-specific deletion of Prdm1, Maf or the combination of Prdm1 and Maf resulted in increased pathology and inflammation in H. hepaticus-infected mice owing to abrogation of IL-10 signaling.To address this question, the T cell-specific transcription-factor-deficient and control mice were infected with H. hepaticus in the presence of an anti-IL-10R blocking antibody (mAb) or isotype-matched mAb control (Extended Data Fig. 1b, right-hand side).Blockade of IL-10R signaling resulted in moderate to severe pathology in the colons of wild-type control mice and resulted in increased pathology in the single Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre H. hepaticus-infected mice.However, the most severe pathology was still observed in the infected double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice in the presence or absence of anti-IL-10R mAb, with no significant increase in pathology observed in mice administered anti-IL-10R mAb compared to those given isotype-control mAb (Extended Data Fig. 1b, right-hand side).These findings suggest that the high level of intestinal pathology observed in the Prdm1 fl/fl Maf fl/fl Cd4 Cre mice results from the effects of both transcription factors on other immune factors in addition to their co-dominant role in Il10 gene regulation.
To dissect the mechanisms underlying the pathology observed in the different T cell-specific transcription-factor-deficient mice, we performed RNA-seq analysis on LPLs from H. hepaticus-infected and uninfected mice (Extended Data Fig. 1c,d, Fig. 1f and Supplementary Table 1).Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre showed a major increase in differentially expressed genes (DEGs) against uninfected control mice, while double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice showed much higher numbers of DEGs compared to infected controls, which showed minimal DEGs (Supplementary Table 2).These formed nine clusters of similarly regulated DEGs, which were annotated using pathway analysis tools (Fig. 1f, Supplementary Table 2 and Extended Data Fig. 1e) with the associated pathology scores shown at the top of the heatmap (Fig. 1f).DEGs in cluster 3 (Metabolism/AhR), cluster 4 (signaling/Wnt/ extracellular matrix), cluster 9 (stem cell/extracellular structure) and clusters 2 and 8 (miscellaneous) were all decreased in the LPLs from the H. hepaticus-infected transcription-factor-deficient mice compared to control mice and largely represented non-immune genes (Fig. 1f).Conversely, DEGs in cluster 1 (T cell activation/T helper cells) and cluster 5 (cell cycle) were partially increased in LPLs from both Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre H. hepaticus-infected mice, and further controlled immune response to pathogens or other triggers 1,[3][4][5][13][14][15] . Given hat transcription factors have multiple gene targets, those that positively regulate Il10 may simultaneously repress proinflammatory cytokine expression in T cells.c-Maf induces Il10 expression directly in multiple T cell subsets, both in vitro and in vivo 1 , while also acting as a negative regulator of Il2 (ref.13) and other proinflammatory cytokines 16 and also exhibiting context-specific effects 13 .Deletion of Maf in T cells or regulatory T cells (T reg ) was reported to not result in spontaneous inflammation 13,17 , whereas in other studies, mice with T cell or T reg -specific deletion of Maf showed signs of intestinal inflammation 18,19 .The transcription factors c-Maf and Blimp-1 are dominant shared coregulators of Il10 gene expression in multiple T cell subsets 14,15 .Although T cell-specific deletion of Prdm1 has been reported to result in spontaneous colitis [20][21][22] associated with increased frequencies of T helper 17 (T H 17) cells 23 , other studies reported no intestinal inflammation in these mice 14,24 .Moreover, although Blimp-1 functions as a molecular switch to prevent inflammatory activity in Foxp3 + RORγt + T reg 25   , deletion of Prdm1 in T reg did not result in severe intestinal inflammation 26 .Spontaneous colitis has been reported in mice with T cell-specific deletion of the combination of both Prdm1 and Maf, and this pathology was associated with a unique cluster of T reg cells and the abrogation of Il10 expression 14 .
We report here that mice with T cell-specific deletion of Prdm1, Maf or both transcription factors do not develop colitis at the steady state.Upon infection with H. hepaticus, the absence of Prdm1 or Maf in T cells resulted in mild to moderate pathology, while the absence of both transcription factors resulted in severe pathology.We interrogated the immune response in the colon LPLs underpinning the pathology in the different T cell-specific transcription-factor-deficient H. hepaticus-infected mice using bulk tissue RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq), complemented by RNA-seq and assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis of purified CD4 + T cells, and we validated key findings using flow cytometry and immunofluorescence staining of colon tissue.Double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre H. hepaticus-infected mice showed a major increase in genes associated with T H 1 and natural killer/innate lymphoid cell 1 (NK/ILC1) effector function including interferon-γ (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF), but this was lower in Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice.By contrast, LPLs from H. hepaticus-infected Maf fl/fl Cd4 Cre mice showed an increased type 17 response with increased expression of Il17a and Il22 and a pronounced signature of innate immunity and neutrophils.Genes identified as over-expressed in human IBD colon biopsies from transcriptomic datasets were differentially perturbed in the LPLs of H. hepaticus-infected mice with T cell-specific deficiencies in either Prdm1, Maf or both transcription factors, potentially reflecting different pathobiological mechanisms relevant to human IBD.

T cell Blimp-1 and c-Maf control colitis via lymphoid and myeloid cells
Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and Prdm1 fl/fl Maf fl/fl Cd4 Cre mice did not develop colitis in the steady state (Extended Data Fig. 1a).Upon infection
To interrogate the gene expression changes further and identify the cellular sources of immune-associated genes, we performed scRNA-seq on LPLs isolated from colons from an independent experiment with H. hepaticus-infected and uninfected mice (Fig. 2 and Extended Data Fig. 2).Data from the LPLs of all groups were first integrated for analysis into a single uniform manifold approximation and projection plot, revealing 17 distinct cell clusters (Fig. 2a) for annotation, using the single-cell Mouse Cell Atlas as in the Methods, the Immgen database (GSE109125) and manual curation (Fig. 2a and Supplementary Table 3).scRNA-seq data from LPLs from H. hepaticus-infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice showed distinct profiles compared to uninfected (fl/fl control mice and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice) and infected control fl/fl mice (Fig. 2b and Extended Data Fig. 2).A similar proportion of immune cell types was identified by scRNA-seq in the LPLs from the uninfected control mice, uninfected double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre+ mice and H. hepaticus-infected control mice, with no intestinal pathology (Fig. 2b and Extended Data Fig. 2).LPL preparations from uninfected double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre+ mice, however, showed some unexplainable increases in the proportion of epithelial cells, although these mice showed no intestinal pathology (Fig. 2b; see Methods).Moreover, no increase in LPLs (Fig. 2c) or CD4 + T cells (Fig. 2d) assessed by flow cytometry was observed at the steady state in these mice.
Increased intestinal pathology (Fig. 2b) and LPL and CD4 + T cell numbers were again observed by flow cytometry (Fig. 2c,d) in H. hepaticus-infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre compared to control fl/fl mice.The scRNA-seq data revealed a similar increase in the proportion of T cells but additionally revealed greater granularity, with an increase in a subset of Ctla4 high T cells in the LPL from Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice and to a greater extent in infected double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Fig. 2b, Extended Data Fig. 2 and Supplementary Table 3).A smaller increase of Icos high T cells but not Cd8 T cells was observed (Fig. 2b and Extended Data Fig. 2).The NK/ILC cell cluster expressed a distinct discrete set of NK cell-specific genes, including Nkg7, Klrb1b and the highest level of Ifng, confirming their identity as NK/ILC1 cells (Fig. 2e and Extended Data Fig. 3a).The Ctla4 high and Icos high T cell subsets, although clustering separately, broadly shared the expression of the top ten marker genes (Fig. 2e and Extended Data Fig. 3a).scRNA-seq data recapitulated an increase in granulocytes in H. hepaticus-infected Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre only, but not in infected Prdm1 fl/fl Cd4 Cre or control mice (Fig. 2b), with increased expression of genes associated with neutrophils, including Acod1, S100a8 and S100a9 (Fig. 2e, Extended Data Fig. 3a and Supplementary Table 3).Populations annotated as macrophages and myeloid leukocytes were increased in the LPLs from H. hepaticus-infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Fig. 2b and Extended Data Fig. 2), with increased expression of genes associated with myeloid or innate immune responses such as Lyz2 (LysM), Csf2rb, Il1r2, Il1b and Cd14 (Fig. 2e, Extended Data Fig. 3a and Supplementary Table 3).These scRNA-seq data thus identified the cellular sources of key gene expression signatures.

Prdm1 and Maf induce Il10 while also disrupting effector T cell gene expression
Expression of Il10 mRNA in purified CD4 + T cells from LPLs was diminished in H. hepaticus-infected Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice, and to the greatest extent in the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Fig. 3a and Supplementary Tables 4 and 5).This was mirrored by similar decreases in IL-10 protein production (Extended Data Fig. 4a,d,e).Conversely, CD4 + T cells showed increased Ifng expression in infected Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice (Fig. 3b), with the greatest increase observed in the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre , mirrored by increased IFNγ protein production (Extended Data Fig. 4a-c,e).Increased Ifng expression was more pronounced in total LPLs by RNA-seq (Fig. 3c) and scRNA-seq (Fig. 3d) than in CD4 + T cells, suggesting that increased numbers of CD4 + T cells, γδ-T cells or NK/ILC1 are contributing to the global increased levels of Ifng expression in the LPLs of the H. hepaticus-infected T cell-specific Fig. 2 | scRNA-seq reveals in-depth colonic gene regulation by Prdm1 and Maf.scRNA-seq was performed on total colon LPL isolated from uninfected Prdm1 fl/fl Maf fl/fl and Prdm1 fl/fl Maf fl/fl Cd4 Cre control mice, and H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice as well as mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.a, Uniform manifold approximation and projection (UMAP) visualization of the integrated scRNA-seq from all conditions, colored by the identified/assigned cell cluster.b, Bar plots representing the proportion of cells in each of the cell clusters per biological replicate within each experimental condition and corresponding histopathological scores (detailed in Methods).c, Colon LPL cell counts for each H. hepaticus-infected group compared to uninfected controls.d, Total CD4 + T cell counts for each group in the experiment for each H. hepaticus-infected group compared to uninfected controls.Each dot within the bar plots represents an individual mouse analyzed.Graph shows means; error bars, s.d.Analyzed by one-way ANOVA followed by Dunnett posthoc test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).Data from n = 5 mice.e, Dot plot of the top ten differentially expressed marker genes of relevant cell clusters from a, colored by the average gene expression across all cell clusters.The dot size represents the percentage of cells per cell cluster expressing the gene in question.Cd4 Cre and Prdm1 fl/fl Cd4 Cre mice (Fig. 3b), with similar findings in LPLs by RNA-seq (Fig. 3c) and scRNA-seq (Fig. 3d).Levels of Il17a were similar in CD4 + T cells in the infected Maf fl/fl Cd4 Cre mice to those in uninfected and infected control mice (Fig. 3b).However, scRNA-seq showed exclusive elevation of Il17a at the level of expression and percentage of Il17a Resource https://doi.org/10.1038/s41590-024-01814-zexpressing cells, suggesting that increased numbers of CD4 + T cells, γδ-T cells or ILC3 contribute to the global increase in Il17a expression in LPLs from H. hepaticus-infected Maf fl/fl Cd4 Cre mice (Fig. 3c,d).Csf2 RNA expression was highest in CD4 + T cells (Fig. 3b) and in scRNA-seq of LPLs from infected double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Fig. 3d), as were T H 1-associated effector molecules Ifng, Il18r1, Il12rb2, Il12rb1 and Cxcr3, and additionally Il23r.Many of these genes were also elevated in the Prdm1 fl/fl Cd4 Cre but not in the Maf fl/fl Cd4 Cre mice (Fig. 3b,d and Extended Data Fig. 4k).
The scRNA-seq data was further interrogated to identify the source of cells expressing Il10 and proinflammatory cytokines in the LPLs from the H. hepaticus-infected T cell-specific transcription-factor-deficient mice.Foxp3 + T reg cells contained within the Ctla4 high cluster of T cells (Fig. 3j) were identified as the main Il10-expressing T cells (Fig. 3e).Expression and percentages of Il10-producing cells were diminished in Foxp3 + T reg cells from both the Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and to the greatest extent in the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre infected mice compared to infected control mice, and reduced similarly in the very low numbers of Foxp3 − CD4 + Il10-expressing T cells (Fig. 3f).Foxp3 + T reg cells showed a graded increase in numbers by flow cytometry in the LPLs from H. hepaticus-infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice, respectively (Extended Data Fig. 5a-c).By contrast, Foxp3 + RORγt + T cells were almost completely abolished in the LPLs from infected Maf fl/fl Cd4 Cre mice (Extended Data Fig. 5b,c) as previously reported 27 , whereas they were increased in LPLs from infected Prdm1 fl/fl Cd4 Cre and to a lesser extent double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice compared to control mice (Extended Data Fig. 5b,c).Despite this, infected double-deficient Prd-m1 fl/fl Maf fl/fl Cd4 Cre mice exhibited the maximum pathology (Fig. 1b,c and Fig. 2b) and expressed the lowest levels of Il10 in CD4 + T cells (Fig. 3a,f).
In contrast to the dominant expression of Il10 in Foxp3 + T reg cells, Ifng and Csf2 were most highly expressed in the Foxp3 − Ctla4 high T cell cluster, while being scarcely detectable in Foxp3 + T reg cells (Fig. 3g,h) and increased in Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre and mostly highly in double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre -infected mice, respectively.Conversely, Il17a and Il22 expression by scRNA-seq, while also expressed largely in Foxp3 − CD4 + T cells, was highest in LPLs from infected Maf fl/fl Cd4 Cre mice (Fig. 3i,j), in keeping with RNA-seq data from flow cytometry-purified CD4 + T cells (Fig. 3b).Diminished expression of Il17a and Il22 in LPLs from infected Prdm1 fl/fl Maf fl/fl Cd4 Cre mice suggest repression of the Il17a and/or Il22 response by Blimp-1 regulated factors (Fig. 3i,j).Collectively, these data suggest that the intestinal pathology resulting from T cell-specific deficiency in Maf differs qualitatively from that of Prdm1 and even Prdm1-Maf double deficiency owing to a type 17-mediated effector cytokine response to H. hepaticus infection rather than the increased type 1 mediated response controlled by both transcription factors.
Global analysis of RNA-seq and ATAC-seq data revealed large unique and overlapping changes in gene expression and differentially accessible sites in CD4 + T cells from both H. hepaticus-infected Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice compared to control mice, with much higher changes in CD4 + T cells from infected double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Extended Data Fig. 6a-g and Supplementary Tables 6 and 7).A combination of ATAC-seq and RNA-seq data from purified CD4 + T cells from colon LPLs of the H. hepaticus-infected T cell-specific transcription-factor-deficient mice was integrated with published chromatin immunoprecipitation with sequencing (ChIP-seq) data and revealed common and distinct binding sites for Blimp-1 and c-Maf in the Il10 locus as previously reported 14 .However, distinct binding sites for Blimp-1 in the Ifng locus were observed, while c-Maf had several binding sites within the Il17 locus and weak binding sites in the Il22 and Csf2 gene loci (Fig. 4a).Our data are supportive of cooperative and independent roles of these transcription factors in the positive regulation of Il10 and additionally in negative regulation of proinflammatory cytokine genes.Pathway analysis applied to the RNA-seq data from CD4 + T cells revealed an increase in IL-12R and downstream Stat1/Stat4 signaling for Ifng induction in the CD4 + T cells from the H. hepaticus-infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice, respectively, with the largest increase in IFNγ observed in the double-deficient mice compared to infected control mice (Fig. 4b).Pathway analysis showed the highest increase of IL17A and IL22 within the T H 17 pathway in the Maf fl/fl Cd4 Cre CD4 + T cells, with a marked decrease in infected double-deficient Prd-m1 fl/fl Maf fl/fl Cd4 Cre mice, suggesting that factors regulated by Blimp-1 may repress expression of the Il17a gene (Fig. 4c).Expression of Csf2 observed within the T H 17 pathway was again increased in CD4 + T cells from LPLs of all the H. hepaticus-infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre although most marked in Prdm1 fl/fl Maf fl/fl Cd4 Cre mice compared to control infected mice (Fig. 4c).

T cell-derived Blimp-1 and c-Maf control innate immunity
The average increased expression of 'innate immunity and myeloidassociated' genes in the bulk tissue RNA-seq data, cluster 6 (Fig. 1f,g) from the LPLs from H. hepaticus-infected Maf fl/fl Cd4 Cre and, to a lesser  a, ATAC-seq was performed on sorted CD4 + T cells from colon LPLs isolated from uninfected Prdm1 fl/fl Maf fl/fl control and H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice as well as mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.Genome browser tracks of ATAC-seq data from each condition together with publicly available c-Maf (green) and Blimp-1 (red) ChIP-seq datasets in the Il10, Ifng, Il17a, Il22 and Csf2 loci.Statistically significant ChIP-seq peaks (q < 0.05) are represented by a green or red bar underneath the normalized read coverage tracks for c-Maf and Blimp-1, respectively.b,c, IPA was used to overlay sorted CD4 + T cells RNA-seq onto the T H 1 (b) and T H 17 (c) pathways.Gene expression fold changes in each H. hepaticus-infected condition relative to the uninfected Prdm1 fl/fl Maf fl/fl control were overlayed onto the T H 1 and T H 17 pathways.A fixed scale of −5 (blue) to 3.5 (red) was kept between all conditions, and Prdm1 was colored black in the Prdm1-deficient T cells.Cre mice compared to controls was confirmed quantitatively (Fig. 5a); for example, for Il1a and Osm, encoding Oncostatin M (Fig. 5b), which was previously associated with colitis 11,28,29 .Granulocyte-associated genes in this cluster, including Csf3r, Lcn2, S100a8 and Cxcr2, were most highly expressed in the LPLs from infected Maf fl/fl Cd4 Cre mice and to a much lesser extent in the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre and Prdm1 fl/fl Cd4 Cre mice (Fig. 5b).Similarly, while the average gene expression in the bulk tissue RNA-seq data granulocyte cluster 7 (Fig. 1f,g) was increased in the LPLs of control mice upon infection with H. hepaticus, a further increase was seen in the Maf fl/fl Cd4 Cre and to a lesser extent in the Prd-m1 fl/fl Cd4 Cre infected mice, but not in the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre infected mice, which showed levels similar to those of the H. hepaticus-infected control mice (Fig. 5c).Genes associated with granulocyte/neutrophil activation, including Ncf2, Itgam, Mmp9 and Padi4, showed the highest expression in the LPLs from infected Maf fl/fl Cd4 Cre mice but not in the Prdm1 fl/fl Cd4 Cre or double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre infected mice, which showed similar or reduced levels to those from infected control mice, the latter suggesting potential counter-regulatory mechanisms provided by Blimp-1 signaling in T cells (Fig. 5d).Additionally, Acod1, a gene encoding enzyme aconitate decarboxylase 1 (Irg1), which produces the metabolite itaconate in myeloid cells 30 , was found to be most highly expressed in LPLs from infected Maf fl/fl Cd4 Cre mice and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre infected mice (Fig. 5d).scRNA-seq analysis showed that the granulocyte/neutrophil and myeloid leukocyte clusters were the source of Il1a and Acod1 in the LPLs from infected Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Fig. 5e).The expression of genes associated with granulocytes/neutrophils, including Lcn2, S100a8, S100a9 and Cxcr2, was exclusively detected in the 'granulocyte/neutrophil' cluster by scRNA-seq (Fig. 5e).Although basal expression of some of these genes was observed in the low number of granulocytes detected by scRNA-seq in the infected control mice, increased expression of these neutrophil-associated genes (Fig. 5e) and granulocyte percentage and numbers (Extended Data Fig. 2b,c) were observed in the LPLs from the Maf fl/fl Cd4 Cre and the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre infected mice.In keeping with the scRNA-seq data, increased percentage and numbers of Ly6G + CD11b + neutrophil cells were observed by flow cytometry analysis in the LPLs from the Maf fl/fl Cd4 Cre but to a lesser extent in double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre H. hepaticus-infected mice compared to uninfected or infected control mice and infected Prdm1 fl/fl Cd4 Cre mice, in which these populations were barely detectable (Fig. 5f,g).This was reinforced by analysis of combined gene expression and percentage in the scRNA-seq data for granulocyte-specific genes, such as Ncf2, Csf3r, S100a8 and S100a9, as well as innate cell genes Il1a, Il1b and Acod1, which were maximal in the LPLs from the infected Maf fl/fl Cd4 Cre mice (Fig. 5h).Ly6G − CD11b + myeloid cells, while present in uninfected and infected control mice at low numbers, showed a significant increase by flow cytometry in the LPLs from the H. hepaticus-infected Prdm1 fl/f Cd4 Cre mice, and to a greater extent in both Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice compared to the uninfected or infected control mice (Fig. 5f,g).This increase in myeloid cells is in keeping with increased expression of Il1a, Il1b, Acod1 and the Csf2ra and Csf2rb in the LPLs from both the infected Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Fig. 5e,h).

Prdm1 and Maf control T cell-myeloid colonic cell interactions
Ligand-receptor pairs identified from the scRNA-seq data using CellChat, as referenced in the Methods, were used to infer putative cell-to-cell crosstalk (Fig. 6 and Extended Data Figs.7 and 8).Analysis of the immune cells of interest from this study revealed outgoing and incoming interactions between the T cell Ctla4 high population and B cells in the LPLs from H. hepaticus-infected control mice, while in the infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice, myeloid leukocyte/macrophage populations delivered increased strength of signals to the T cell Ctla4 high population, which in turn signaled back to these myeloid cells (Fig. 6a).These findings were validated by immunofluorescent staining of colon sections (Fig. 6b and Extended Data Fig. 9).CD4 + T cells (white) and CD68 + mononuclear phagocytes (magenta) were found to be increased and in close proximity in H. hepaticus-infected Prdm1 fl/fl Cd4 Cre mice, and to a greater extent in Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre -infected mice compared to control mice (Fig. 6b).Neutrophils staining positive for MPO were most abundant in Maf fl/fl CD4 Cre -infected mice (Fig. 6b), in keeping with the RNA-seq and flow cytometry data, and appeared to co-localize for the most part with CD68 + mononuclear phagocytes and CD4 + T cells (Fig. 6b).The largest inferred increase in both outgoing and incoming signals observed in myeloid leukocytes was in the LPLs from infected Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/f Maf fl/fl Cd4 Cre mice, which included strong outgoing CCL and CXCL signaling.The T cell Ctla4 high population was inferred to deliver a strong Ccl5 signal to Ccr1/Ccr5 on myeloid leukocytes (Fig. 6c,d), whereas the myeloid leukocyte population was predicted to deliver a strong Cxcl16/Cxcl10 signal largely to Cxcr6/Cxcr3 on the T cell Ctla4 high population, highlighting potential axes contributing to CD4 T cell accumulation in the LPLs of these mice (Fig. 6c,d).Consistent with the well-established role of Cxcl2-Cxcr2 in neutrophil responses, inferred interactions between the myeloid leucocyte population and granulocytes through the Cxcl2-Cxcr2 axis-and additionally, strong Ccl interactions within the myeloid leukocyte population and from myeloid leukocytes to granulocytes-were detected in the infected Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/f Cd4 Cre mice (Fig. 6c,d), potentially contributing to the increased neutrophil and myeloid numbers in these mice.While IFNγ signaling in infected control mice was inferred to be delivered from NK cells to B cells, an outgoing signal from T cell Ctla4 high cells to myeloid cells and B cells was observed in the infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice (Extended Data Fig. 8c).Inferred Csf1 signaling between myeloid populations detected in both Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/f Maf fl/f Cd4 Cre mice (Extended Data Fig. 8d) potentially contributed to the increased myeloid cells and neutrophils.Thus, dysregulated cytokine and chemokine cell-cell interaction networks downstream of disrupted transcriptional regulation in T cells by Blimp-1 and c-Maf may contribute to pathology during H. hepaticus infection.Transcriptomic data from colon biopsies of patients with IBD, including Crohn's disease and ulcerative colitis, were obtained from the Gene Expression Omnibus (GEO) (GSE193677 and GSE126124).Modular analysis was performed to display clusters of up-regulated or down-regulated genes compared to controls.Modules of interest were annotated and selected to interrogate our mouse LPL scRNA-seq dataset from the H. hepaticus-infected mice.(Fig. 7a,b,c and Supplementary Table 8).Genes within the human IBD 'Immune effector' module were found to be largely expressed in the 'B cells 1', 'macrophage/myeloid leukocyte', 'T cells Ctla4 high' and 'T cells Icos high' in the mouse LPL scRNA-seq clusters from infected Prdm1 fl/f Cd4 Cre mice and to a greater extent the Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/f Maf fl/fl Cd4 Cre mice (Fig. 7d).Expression of genes from the human 'granulocyte, myeloid' module, which were more highly expressed in adult ulcerative colitis, which usually manifests as inflammation of the colon, was largely confined to the mouse scRNA-seq 'epithelial', 'granulocyte' and 'macrophage/ myeloid leukocyte' clusters, and expressed at the highest level in the Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/f Maf fl/f Cd4 Cre mice (Fig. 7e,f).T cell/ILC-related genes shown earlier to be expressed at the highest level in the LPLs from H. hepaticus double-deficient Prdm1 fl/f Maf fl/f Cd4 Cre -infected mice (Fig. 3) were contained within these human IBD modules and IFNG, CSF2, IL18R1, IL-12RB1, IL12RB2 and IL23R expression was increased in Crohn's disease and to a greater extent in ulcerative colitis (Fig. 7f).Additionally, identified within the human IBD 'Immune effector module', IL21 was over-expressed in ulcerative colitis (Fig. 7f) and showed the highest abundance in the LPLs from H. hepaticus double-deficient Prdm1 fl/f Maf fl/f Cd4 Cre -infected mice (Fig. 7g), suggesting that IL-21 is associated with a type 1 response.T cell/ILC-associated genes most highly expressed in LPLs from infected Maf fl/fl Cd4 Cre mice (Fig. 3), including IL17A and IL17F, were highly expressed in human IBD, again to a higher extent in ulcerative colitis (Fig. 7f,g and Supplementary Table 8).Multiple genes associated with myeloid cells and innate immunity, which we showed earlier were most highly expressed in the LPLs from infected Maf fl/fl Cd4 Cre mice (Fig. 5h), were over-expressed in human IBD, including IL1B, IL1A, CSF3, ACOD1, OSM, S100A8, S100A9 and CXCR2 (Fig. 7f).However, ALPK2, over-expressed in human IBD and mostly in ulcerative colitis (Fig. 7f), was equally expressed in Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/f Maf fl/f Cd4 Cre mice (Fig. 7g), while CXCL10 was increased in both ulcerative colitis and Crohn's disease (Fig. 7f), and was highest in the double-deficient LPLs (Fig. 7g).Other genes identified within the human IBD 'granulocyte, myeloid' module, including NOD2, FCGR3 and PROK2, which were most highly expressed in the biopsies from ulcerative colitis (Fig. 7f), were most highly abundant in the Maf fl/fl Cd4 Cre mice (Fig. 7g).Our findings indicate that genes that show increased expression in human IBD colon biopsies, including those with mutations that have been linked to an increased risk of Crohn's disease and/or ulcerative colitis, such as NOD2, IL23R, IL21 and IFNG 11 , are differentially abundant in the LPLs from H. hepaticus-infected mice with T cell-specific deletion of Prdm1, Maf or both transcription factors, suggesting that these mouse models may reflect different pathobiological mechanisms relevant in human IBD.

Discussion
IL-10 production by CD4 + T cells is critical in regulating immune responses to avoid intestinal immune pathology in response to any potentially disease-causing microorganisms.Here, we show that during oral infection with H. hepaticus, Blimp-1 and c-Maf cooperate to positively regulate Il10 gene expression and differentially negatively regulate a large network of proinflammatory effector genes.H. hepaticus infection of double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice resulted in a high-level type 1 immune response in LPLs with increased Ifng, Csf2, Il23r, ll12rb1 and Il12rb2 expression, which were also increased (but to a lesser extent) in LPLs from infected Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice.Conversely, H. hepaticus infection of Maf fl/fl Cd4 Cre mice resulted in a Type 17 response, with increased Il17a and Il22 expression and a pronounced signature of neutrophils, myeloid cells and innate immunity.Thus, Blimp-1 and c-Maf cooperate to control common and distinct gene networks in T cells by specific, direct and shared actions on proinflammatory cytokines, over and above their direct stimulatory effects on Il10.Genes over-expressed in human IBD showed differential expression in the LPLs from H. hepaticus-infected mice with T cell-specific deletion of Prdm1 or Maf, potentially revealing T cell-regulated mechanisms relevant to human disease.
Our findings that T cell-specific deletion of Maf did not result in overt inflammation of the colon in the steady state are in keeping with most reports that Maf fl/fl Cd4 Cre or Maf fl/fl Foxp3 Cre mice do not develop colitis 13,14,17,18 in the absence of challenge, although there are a few conflicting reports of spontaneous mild colitis 18,19 .Likewise, there are conflicting reports in T cell-specific Prdm1-deficient mice, with some indicating spontaneous colitis 22,23,31 whereas in other reports no signs of colitis were observed 24 , the latter in keeping with our findings.Spontaneous colitis in double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice has been reported 14 , in contrast to our findings in the same mice, probably reflecting the controlled microbiota in our vivarium.Spontaneous colitis in mice with T cell-specific deletion of Prdm1, Maf or both transcription factors has been associated with either T reg cells losing their immunosuppressive function 14 or increased frequencies of T H 17 cells 23 , probably triggered by undefined microbiota or infection with pathobionts.Microbiota or pathobionts may be maintaining T H 17 cells at the steady state, as has been previously described 17,32,33 and very recently shown to be under c-Maf-dependent T reg control 17 .
Given that mice with T cell-specific deletion of Prdm1, Maf or both transcription factors did not exhibit any signs of intestinal inflammation, this provided us with an excellent baseline to determine their role in regulating the intestinal immune response to a defined pathobiont.No intestinal pathology was observed in the control H. hepaticus-infected mice unless they were co-administered anti-IL-10R mAb, in keeping with previous reports 8, [34][35][36] .As no increase in pathology was observed in the Prdm1 fl/fl Maf fl/fl Cd4 Cre H. hepaticus-infected mice when co-administered anti-IL-10R mAb, this suggests that the high level a, Modules of co-expressed genes were derived from human adult IBD colonic biopsies (GSE193677) using the R package WGCNA and b, tested in an independent human pediatric IBD dataset (GSE126124).CD, Crohn's disease; UC, ulcerative colitis.In the dot plots, the color and size of the dots represent the fold enrichment in disease compared to controls.Re-named modules indicate biological processes associated with the genes within a module.Fold-enrichment scores were derived using QuSAGE software; red and blue colors indicated over-abundance and under-abundance, respectively, of genes within a module (compared to control samples).Size of the dots represents the relative degree of perturbation (larger dots represent a higher degree of perturbation), and only modules with an adjusted P < 0.05 were considered significant and depicted in the plot.c, Enrichment of genes within the 'immune effector cells' and the 'granulocyte, myeloid and innate cells' modules were then tested in our mouse colon LPL scRNA-seq dataset.d,e, Scoring of the 'immune effector cells' (d) and 'granulocyte, myeloid and innate cells' modules (e) were projected into our scRNA-seq UMAP.f, Dot plot of over-expressed T cell-associated or myeloid cell-associated genes from human IBD colon biopsies found within the modules from a and b versus controls.The dot size represents the P value and the log 2 (fold change) is indicated by the color scale.g, Dot plot of gene expression of selected genes found to be over-expressed in human IBD as in f, in colon LPL scRNA-seq from H. hepaticus-infected control Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cremediated deletion of either Prdm1, Maf or both Prdm1 and Maf.The dot size represents the percentage of cells per cell cluster expressing the gene in question and the expression level indicated by the color scale.  of intestinal pathology resulted from the effects of both Prdm1 and Maf on other immune factors in addition to their co-dominant and direct role in Il10 gene regulation [3][4][5]13,14,37 . This is upported by our findings of distinct Blimp-1 binding sites for Ifng and unique c-Maf binding sites for Il17a and Il22, coinciding with open chromatin (ATAC-seq) sites from sorted CD4 + T cells from H. hepaticus-infected mice.
Our findings that T cell-associated and ILC-associated genes such as IFNG, IL12RB2, IL23R, IL18R, CSF2, IL17A and IL17F that were highly expressed in colon biopsies from human IBD 11,38,39 showed differential expression in the LPLs from double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice and were enriched in Foxp3 − effector T cells and ILC may reveal distinct pathological mechanisms of human IBD.Many of these genes have been reported as susceptibility loci for IBD and have highlighted shared genetic risk across populations 11,39 .For example, Prdm1 has been reported to harbor rare missense mutations in PR domain-containing 1 (PRDM1) associated with IBD, and these mutations resulted in increased T cell proliferation and production of proinflammatory cytokines such as IFNγ 12 .Moreover, the Notch-STAT3 − -Blimp-1-c-Maf axis, shown to be a common anti-inflammatory pathway in T cells, has been shown to be defective in effector CD4 + T cells from patients with Crohn's disease 40 .
The increased Ifng and Csf2 expression accompanying the intestinal pathology in the Prdm1 fl/fl Maf fl/fl Cd4 Cre H. hepaticus-infected mice, and to a lesser extent in the single T cell-specific transcription-factor-deficient mice, supports earlier studies in different models of colitis, implicating IFNγ 41 and GM-CSF 42 in exacerbating intestinal pathology.Although IL-23 receptor signaling has been strongly linked with induction of a T H 17 response 43 , expression of Il23r in LPLs from Prdm1 fl/fl Cd4 Cre and Prdm1 fl/fl Maf fl/fl Cd4 Cre mice accompanying high levels of Ifng, but not in the Type 17-dominated response in Maf fl/fl Cd4 Cre mice, supports earlier reports of IL-23 in promoting IFNγ production and intestinal inflammation 35,44 .The significant increase in Il17a expression in the LPLs from H. hepaticus-infected Maf fl/fl Cd4 Cre mice supported a major role for c-Maf in negatively regulating IL-17 responses to pathobionts, in keeping with previous in vitro reports highlighting c-Maf as a repressor of T H 17 (Il17a and Il22) responses 16,45 .Given that CD4 + T cells from LPLs of H. hepaticus-infected control mice showed a similar level of expression of Il17a, although not Il22, to that in Maf fl/fl Cd4 Cre mice, this suggests that microbiota may be maintaining Il17a expression at the steady state as has been previously described 17,32,33 .The source of the increased Il17a and Il22 in the total LPLs from H. hepaticus Maf fl/fl Cd4 Cre mice above that of infected control mice may be attributed to the increased abundance of T H 17 cells or to other IL-17A producers such as ILC3 or γδ-T cells 34 and to the heterogeneity of T H 17 cells.
Increased Il17a expression in the Maf fl/fl Cd4 Cre mice may explain the increased numbers of neutrophils in the colon, as IL-17 has been reported to promote neutrophil recruitment and function during infections, and supports the role of neutrophils in intestinal pathology and their association with human IBD 29 .Other neutrophil/myeloid-associated genes, PROK2 (prokineticin 2) and FCGR3, which were most highly expressed in the biopsies from ulcerative colitis patients, were most abundant in the LPLs from H. hepaticus-infected Maf fl/fl Cd4 Cre mice, potentially underpinning a pathobiologic neutrophil-driven mechanism for human IBD.Cytokines previously associated with IBD, such as Il1a 28 , Osm 11,46 and other myeloid-associated genes were elevated in the LPLs from H. hepaticus-infected Maf fl/fl Cd4 Cre but to a lesser extent in double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre infected mice.Given that neutrophil numbers, neutrophils and innate immunity-associated genes were increased to a lesser extent in the double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre infected mice, this may suggest cross-regulation of the IL-17 responses and neutrophil function by IFNγ 47 or by other factors elevated in the absence of Prdm1 in T cells.
Our findings support previous reports that Foxp3 + T reg cells are the major source of Il10 during H. hepaticus infection and are most likely to control intestinal inflammation.Foxp3 + RORγt + T reg cells were almost completely abolished in LPLs from infected Maf fl/fl Cd4 Cre mice, as previously reported 27 , but not in the infected double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice, which exhibited the maximum pathology and expressed the lowest levels of Il10 in CD4 + T cells, indicating that Foxp3 + RORγt + T cells are not the only IL-10 producing Foxp3 + T reg population regulating intestinal pathology.The source of low levels of Blimp-1 and c-Maf-dependent Il10 expression by Foxp3 − T cells could potentially be T H cells or Tr1 cells 18,48 , which could have arisen by chronic stimulation of T H 17 or T H 1 cells as previously reported 49 or from peripherally induced T reg cells that have lost Foxp3 expression 50 .The CD4 + T cell source of IL-10 controlling immune responses to limit host damage is likely to be dictated by whether the immune response is to intestinal microbiota, pathobionts, different pathogens or different isolates of the same pathogen, and may change at different stages of infection and/or anatomical locations, as we previously discussed 4 .
Intestinal IL-10-producing T reg cells, which exert their effects in lymphoid aggregates in the lamina propria, have been recently referred to as Foxp3 + T reg effector cells, expressing Areg, Gzmb, Icos, Tigit, Tnfrsf4 (OX40) and Tnfrsf18 (GITR) 34 .Although our findings support the increased expression of these effector genes in Foxp3 + T reg cells, these genes were increased to a much greater extent in Foxp3 − CD4 + T cells in LPLs from H. hepaticus-infected Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice compared to controls (data not shown).Our findings are in keeping with previous reports that c-Maf controls T reg cell-derived IL-10 and intestinal T H 17 responses 17,18 .However, given that Foxp3 − c-Maf-deficient T cells expressed the maximal levels of Il17 in the LPLs from H. hepaticus-infected Maf fl/ fl Cd4 Cre mice, our data suggest that c-Maf is controlling Il17 expression in non-T reg IL-17-producing effector T cells.
Collectively, our findings show that Blimp-1 and c-Maf cooperate to positively regulate Il10 expression and to directly control genes encompassing the type 1 effector cell responses to prevent severe H. hepaticus-induced colitis.However, Maf uniquely regulated increased Il17a and Il22 expression and an accompanying pronounced signature of neutrophil activation and increased neutrophil numbers.Thus, Blimp-1 and c-Maf control common and distinct gene networks in T cells that regulate qualitatively different CD4 + T cell effector and innate immune responses and subsequent intestinal pathology.DEGs in the LPL of H. hepaticus-infected mice with T cell-specific deletion of Prdm1 or Maf were elevated in colon biopsies from patients with IBD and may help delineate pathways to reveal novel pathobiologic mechanisms of human disease.

Online content
Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41590-024-01814-z.

RNA-seq of colon LPLs
RNA was extracted from colon LPLs of individual mice using the QIAShredder and RNeasy Mini Kit with on-column DNase digestion, according to the manufacturer's instructions (Qiagen).RNA-seq libraries were then made with total RNA using the KAPA RNA HyperPrep with RiboErase and unique multiplexing indexes, according to the manufacturer's instructions (Roche).All libraries were sequenced using the HiSeq 4000 system (Illumina) with paired-end read lengths of 100 bp and at least 25 million reads per sample.

scRNA-seq of colon LPLs
Isolated colon LPLs from individual mice (as detailed above) were filtered using a 70 μm filter, and cells were suspended in PBS 0.04% BSA (UltraPure BSA, Invitrogen).For each sample, an aliquot of cells was stained with AO/PI Cell Viability Kit (Logos Biosystems) and counted with the LunaFx automatic cell counter.For all samples, cell viability before loading was >80%.As per the manufacturer's instructions, the Master Mix was prepared as detailed in the Chromium Next GEM Single Cell 3′ Reagent Kit v.3.1 (Dual Index) manual, and 10,000 cells per sample were loaded into the 10× Chromium chips (10× Genomics).The 10× Chromium libraries were prepared and sequenced (paired-end reads) using the NovaSeq 6000 (Illumina).

RNA-seq of sorted CD4 + T cells from colon lamina propria
RNA was extracted from flow-sorted CD4 + T cells isolated from the colon lamina propria of individual mice using the QIAShredder and RNeasy Mini Kit with on-column DNase digestion, according to the manufacturer's instructions (Qiagen).RNA-seq libraries were then made with total RNA using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina and unique multiplexing indexes, according to the manufacturer's instructions (New England Biolabs).All libraries were sequenced using the HiSeq 4000 system (Illumina) with paired-end read lengths of 100 bp and at least 25 million reads per sample.

Statistical analysis
All figure legends show the number of independent biological experiments performed for each analysis and all replicates.Flow cytometry percentages and associated cell numbers were analyzed as a one-way ANOVA with Tukey's multiple comparisons test and 95% confidence intervals for statistical analysis.All statistical analyses, apart from sequencing, were carried out with Prism8 software (GraphPad), and the following P values were considered statistically significant: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.Analyses for RNA-seq and ATAC-seq data were performed with R version 3.6.1 and Bioconductor version 3.9.Analyses for scRNA-seq data were performed with R ver-

RNA-seq data processing and analysis
For bulk tissue LPL RNA-seq, adaptors were trimmed using Skewer software version 0.2.2 (ref.54) with the following parameters: '-m pe -q 26 -Q 28 -e -l 30 -L 100', specifying the relevant adaptor sequences.For sorted CD4 + T cell RNA-seq, adaptors were trimmed using FLEXBAR software 55 , as recommended by the manufacturer (NEB) when using the NEBNext Single Cell/Low input RNA Library Prep Kit for Illumina.FLEXBAR was run following the provider's suggested pipeline found at https://github.com/nebiolabs/nebnext-single-cell-rna-seq.For both bulk tissue LPL RNA-seq and sorted CD4 + T cell RNA-seq, reads were aligned to the mm10 genome and the GENCODE reference transcriptome version M22 using STAR software version 2.7.1 (ref.56), excluding multi-mapping reads by setting the parameter 'outFilterMultimapNmax' to 1.To increase read mapping to novel junctions, the parameter 'twopassMode' was set to 'Basic'.Raw gene counts were retrieved using QoRTs software version 1. 1.8 (ref.57).Normalized read counts were retrieved using DeSeq2 version 1.24.0 (ref.58) and were rlog transformed to visualize gene quantifications.

Cell enrichment and biological pathway annotation
The identified DEGs (in any condition) were subjected to k-means clustering using k = 9; the expression values for the DEGs were standardized into z-scores and visualized in a heatmap (Fig. 1f).To provide a biological interpretation of these clusters, each cluster was subjected to 'cell-type enrichment' and 'biological pathways' annotation.The cell-type enrichment analysis used the cell-type signatures from a previous publication 51 , and a Fisher's exact test was performed to identify cell-type signatures enriched in each of the clusters.Adjusted P values were obtained using the BH correction.Cell-type signatures that are statistically significantly enriched (adjusted P < 0.05) are shown in Fig. 1g.The R package topGO 59 was used to obtain the enriched biological processes in each cluster (Extended Data Fig. 1e).Additionally, an ingenuity pathway analysis (IPA) 'core analysis' (Qiagen, www.qiagen.

ATAC-seq data processing and analysis
Paired-end ATAC-seq reads from sorted CD4 + T cells were quality controlled and adaptors were trimmed using Skewer software version 0.2.2 (ref.54) with the following parameters: '-m pe -q 26 -Q 30 -e -l 30 -L 50', specifying 'CTGTCTCTTATACAC' as reference adaptor sequence to remove.The reads were then aligned to the mm10 genome using BWA-MEM 60 , duplicate reads were removed with Picard 61 , and SAMtools 1.3.1 (ref.62) was used to discard discordant alignments and/or low mapping qualities (mapQ < 30).To account for transposase insertion, reads were shifted +4 bp in the forward and −5 bp in the reverse strand; moreover, read-pairs that spanned >99 bp were excluded from further analyses as they would span nucleosomes 53 .MACS2 (version 2.1.1)was used to identify ATAC-seq peaks using the following parameters: 'parameters-keep-dup all-nomodel-shift -100-extsize 200; q-value < 0.01', to identify enrichment of Tn5 insertion sites 63 .Diff-Bind software version 2.0.2 (ref.64) was used to generate raw counts underlying each ATAC-seq peak.Furthermore, batch correction was performed on raw counts using the RUVSeq R package 65 to remove batch effects that resulted from independent experiments.BeCorrect software 66 was used to generate batch-corrected bigwig files, using the outputs from RUVseq software.The resulting batch-corrected and normalized counts were used for visualization.The R package ggbio was used to plot the genome browser tracks 67 .To identify differentially accessible sites of interest for each genotype, differentially accessible sites were subjected to k-means clustering using k = 7; the normalized read counts for the differentially accessible sites were standardized into z-scores and visualized in a heatmap.

ChIP-seq data processing and analysis
Publicly available c-Maf ChIP-seq raw fastq files were obtained from GSE40918 (ref.16) and Blimp-1 ChIP-seq raw fastq files were obtained from GSE79339 (ref.68).Trimmomatic (version 0.36) was used for quality control and to trim adaptor sequences using the following parameters: 'HEADCROP:2 TRAILING:25 MINLEN:26' (ref.69).Trimmed reads were aligned to the mouse genome mm10 with Bowtie version 1.1.2(ref.70), with the parameters: 'y -m2-best-strata -S'.MACS2 (version 2.1.1)was used with default parameters to identify ChIP-seq peaks, and peaks with a q-value of <0.01 were defined as statistically significant binding sites.'bamCoverage' from DeepTools (version 2.4.2) was used to normalize ChIP-seq data to RPKMs, and the R package ggbio was used to visualize the genome browser tracks 67 together with the ATAC-seq data.

IPA pathways
T H 1 and T H 17 pathways were constructed in and obtained from the IPA signaling pathways library.Log 2 (fold changes) from the differential expression analyses outlined above were overlaid on the T H 1 and T H 17 pathways, all with a fixed scale of −5 (blue) to 3.5 (red).

scRNA-seq data processing and analysis
Fastq files were aligned to the mm10 transcriptome, and count matrices were generated, filtering for GEM cell barcodes (excluding GEMs with free-floating mRNA from lysed or dead cells) using Cell Ranger (version 6.1.2).Count matrices were imported into R and processed

Fig. 1 |
Fig. 1 | T cell-specific Blimp-1 and c-Maf control intestinal responses.a, Schematic of experimental method used to infect mice with H. hepaticus by oral gavage.b-e, Representative H&E colon sections from each genotype following infection with H. hepaticus for 14 days (b) with the corresponding colon histopathology scores (detailed in Methods) (c), colon LPL cell counts for each H. hepaticus-infected group compared to uninfected controls (d) and total CD4 + T cell counts for each H. hepaticus-infected group compared to uninfected controls (e).Each dot within the bar plots represents an individual mouse analyzed.Graph shows means, error bars, s.d.Analyzed by one-way ANOVA followed by Dunnett post-hoc test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).Scale bar, 100 μm.Bulk tissue RNA-seq was performed on total Resource https://doi.org/10.1038/s41590-024-01814-zPrdm1 l s γ δ -T c e l l s M a c r o p h a g e s D e n d r it ic c e l l s G r a n u l o c y t e s I n n a t e l y m p h o id c e l l s M a s t c e l l s B c e l l s S t r o m a l c e l l s S t i b r o g e n i c p r o g e n i t o r c e l l E n d o t h e l i a l c e l i b r o g e n i c p r o g e n i t o r c e l l E n d o t h e l i a l c e l

Fig. 3 |
Fig. 3 | Prdm1 and Maf induce Il10 and control T effector cytokines.a,b, RNA-seq gene expression of Il10 (a) and Ifng, Il17a, Il22 and Csf2 (b) in sorted CD4 + T cells from colon LPLs isolated from uninfected Prdm1 fl/fl Maf fl/fl mice and H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice as well as mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.c, Gene expression of Ifng, Il17a and Il22 in bulk tissue total colon LPLs isolated from uninfected and H. hepaticus-infected mice.In a-c, DEGs in each condition against uninfected Prdm1 fl/fl Maf fl/fl mice were marked for statistical significance as follows, *BH-adjusted P ≤ 0.05; **BH-adjusted P ≤ 0.01; ***BH-adjusted P ≤ 0.001; ****BH-adjusted P ≤ 0.0001.d, Dot plot of scRNA-seq gene expression of selected genes Ifng, Il17a, Il22, Il18r1, Il12rb2, Il12rb1, Il23r, Cxcr3 and Csf2 in colon LPLs from H. hepaticus-infected Prdm1 fl/fl Maf fl/fl or mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.The dot size represents the percentage of cells per cell cluster expressing the gene in question and the expression level indicated by the color scale.e, Expression of Foxp3 as assessed by scRNA-seq within the UMAP visualization of annotated scRNA-seq datasets within each of H. hepaticus-infected genotypes.f-j, Expression at the single-cell level of Il10 (f), Ifng (g), Csf2 (h), Il17a (i) and Il22 (j) in the 'Ctla4 high' cells expressing Foxp3 (Foxp3 + ) or not (Foxp3 − ).The distribution of expression in cells within each condition is shown by the violin plots; the dot plot (top panels) displays the proportion of cells expressing the gene in question, with the expression level indicated by the color scale.

Fig. 5 |
Fig. 5 | Increased myeloid gene expression in the absence of Maf in T cells.a-d, Violin plots summarizing the expression values (quantified as z-scores) and boxplots of example genes of innate immunity-associated genes found in cluster 6 (a,b) and neutrophil activation associated genes found in cluster 7 (c,d) of the bulk tissue LPL RNA-seq data analysis from Fig. 1f.DEGs in each condition against uninfected Prdm1 fl/fl Maf fl/fl mice were marked as follows (all P values BH-adjusted): *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.e, Expression of selected innate immunity and granulocyte-associated genes queried in the granulocyte, macrophage and myeloid leukocyte cell clusters across all H. hepaticus-infected conditions within the colon LPL scRNA-seq dataset.f, Representative flow plots and gating strategy used for the flow cytometry analysis of neutrophils (Live CD90.2 − TCR-β − CD19 − CD11 + Ly6G + ) across uninfected Prdm1 fl/fl Maf fl/fl and Prdm1 fl/fl Maf fl/fl Cd4 Cre , and H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice as well as mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.g, Bar plots of percentage from live (top panels) and absolute cell counts (bottom panels) of neutrophils and Ly6G -CD11b + cells from lamina propria of the colon.Each dot within the bar plots represents an individual mouse analyzed.Graph shows means, error bars, s.d.Analyzed by one-way ANOVA followed by Dunnett post-hoc test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).Data from n = 4-5 mice.h, Dot plot of scRNA-seq gene expression of selected genes Il1b, Ncf2, Acod1, Csf2ra, Csf2rb, Csf3r, Il1a, Osm, S100a8, S100a9, Csf3 and Cxcr2, in the colon LPLs from H. hepaticus-infected control Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.The dot size represents the percentage of cells per cell cluster expressing the gene in question, and the expression level is indicated by the color scale.

Fig. 4 |
Fig.4| Multi-omic integration identifies binding sites for Blimp-1 and c-Maf.a, ATAC-seq was performed on sorted CD4 + T cells from colon LPLs isolated from uninfected Prdm1 fl/fl Maf fl/fl control and H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice as well as mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.Genome browser tracks of ATAC-seq data from each condition together with publicly available c-Maf (green) and Blimp-1 (red) ChIP-seq datasets in the Il10, Ifng, Il17a, Il22 and Csf2 loci.Statistically significant ChIP-seq peaks (q < 0.05)

Fig. 6 |
Fig. 6 | Increased interaction between T cells, macrophages and neutrophils.Cell-to-cell communication networks inferred using CellChat software from gene expression of ligands and their receptors in immune cell clusters of interest from the colonic LPL scRNA-seq dataset.a, Strength of interaction of cell-to-cell interactions, represented in the edge width, in H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.b, Representative images (n = 4-5) of colon sections by immunofluorescence, staining for CD4 + T cells (CD4, white), mononuclear phagocytes (CD68, magenta), neutrophils (MPO, green) and nuclear staining (DAPI, blue) from H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.Scale bar, 20 μm.c,d, Cell-to-cell communication networks underlying CCL (c) and CXCL (d) pathways across all H. hepaticus-infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1, Maf or both Prdm1 and Maf.The chord plot has receiver cells at the top (incoming signaling) and transmitter cells (outgoing signaling) at the bottom.The edges are colored based on the cell clusters expressing the outgoing signals.In a,c and d, node size is proportional to the number of cells in each experimental group, and the edges are colored based on the cell clusters expressing the outgoing signals.

Fig. 7 |
Fig. 7 | IBD patient gene signatures are regulated by Prdm1 and Maf.a, Modules of co-expressed genes were derived from human adult IBD colonic biopsies (GSE193677) using the R package WGCNA and b, tested in an independent human pediatric IBD dataset (GSE126124).CD, Crohn's disease; UC, ulcerative colitis.In the dot plots, the color and size of the dots represent the fold enrichment in disease compared to controls.Re-named modules indicate biological processes associated with the genes within a module.Fold-enrichment scores were derived using QuSAGE software; red and blue colors indicated over-abundance and under-abundance, respectively, of genes within a module (compared to control samples).Size of the dots represents the relative degree of perturbation (larger dots represent a higher degree of perturbation), and only modules with an adjusted P < 0.05 were considered significant and depicted in the plot.c, Enrichment of genes within the 'immune effector cells' and the
sion 4.1 and Seurat version 4.1.1.Error bars and sample sizes used are described in the figure legends.
LPLs by RNA-seq DeSeq2 (ref.58) was used to obtain DEGs for each of the four H. hepaticus-infected groups: Prdm1 fl/fl Maf fl/fl , Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and Prdm1 fl/fl Maf fl/fl Cd4 Cre against the uninfected Prdm1 fl/fl Maf fl/fl control.A gene was considered to be statistically differentially expressed if the fold change was ≥1.5 and the BH-adjusted P value was <0.05, resulting in: Prdm1 fl/fl Maf fl/fl infected vs uninfected Prdm1 fl/fl Maf fl/fl : 5 DEGs Prdm1 fl/fl Cd4 Cre vs uninfected Prdm1 fl/fl Maf fl/fl : 1,207 DEGs Maf fl/fl Cd4 Cre vs uninfected Prdm1 fl/fl Maf fl/fl : 1,740 DEGs Prdm1 fl/fl Maf fl/fl Cd4 Cre vs uninfected Prdm1 fl/fl Maf fl/fl : 3,392 DEGs.
com/ingenuity) was performed to identify the IPA pathways enriched in each cluster.The Prdm1 fl/fl Maf fl/fl Cd4 Cre versus uninfected Prdm1 fl/fl Maf fl/fl expression values served as input for the calculation of the z-scores in the bar plots depicted in Extended Data Fig.1e, as this was the comparison that resulted in the highest amount of DEGs.Differential gene expression in sorted colonic CD4 + T cellsDeSeq2 (ref.58) was used to obtain DEGs for the uninfected Prdm1 fl/fl Maf fl/fl Cd4 Cre and each of the four H. hepaticus-infected groups: Prdm1 fl/fl Maf fl/fl , Prdm1 fl/fl Cd4 Cre , Maf fl/fl Cd4 Cre and Prdm1 fl/fl Maf fl/fl Cd4 Cre against the uninfected Prdm1 fl/fl Maf fl/fl control (Extended Data Fig.6b).A gene was considered to be a statistically differentially expressed if the fold change was ≥1.5 and the BH-adjusted P value was <0.05.

Extended Data Fig. 1 |Extended Data Fig. 4 |Extended Data Fig. 6 |Extended Data Fig. 7 |
Changes in gene expression regulated by Blimp-1 and c-Maf.a) Representative H&E colon sections from steady state mice aged to 24-30 weeks, scale bar = 100μm.Data representative of multiple female and male mice: Prdm1 fl/fl (n = 8), Prdm1 fl/fl Cd4 Cre (n = 19), Maf fl/fl (n = 10), Maf fl/fl Cd4 Cre (n = 22), Prdm1 fl/fl Maf fl/fl (n = 36), Prdm1 fl/fl Maf fl/fl Cd4 Cre (n = 38) b) Schematic of experimental method used to infect mice with H. hepaticus by oral gavage and treatment with anti-IL-10R blocking antibody.Representative H&E colon sections (scale bar = 100μm) from each genotype following infection with H. hepaticus and treatment with anti-IL-10R antibody and harvested on Day 14 with the corresponding colon histopathology scores (bottom panels).Each dot within the barplots represents an individual mouse analyzed.Graph shows mean ±s.d., analyzed by one-way ANOVA followed by Tukey's post-hoc test (*=p value ≤ 0.05, **=p value ≤ 0.01, ***=p value ≤ 0.001, ****=p value ≤ 0.0001).c) Unsupervised hierarchical clustering of a pair-wise Spearman correlation and d) PCA plots showing on PC1 the separation of floxed control mice (uninfected and infected) versus H. hepaticus-infected mice with Cd4 Cre -mediated deletion of Prdm1, Maf, or Prdm1 and Maf.e) Enriched IPA canonical pathways (left) and gene ontology biological processes (right) were obtained for the clusters of differentially expressed genes arising upon infection and Cd4 Cre -mediated deletion of either Prdm1, Maf, or both Prdm1 and Maf (related to Fig. 1f).Data from n = 3 − 5 mice.Extended Data Fig. 3 | Top genes in colon scRNAseq clusters in infected mice.a) Heatmap and b) dotplot (of cell clusters not in Fig. 2e) of the top 10 differentially expressed marker genes of the identified cell clusters in our colon LPL scRNA-seq.Colored by the average gene expression across all cell clusters.In the dotplots, the dot size represents the percentage of cells per cell cluster expressing the gene in question.Flow cytometry and RNAseq analysis of colon in infected mice.a) Gating strategy for flow cytometry analysis of colon LPLs.b-d) Percentages from live cells of b) total lymphocytes and c-d) total CD4 + T cells (Live CD90.2 + TCR-β + CD4 + CD8-)) producing IFN-γ, GM-CSF, IL-17A and IL-10.Flow plots of CD4 + T-cells for IFN-γ production versus e) IL-10, f) GM-CSF and g) IL-17A.h) Gating of total CD4 + GM-CSF+ lymphocytes with corresponding flow plots of IFN-γ versus IL-17A.i) Gating of total GM-CSF+ lymphocytes with corresponding flow plots of IFN-γ versus IL-17A with bar plots of j) percentages (from live population) of lymphocytes producing GM-CSF alongside IL-17A, IFN-γ, or both.All flow analyses were performed on from H. hepaticus infected mice.k) Gene expression of Il12rb2, Il12rb1, Il23r, Stat4, Il18r1, Cxcr3 and Csf2 in bulk tissue total colon LPL isolated from uninfected and H. hepaticus infected mice.Differentially expressed genes in each condition against uninfected Prdm1 fl/fl Maf fl/fl mice were marked as follows: *=BH adjusted p value ≤ 0.05, **= BH adjusted p value ≤ 0.01, ***= BH adjusted p value ≤ 0.001, ****= BH adjusted p value ≤ 0.0001.Extended Data Fig. 5 | c-Maf regulates colon RORγt+Foxp3+ cells during infection.a) Boxplots of log2(normalized read counts) of Rorc and Foxp3 in bulk tissue LPL RNA-seq.b) Flow cytometry analysis of RORγt and Foxp3 transcription factor expression with c) percentages (top) and numbers (bottom) of CD4 + T cells producing of RORγt and/or Foxp3.d) Boxplots of log2(normalized read counts) of Rorc and Foxp3 in sorted CD4 + T cells from colon LPL isolated from uninfected and H. hepaticus infected mice.Differentially expressed genes in each condition against uninfected Prdm1 fl/fl Maf fl/fl mice were marked as follows: *=BH adjusted p value ≤ 0.05, **= BH adjusted p value ≤ 0.01, ***= BH adjusted p value ≤ 0.001, ****= BH adjusted p value ≤ 0.0001.Changes in T-cell gene expression and chromatin accessibility.a) Unsupervised hierarchical clustering of a pair-wise Spearman correlation of RNA-seq on sorted CD4 + T cells from colon LPL isolated from uninfected Prdm1 fl/fl Maf fl/fl and Prdm1 fl/fl Maf fl/fl Cd4 Cre , and H. hepaticus infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1, Maf, or both Prdm1 and Maf.b) Number of differentially expressed genes identified in H. hepaticus infected mice compared to uninfected Prdm1 fl/fl Maf fl/fl controls (fold change >=1.5 and BH adjusted p value < 0.05).c) Euler diagram of distinct and overlapping differentially expressed genes between transcription factordeficient T cells from H. hepaticus infected mice.d) Unsupervised hierarchical clustering of a pair-wise Spearman correlation of ATAC-seq on sorted CD4 + T cells from colon LPL isolated from uninfected Prdm1 fl/fl Maf fl/fl and Prdm1 fl/fl Maf fl/fl Cd4 Cre , and H. hepaticus infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1, Maf, or both Prdm1 and Maf.e) Number of differentially accessible sites identified in H. hepaticus infected mice compared to uninfected Prdm1 fl/fl Maf fl/fl controls (fold change >=1.5 and BH adjusted p value < 0.05).f) Euler diagram of distinct and overlapping differentially accessible sites between transcription factor-deficient T cells from H. hepaticus infected mice.g) Heatmap of accessibility values (represented as z-scores) of accessible sites identified in CD4 + T cells from H. hepaticus infected mice compared to uninfected Prdm1 fl/fl Maf fl/fl controls (fold change >=1.5 and BH adjusted p value < 0.05), partitioned into 7 clusters using k-means clustering.Pathology scores associated to each mouse are shown at the top of the heatmap.Inference of cell-to-cell communication networks in scRNA-seq.Cell-to-cell communication networks inferred using CellChat software from gene expression of ligands and their receptors in immune, stromal, epithelial and endothelial cell clusters from the colonic LPL scRNA-seq dataset.a) number and b) strength of interaction of cell-to-cell interactions, represented in the edge width, in and H. hepaticus infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1, Maf, or both Prdm1 and Maf.c) Plot of "Outgoing interaction strength" against "Incoming interaction strength" across all H. hepaticus infected conditions.In a-b) node size is proportional to the number of cells in each experimental group, and the edges are colored based on the cell clusters expressing the outgoing signals.