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. Author manuscript; available in PMC: 2026 Jul 18.
Published in final edited form as: J Exp Med. 2026 Apr 24;223(6):e20251094. doi: 10.1084/jem.20251094

B7 co-stimulation antagonizes RORγt+ regulatory T cells and immune tolerance in the intestine

Mengze Lyu 1,2,3,*, Gregory F Sonnenberg 1,2,3,*
PMCID: PMC13378601  NIHMSID: NIHMS2190463  PMID: 42030099

Abstract

Regulatory T (Treg) cells that recognize dietary- or microbiota-derived antigens express RORγt and are essential for immune tolerance in the intestine. A recent paradigm shift found these cells require major histocompatibility complex class II (MHCII) on RORγt+ antigen-presenting cells (APCs) rather than conventional dendritic cells (cDCs) for signal one. Here, we evaluate signal two and unexpectedly find that co-stimulatory molecules B7–1 (CD80) and B7–2 (CD86) antagonize the generation of microbiota-specific RORγt+ Treg cells. Gain-of-function or loss-of-function therapeutics targeting B7 via CTLA-4 exert reciprocal effects on the generation of microbiota-specific RORγt+ Treg cells. This axis was independent of B7 on RORγt+ APCs but required MHCII on this cell type. Finally, CTLA4-Ig treatment restores microbiota-specific RORγt+ Treg cell generation and protects from experimental intestinal inflammation induced by pathobiont colonization with IL-10R signaling blockade. These results define that RORγt+ Treg cells are uniquely restrained by B7 co-stimulation, while CTLA4-Ig enhances immune tolerance in the intestine when acting cooperatively with RORγt+ APCs.

Graphical Abstract

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Introduction

Regulatory T (Treg) cells co-expressing the lineage-defining transcription factors Foxp3 and RORγt represent a specialized subset essential for maintaining immune tolerance within the gastrointestinal tract (Ohnmacht et al., 2015; Sefik et al., 2015; Yang et al., 2016). This unique type of RORγt+ Treg cell is peripherally induced, enriched in the intestinal lamina propria, requires and produces abundant IL-10, exhibits superior suppressive capabilities over conventional Treg cells. Additionally, RORγt+ Treg cells share some overlapping requirements with T helper 17 (TH17) cells for differentiation, such as IL-6 and IL-23, as well as the presence of gut microbiota (Ohnmacht et al., 2015; Sefik et al., 2015; Yang et al., 2016). Many of these peripherally-induced RORγt+ Treg cells recognize antigens derived from gut microbiota or dietary cues (Abdel-Gadir et al., 2019; Al Nabhani et al., 2019; Kim et al., 2016; Knoop et al., 2020; Ramanan et al., 2020; Xu et al., 2018) indicating there are key roles and therapeutic relevance for RORγt+ Treg cells in states of health, inflammatory bowel disease (IBD), or food allergy.

Understanding the pathways that promote the generation of antigen-specific RORγt+ Treg cells has generated considerable interest, with a recent paradigm shift indicating clear distinctions from more conventional Treg cell subsets. This started in part with the discovery of RORγt+ antigen-presenting cells (APCs) in the intestine and associated draining lymph nodes of both mice and humans, which were first characterized as lymphoid tissue inducer (LTi)-like group 3 innate lymphoid cells (ILC3s), and experimental deletion of MHCII on RORγt+ cells revealed an essential role in restraining inflammatory T cell responses to gut microbiota and preventing intestinal inflammation (Hepworth et al., 2015; Hepworth et al., 2013). With the development of additional models and single cell RNA-sequencing (scRNA-seq), subsequent studies revealed a broader family of RORγt+ APC subsets that are necessary and sufficient to instruct the differentiation of microbiota-specific RORγt+ Treg cells (Akagbosu et al., 2022; Kedmi et al., 2022; Lyu et al., 2022; Wang et al., 2021; Yamano et al., 2019). Strikingly, and in contrast to conventional Treg cells, the generation of antigen-specific RORγt+ Treg cells occurred independent of MHCII on cDCs (Akagbosu et al., 2022; Kedmi et al., 2022; Lyu et al., 2022). There are also critical roles for RORγt+ APCs in establishing oral tolerance to dietary antigens via RORγt+ Treg differentiation and by restraining allergen-specific T helper 2 (TH2) cell responses (Cabric et al., 2025; Fu et al., 2025; Narasimhan et al., 2025; Rodrigues et al., 2025; Sun et al., 2025; Teng et al., 2021), or impacting other diverse immune responses in neuroinflammation, colon cancer, or following fungal infection (Dobes et al., 2022; Goc et al., 2021; Grigg et al., 2021). Importantly, there is evidence of altered RORγt+ Treg cells and RORγt+ APCs in the inflamed intestine of IBD patients or food allergy patients (Abdel-Gadir et al., 2019; Hepworth et al., 2015; Lyu et al., 2022), indicating that these cell types support immune tolerance and gut health in humans.

These collective studies demonstrate the necessity of RORγt+ APCs in the generation of RORγt+ Treg cells via MHCII or signal one, but the requirements for signal two remain poorly defined. CD80 and CD86 (or B7 co-stimulation) are expressed on professional APCs and are pivotal in the activation of naïve T cells by engaging CD28 (Azuma et al., 1993; Burke et al., 2024; Caux et al., 1994; Freeman et al., 1993; Green et al., 1994; Lenschow et al., 1996; Linsley & Ledbetter, 1993; Sharpe & Freeman, 2002). CTLA-4, another counter-receptor for B7 molecules on T cells and enriched on Treg cells, acts as a critical negative regulator, maintaining immune homeostasis by attenuating T cell responses (Leach et al., 1996; Tivol et al., 1995; Wing et al., 2008). Together with antigen engagement by the T cell receptor (TCR) binding to MHCII with a cognate peptide, CD28 binds to B7 and generates a synergistic signal that activates PI3K and subsequently phosphorylates Akt to drive T cell survival and proliferation (Appleman et al., 2002; Barata et al., 2004). Beyond PI3K-Akt, CD28 engagement also activates NF-κB, MAPK, and mTOR pathways, reinforcing IL-2 transcription, metabolic reprogramming, and sustained effector differentiation (Acuto & Michel, 2003; Chi, 2012; Esensten et al., 2016; Zhang et al., 1999). While it has long been acknowledged that conventional and thymic-derived Treg cells require B7-engagement of CD28 co-stimulatory signaling (Salomon et al., 2000; Tang et al., 2003), the role of this pathway in the development of RORγt+ Treg cells remains unexplored and should be considered given the unique requirements of this subset.

Here, we demonstrate that CD28 activation through CD80 and CD86 engagement unexpectedly antagonizes antigen-specific RORγt+ Treg cells in the gut. This could be driven by blocking CD80 and CD86 with CTLA4-Ig (Abatacept), which is a FDA approved drug for treating multiple human inflammatory or autoimmune diseases (Moreland et al., 2006). Surprisingly, generation of microbiota-specific RORγt+ Treg cells was not impacted by B7 on RORγt+ APCs, whereas CTLA4-Ig mediated expansion required MHCII on these APCs. Further, in a mouse colitis model of Helicobacter hepaticus colonization combined with anti-IL-10R blockade that recapitulates microbiota-driven gut inflammation and features of human IBD (Kullberg et al., 2006; Kullberg et al., 2001; Kullberg et al., 1998), we demonstrate that blocking B7 co-stimulation enhances RORγt+ Treg cell generation to provide striking protection, but only in the presence of signal one from RORγt+ APCs, provoking a new strategy to drive effective immune tolerance in the gut.

Results and discussion

B7 co-stimulation antagonize microbiota-specific RORγt+ Treg cells in the gut

To investigate the role of co-stimulation on RORγt+ Treg cell responses in the gut, we utilized a previously established model to track the development of antigen-specific T cel responses to Helicobacter hepaticus (Hh) colonization in the intestines of wild-type (WT) C57BL/6J mice (Xu et al., 2018). In brief, congenically marked naïve CD4+ T cells from Hh-specific TCR transgenic (HH7–2tg) mice were adoptively transferred into Hh-colonized recipients with or without B7 co-stimulation blockade. Two weeks post transfer, we analyzed Hh-specific T cells in the mesenteric lymph nodes (mLN) and large intestine (LI) (Fig. S1 A). As expected, Hh-specific T cells efficiently differentiated into RORγt+ Treg cells in control mice, but surprisingly, this was significantly enhanced in mice treated with anti-CD80 and anti-CD86 blocking antibodies (Fig. 1, A–C). The administration of blocking antibodies had a minor effect on the endogenous RORγt+ Treg cell population in the mLN and LI of the recipients (Fig. S1, B and C). We next assessed CD80 and CD86 knockout mice (Borriello et al., 1997) relative to littermates that were complemented with a floxed CD80 transgene (CD80/86KO; CD80Tgfl/fl), which rescues B7 expression and permits deletion in a lineage-specific manner (Watanabe et al., 2017). Consistent with our antibody blockade studies, mice with a complete knockout of CD80 and CD86 exhibited significantly expanded Hh-specific and endogenous RORγt+ Treg cells in both mLN and LI relative to littermates that were complemented to restore CD80 expression in all cell types (Fig. 1, D–I). In contrast, endogenous RORγt− conventional Treg cells were significantly decreased in CD80 and CD86 knockout mice (Fig. S1 D), which is consistent with previous reports (Salomon et al., 2000; Tang et al., 2003). Given the critical role of RORγt+ APCs in instructing RORγt+ Treg cells via signal one (Akagbosu et al., 2022; Kedmi et al., 2022; Lyu et al., 2022), we investigated whether conditional deletion of B7 in RORγt+ APCs impacts RORγt+ Treg cell induction by crossing the above mice with RORγtCre mice. In this context, we found no significant impact on Hh-specific and endogenous RORγt+ Treg cells in both mLN and LI (Fig. 1, D–I). In addition, we examined RORγt− conventional Treg cells in these mice and observed no changes (Fig. S1 D). Collectively, these findings identify that in contrast to conventional Treg cells, the generation of RORγt+ Treg cells does not require B7 co-stimulation, and rather B7 co-stimulation on cell types other than RORγt+ APCs antagonizes the development of RORγt+ Treg cells.

Figure 1. B7 co-stimulation antagonizes RORγt+ Treg cell responses in the gut.

Figure 1.

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(A-C) (A) Flow cytometry plots of Hh-specific CD4+ T cells (mLN, top panel; LI, bottom panel) and frequency of RORγt+ Treg cells in (B) mLN and (C) LI of C57BL/6J WT mice treated with IgG2a/2b (n = 11) or αCD80/αCD86 (n = 11). RORγt+ Treg cells were gated as CD4+ Foxp3+ RORγt+ T cells, and this strategy was applied consistently in all experiments. (D-F) (D) Flow cytometry plots, (E) frequency, and (F) cell number of Hh-specific RORγt+ Treg cells in the mLN (top panel) and LI (bottom panel) of Cd80−/−Cd86−/−; CD80Tgfl/fl mice (n = 4), Cd80−/−Cd86−/− mice (n = 5), and RORγtCre Cd80−/−Cd86−/−; CD80Tgfl/fl mice (n = 5). (G-I) (G) Flow cytometry plots, (G) frequency, and (I) cell number of endogenous RORγt+ Treg cells in mLN (top panel) and LI (bottom panel) of Cd80−/−Cd86−/−; CD80Tgfl/fl mice (n = 4), Cd80−/−Cd86−/− mice (n = 5), and RORγtCre Cd80−/−Cd86−/−; CD80Tgfl/fl mice (n = 5). The data in (Fig. 1) are pooled from three independent experiments. The data are shown as means ± s.e.m.; the statistics shown in (E, F, H, and I) were obtained by ordinary one-way ANOVA analysis of variance with Tukey’s multiple comparisons test. ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001.

Therapeutics targeting CTLA-4 can be harnessed to modulate RORγt+ Treg cells

CTLA-4 has a greater affinity for CD80 and CD86 than CD28, thus permitting a block in activation of T cells (Collins et al., 2002; Schwartz et al., 2001; Stamper et al., 2001). Therefore, we next examined the impact of CD80 and CD86 blockade on Hh-specific RORγt+ Treg cell generation by harnessing the CTLA4-Ig fusion protein (Abatacept) (Fig. S1 E), an FDA-approved molecule that is used in autoimmune conditions but failed to demonstrate efficacy in IBD patients (Davenport et al., 2002; Kremer et al., 2005; Kremer et al., 2006; Kremer et al., 2003; Linsley et al., 1991; Moreland et al., 2006; Read et al., 2000; Ruperto et al., 2008; Sandborn et al., 2012). Consistent with the results obtained with B7 co-stimulatory blocking antibodies, mice treated with CTLA4-Ig exhibited a significantly increased Hh-specific RORγt+ Treg cell population, accompanied by a significant increase in the endogenous RORγt+/RORγt− Treg ratio in the mLN and LI relative to those treated with the isotype control antibody (Fig. 2, A–C, and Fig. S1, F and G). Thus, it is possible to harness a gain-of-function CTLA-4 therapeutic to boost the abundance of RORγt+ Treg cell population in the gut.

Figure 2. CTLA-4 modulation engages B7-dependent regulation of RORγt+ Treg cells.

Figure 2.

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(A-C) (A) Flow cytometry plots of the Hh-specific CD4+ T cells (mLN, top panel; LI, bottom panel) and frequency of RORγt+ Treg cells in (B) mLN and (C) LI of Hh-colonized WT mice treated with IgG1 Fc (n = 15) or CTLA4-Ig (n = 15). (D-F) (D) Flow cytometry plots of the Hh-specific CD4+ T cells (mLN, top panel; LI, bottom panel) and frequency of RORγt+ Treg cells in (E) mLN and (F) LI of Hh-colonized WT mice treated with IgG2b (n = 14) or anti-CTLA4 (n = 14). The data in (Fig. 2) are pooled from three independent experiments. The data are shown as means ± s.e.m.; the statistics shown in (B, C, E, and F) were obtained by unpaired two-tailed Student’s t-test. ***P < 0.001; ****P < 0.0001.

To examine for cellular pathways involved in the effect of CTLA4-Ig on RORγt+ Treg cell, we isolated cells from the mLN and LI of C57BL/6J mice and notably detected robust binding of CTLA4-Ig to cDCs and macrophages (Fig. S2, A–D). To distinguish subsets within RORγt+ APCs, we developed a refined gating strategy informed by our previously published scRNA-seq data from healthy mouse mLN (Lyu et al., 2022), based on the mutually exclusive expression of Klrb1b and Spi1, enabling discrimination of ILC3s and extrathymic AIRE-expressing cells (eTACs), respectively (Fig. S2 A). Using this approach, CTLA4-Ig binding was observed on RORγt+ APCs in the mLN and LI, with a particular enrichment in eTACs within the mLN, while minimal CTLA4-Ig binding was observed on T cells (Fig. S2, B–D). We next examined how CTLA4-Ig treatment changes the frequency and number of each of these APC populations. We observed that CTLA4-Ig treatment decreased the numbers of cDCs and macrophages in the LI, but not in the mLN (Fig. S2, E and F). In addition, CTLA4-Ig treatment increased the frequency of RORγt+ APCs in the mLN and LI, and increased their absolute number in the LI, providing an expanded pool of RORγt+ APCs that may support RORγt+ Treg cell differentiation (Fig. S2, E and F), which is likely through signal one they provide (Akagbosu et al., 2022; Kedmi et al., 2022; Lyu et al., 2022), as CD80 expression on RORγt+ APCs did not impact endogenous or Hh-specific RORγt+ Treg cell responses compared to those in littermate control mice (Fig. 1, D–I). Moreover, we examined CD80 and CD86 levels on these APC subsets following in vivo CTLA4-Ig treatment, and found significantly increased CD80 and CD86 levels on macrophages and RORγt+ APCs in the mLN but significantly decreased CD80 and CD86 levels on RORγt+ APCs in the LI, with no detectable changes in other populations from either tissue (Fig. S2, E and F).

To determine whether B7 co-stimulation provided by cDCs is responsible for the suppression of RORγt+ Treg cells, we generated mice with conditional deletion of CD80 in cDCs by crossing Clec9aCre mice with CD80/86KO; CD80Tgfl/fl mice. However, we did not observe changes in Hh-specific or endogenous RORγt+ Treg cells (Fig. S1, H and I), nor did we see a change in RORγt− conventional Treg cells in the mLNs and LI (Fig. S1, J and K). These data suggest that CD80 on cDCs is at least not solely responsible for the suppressive effect on RORγt+ Treg cells and indicate that other specific APC subsets act dominantly or redundantly in regulating this cellular program.

Collectively, these data indicate that CTLA4-Ig acts primarily by binding to B7 on multiple types of APCs, which then promotes RORγt+ Treg generation primarily through inhibiting the engagement of CD28-signaling. In support of this conclusion, we pursued a loss-of-function approach by blocking CTLA-4 with an antibody (Fig. S1 E) and observed a significant reduction in the generation of Hh-specific RORγt+ Treg cells in the mLN and LI of mice relative to those treated with the isotype control antibody (Fig. 2, D–F).

CTLA4-Ig enforces RORγt+ Treg cells and protects from experimental gut inflammation

Treg cells are impaired in the context of IL-10R deficiency, a common mutation occurring in very-early-onset IBD patients (Glocker et al., 2009; Kelsen et al., 2015). Further, RORγt+ Treg cells have been identified as a key mediator of antigen-specific immune tolerance to commensal microbiota in the intestine and as a critical suppressor of colitis (Ohnmacht et al., 2015; Sefik et al., 2015; Yang et al., 2016). Therefore, we next sought to determine whether CTLA4-Ig administration impacts RORγt+ Treg cell generation or provides protection in a mouse colitis model induced following Hh colonization combined with IL-10R blockade (anti-IL-10R) (Fig. 3 A). In particular, Hh colonization combined with anti-IL-10R prevents the differentiation of Hh-specific RORγt+ Treg cells and rather drives the expansion of Hh-specific effector T cells that promote intestinal inflammation (Xu et al., 2018). Strikingly, in this context, CTLA4-Ig administration overcomes a block in RORγt+ Treg cell generation, dramatically expanding Hh-specific RORγt+ Treg cells and reducing Hh-specific TH17 cells in both the mLN and LI relative to mice treated with IgG1 Fc control (Fig. 3, B–D). Consistent with previous studies demonstrating that the transcription factor c-Maf is critical for RORγt+ Treg cell differentiation (Wheaton et al., 2017; Xu et al., 2018), we observed that the expression of c-Maf was significantly upregulated in Hh-specific T cells of mice treated with CTLA4-Ig compared to IgG1 Fc (Fig. S3, A and B). Moreover, in CTLA4-Ig-treated mice, Hh-specific T cells significantly downregulated T-bet and produced significantly less interferon-γ (IFNγ) and tumor necrosis factor (TNF) relative to controls (Fig. 3, E–H). Importantly, CTLA4-Ig administration significantly ameliorated parameters of intestinal inflammation, including typhlocolitis, enlarged mLNs, inflammation-associated pathology, and neutrophil infiltration in the LI (Fig. 3, I–L). Beyond antigen-specific T cell responses, CTLA4-Ig-treated mice exhibited a marked increase in endogenous RORγt+ Treg cells, elevated c-Maf levels, and a concurrent reduction in TH17 cells (Fig. S3, C–G). These endogenous TH17 cells displayed a reduced pro-inflammatory phenotype, characterized by diminished T-bet expression and reduced production of IFNγ, TNF, and IFNγ+IL-17A+ co-producing cells (Fig. S3, H–M). These findings demonstrate that CTLA4-Ig treatment is sufficient to overcome a block in microbiota-specific RORγt+ Treg cell generation due to a deficiency in IL-10R signaling, as well as provide significant protection from gut inflammation.

Figure 3. CTLA4-Ig enforces microbiota-specific RORγt+ Treg cells and protects from gut inflammation.

Figure 3.

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(A) C57BL/6J WT mice were colonized with Hh on day 0, 2, and 4, followed by Hh-specific naïve CD4+ T cell transfer retro-orbitally on day 7, mice were intraperitoneally injected with anti-IL-10R on day 0, 6, 12, and 18, and were intraperitoneally injected with CTLA4-Ig on day 5, 10, 13, 16, 19 and 22. Mice were euthanized for analysis on day 24. (B-D) (B) Flow cytometry plots of the Hh-specific CD4+ T cells (mLN, top panel; LI, bottom panel) and frequencies of (C) Hh-specific RORγt+ Treg cells and (D) Hh-specific TH17 cells in mLN (top panel) and LI (bottom panel) of Hh-colonized WT mice treated with IgG1 Fc (n = 5), or CTLA4-Ig (n = 4). TH17 cells were gated as CD4+ Foxp3− RORγt+ T cells, and this strategy was applied consistently in all experiments. (E and F) The frequencies of (E) T-bet+ Hh-specific TH17 cells and (F) IFNγ+ Hh-specific CD4+ T cells in LI of the WT mice in (B). (G and H) (G) Flow cytometry plots and (H) frequency of TNF+ Hh-specific TH17 cells in LI of the WT mice in (B). (I-K) Images of (I) LI showing typhlocolitis, (J) enlarged mLN, and (K) H&E staining of the distal colon in the WT mice in (B). Scale bars, 100 μm. (L) Cell number of neutrophil infiltration in LI of the WT mice in (B). The data in (Fig. 3) are representative of two independent experiments. The data are shown as means ± s.e.m.; the statistics shown in (C, D, E, F, H, and L) were obtained by unpaired Student’s t-test (two-tailed). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

RORγt+ APCs are necessary for CTLA4-Ig driven immune tolerance in the gut

CTLA4-Ig, also known as abatacept or Orencia, was tested in treating multiple human inflammatory or autoimmune diseases such as rheumatoid arthritis (RA), psoriatic arthritis, type 1 diabetes, multiple sclerosis, alopecia totalis, systemic lupus erythematosus, and IBD (Kremer et al., 2003; Mease et al., 2011; Merrill et al., 2010; Orban et al., 2011; Rosenblum et al., 2012; Viglietta et al., 2008). While promising and currently used to treat several disorders, CTLA4-Ig lacked efficacy in moderate-to-severe human IBD (Sandborn et al., 2012). Indeed, RORγt+ Treg cells are reduced in the intestine of patients with food allergy or IBD (Abdel-Gadir et al., 2019; Hepworth et al., 2015; Lyu et al., 2022), suggesting that CTLA4-Ig should be effective. However, RORγt+ APCs expressing MHCII (signal one) are also numerically and functionally compromised in the inflamed gut environment of human IBD patients (Hepworth et al., 2015; Lyu et al., 2022). Given the key role of RORγt+ APCs in instructing RORγt+ Treg cells through signal one (Akagbosu et al., 2022; Kedmi et al., 2022; Lyu et al., 2022), we next sought to determine whether this CTLA4-Ig mediated expansion of RORγt+ Treg cells requires interplay with this pathway. To address this, we utilized RORγtCre H2-Ab1fl/fl mice with a selective deletion of MHCII on RORγt+ APCs (Hepworth et al., 2015; Hepworth et al., 2013), and found that in contrast to a blockade in IL-10 signaling, CTLA4-Ig was unable to overcome a defect in the generation of RORγt+ Treg cells in these mice (Fig. 4, A–C, and Fig. S1 E). Furthermore, we employed the mouse colitis model induced following Hh colonization and combined with anti-IL-10R in RORγtCre H2-Ab1fl/fl mice versus littermate controls (Fig. S3 N). Strikingly, CTLA4-Ig treatment failed to restore Hh-specific RORγt+ Treg cells in both mLN and LI of RORγtCre H2-Ab1fl/fl mice relative to that observed in littermate controls (Fig. 4, D–F). Rather, in this context and in contrast to littermate controls, mice lacking MHCII on RORγt+ APCs exhibited a significant increase in Hh-specific TH17 cells and in their TNF production (Fig. 4, G–I). Importantly, CTLA4-Ig administration failed to ameliorate intestinal inflammation in RORγtCre H2-Ab1fl/fl mice, as evidenced by colon shortening, mLN enlargement, and significant neutrophil infiltration in the LI (Fig. 4, J–L). These findings demonstrate that MHCII-dependent signal one from RORγt+ APCs is required to enforce RORγt+ Treg cell generation when blocking co-stimulatory B7 signal two, and for associated protection from intestinal inflammation.

Figure 4. RORγt+ APCs are required for CTLA4-Ig-driven immune tolerance.

Figure 4.

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(A-C) (A) Flow cytometry plots of the Hh-specific CD4+ T cells (mLN, top panel; LI, bottom panel) and frequencies of (B) Hh-specific RORγt+ Treg cells and (C) Hh-specific TH17 cells in mLN (top panel) and LI (bottom panel) of H2-Ab1fl/fl mice (n = 6) and RORγtCre H2-Ab1fl/fl mice (n = 6). (D-F) (D) Flow cytometry plots of the Hh-specific CD4+ T cells (mLN, top panel; LI, bottom panel), (E) frequency, and (F) cell number of Hh-specific RORγt+ Treg cells in mLN (top panel) and LI (bottom panel) of H2-Ab1fl/fl mice (n = 4) and RORγtCre H2-Ab1fl/fl mice (n = 5). (G) Frequency of Hh-specific TH17 cells in LI of the mice in (D). (H and I) (H) Flow cytometry plots and (I) frequency of TNF+ Hh-specific TH17 cells in LI of the mice in (D). (J-L) Images of (J) LI showing typhlocolitis, (K) enlarged mLN, and (L) cell number of neutrophil infiltration in the LI of the mice in (D). The data in (A-C) are pooled from two independent experiments. The data in (D-L) are representative of two independent experiments. The data are shown as means ± s.e.m.; the statistics shown in (B, C, E, F, G, I, and L) were obtained by unpaired Student’s t-test (two-tailed). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

In summary, our collective findings identify that RORγt+ Treg cell generation undergoes a unique pathway independent of B7 co-stimulation, and rather these signals antagonize this developmental program. This strikingly contrasts prior paradigms for conventional and thymic-derived Treg cells, which require B7-dependent co-stimulation for development, homeostasis and function (Salomon et al., 2000; Tang et al., 2003). At the signaling level, CD28 ligation initiates multiple intracellular pathways that regulate T cell activation and differentiation. In addition to the well-characterized PI3K-Akt-Foxo1 axis (Brunet et al., 1999; Burke et al., 2024; Fabre et al., 2005; Ouyang et al., 2009; Shrestha et al., 2015), CD28 engagement activates NF-κB, MAPK, and mTOR signaling, which together control effector T cell survival, cytokine production, and metabolic reprogramming (Acuto & Michel, 2003; Chi, 2012; Esensten et al., 2016; Zhang et al., 1999). These pathways are also likely to influence RORγt+ Treg cell biology, given their close developmental and functional relationship to effector TH17 cells, but how these signaling modules are mechanistically integrated in RORγt+ Treg cells remains incompletely understood and warrants further investigation. Separately, c-Maf is induced downstream of TCR, costimulatory, and cytokine signals and is essential for the development and suppressive function of RORγt+ Treg cells (Wheaton et al., 2017; Xu et al., 2018). How c-Maf is regulated downstream of B7-CD28 engagement in RORγt+ Tregs, and how it in turn contributes to their differentiation and suppressive function, remains an important question for future studies.

Our findings also reveal that it is possible to harness existing therapeutics, such as gain-of-function or loss-of-function approaches with CTLA-4, to impact B7-dependent regulation of RORγt+ Treg cells in the gut. This may explain why intestinal inflammation is a frequent adverse event associated with CTLA-4 immune checkpoint blockade in cancer therapy (Bamias et al., 2017; Braun et al., 2025; Lo et al., 2024; Marthey et al., 2016). Further, our data demonstrate that CLTA4-Ig can be harnessed to enforce antigen-specific RORγt+ Treg cell responses and provide protection from experimental intestinal inflammation, which complements a recent study showing the therapeutic potential of this compound for enhancing oral tolerance to dietary antigens (Arai et al., 2026). In both contexts, the relevant cellular sources of co-stimulation that antagonizes immune tolerance remain unclear, where our new data reveal that it is not solely driven by B7 on either cDCs or RORγt+ APCs. Thus, it is likely dependent on multiple redundant sources, or on a unique source that has not yet been extensively explored, such as macrophages. Moreover, CTLA4-Ig likely acts through dual, complementary mechanisms to restore immune tolerance in the gut. On one hand, CTLA4-Ig blocks B7-CD28 co-stimulation, limiting effector T-cell activation and cytokine production (Tivol et al., 1995; Walunas et al., 1996; Walunas et al., 1994). While on the other hand, our new data demonstrate that CTLA4-Ig increases the abundance of RORγt+ Treg cells, a cell type specialized in enforcing tolerance to gut microbiota and dietary antigens (Ohnmacht et al., 2015; Sefik et al., 2015). These observations align with prior findings that CTLA-4 regulates both effector and regulatory T cell compartments (Peggs et al., 2009), indicating that CTLA4-Ig promotes intestinal immune homeostasis by both restraining effector responses and enhancing RORγt+ Treg cell-mediated tolerance.

Intriguingly, in both our new data on intestinal inflammation, and in the recent study on oral tolerance to dietary antigens (Arai et al., 2026), CTLA4-Ig is effective only when signal one is simultaneously provided either from antigen-presentation by RORγt+ APCs or continuous antigen feeding. This indicates that the key to driving optimal antigen-specific RORγt+ Treg responses and immune tolerance in the gut is to preserve a necessary MHCII-dependent signal one from RORγt+ APCs in the context of B7 co-stimulation blockade. Importantly, this provides one mechanistic explanation for the limited efficacy of CTLA4-Ig in clinical trials of human IBD with moderate-to-severe disease (Mayer et al., 2012; Sandborn et al., 2012). We propose that in the chronically inflamed human intestine, there is a previously reported dysregulation of RORγt+ APCs (Hepworth et al., 2015; Lyu et al., 2022), which restricts the availability of signal one and thereby uncouples co-stimulatory blockade from immune tolerance. In this setting, blockade of B7-CD28 interactions alone is insufficient to drive the expansion of antigen-specific RORγt+ Treg cells, despite effectively limiting effector T cell activation. Our data demonstrate that blockade of the B7 family of co-stimulatory molecules must act in concert with MHCII on RORγt+ APCs to promote RORγt+ Treg cell responses, providing a framework for understanding why CTLA4-Ig fails once gut inflammation is established. We previously showed that RORγt+ APCs can be therapeutically harnessed to drive antigen-specific Treg cells and enforce immune tolerance (Grigg et al., 2021). Thus, there is great promise that when combined with strategies to boost RORγt+ APCs, then B7 co-stimulatory signaling blockade with CTLA4-Ig or other approaches will be highly effective at driving antigen-specific RORγt+ Treg cell response and enforcing immune tolerance in the intestine.

Materials and methods

Mice

C57BL/6J mice, CD45.1 transgenic mice, Cd80−/−/Cd86−/− mice (Borriello et al., 1997), CD80 Tgfl/fl mice (Watanabe et al., 2017), H2-Ab1fl/fl mice (Hashimoto et al., 2002), H. hepaticus (HH7–2) TCR transgenic mice (Xu et al., 2018), Clec9aCre mice (Schraml et al., 2013) were purchased from Jackson Laboratories. RORγtCre mice (Lochner et al., 2008) were provided by Gérard Eberl (Institut Pasteur, Paris, France). All mice were on a C57BL/6 background and maintained in specific-pathogen-free facilities in Weill Cornell Medicine. Male and female mice were used at 5 to 12 weeks of age. All protocols were approved by the Institutional Animal Care and Use Committee at Weill Cornell Medicine, and all experiments were performed in accordance with its guidelines.

Flow cytometry and cell sorting

Single-cell suspensions were incubated on ice with conjugated antibodies in PBS containing 2% FBS and 1 mM EDTA. Unlabeled anti-CD16/32 (clone 2.4G2, BD Biosciences) was used to block Fc receptors when analyzing myeloid cells. Dead cells were excluded with Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific). The staining antibodies for flow cytometry were mainly purchased from Thermo Fisher Scientific, BioLegend or BD Biosciences. The following were used for mouse cell-surface staining: CD3ε (145–2C11), CD4 (RM4–5, GK1.5), CD8a (53–6.7), CD5 (53–7.3), CD11b (M1/70), CD11c (N418), CD19 (1D3), CD64 (X54–5/7.1), GR1 (RB6–8C5), LY6G (1A8), CD45R (also known as B220; RA3–6B2), CD45.1 (A20), CD45.2 (104), CD45 (30-F11), CD90.2 (30-H12), CD127 (A7R34), MHCII (M5/114.15.2), NK1.1 (PK136), TCRγδ (GL3), CD44 (IM7), CD62L (MEL-14), CD25 (PC61), CXCR6 (SA051D1), NKR-P1B (2D12), and TCR Vβ6 (RR4–7). The following were used for mouse intracellular staining: FOXP3 (FJK-16S), RORγt (B2D), PU.1 (7C2C34), T-bet (4B10), IL-17A (17B7), IFNγ (XMG1.2), c-MAF (sym0F1), and TNFα (MP6-XT22). Lineage markers for mouse were as follows: CD3ε, CD5, CD19, B220, GR1, NK1.1, and TCRγδ, unless otherwise indicated.

For intracellular staining, cells were fixed and permeabilized with FoxP3/Transcription Factor Staining Buffer Set following the manufacturer’s instructions (Thermo Fisher Scientific). Briefly, cells were incubated with FoxP3 Fixation/Permeabilization working solution for 30 minutes at room temperature or overnight at 4°C and then stained for intracellular targets by incubating with conjugated antibodies in 1X permeabilization buffer for 45 minutes at room temperature. For intracellular cytokine staining, cells were first incubated for 4 hours in RPMI with 10% FBS, 50 ng/ml phorbol 12-myristate 13-acetate, 750 ng/ml ionomycin and 10 μg/ml brefeldin A, all obtained from Sigma-Aldrich. Antibodies for flow cytometry were purchased from BioLegend, Thermo Fisher Scientific or BD Biosciences. Flow cytometry data were collected using an LSR Fortessa (BD Biosciences) and analyzed with FlowJo V10 software (Tree Star). Cell sorting was performed with an Aria II (BD Biosciences).

Preparation of single-cell suspensions from intestine or lymph nodes

Large intestine including cecum and colon were removed, opened longitudinally and rinsed with ice-cold PBS. Dissected intestinal tissues were cut into pieces of approximately 0.5 cm, and intestinal epithelial cells were dissociated by incubating in HBSS (Sigma-Aldrich) containing 5 mM EDTA (Thermo Fisher Scientific), 1 mM dithiothreitol (Sigma-Aldrich) and 2% FBS with shaking at 200 rpm for 20 minutes at 37°C. Dissociation of epithelial cells was performed twice. Samples were vortexed and rinsed with PBS after each step. The epithelial cell fraction was discarded. Remaining tissues were minced and enzymatically digested in RPMI containing 0.4 U/ml dispase (Thermo Fisher Scientific), 1 mg/ml collagenase III (Worthington), 20 μg/ml DNase I (Sigma-Aldrich) and 10% FBS on a shaker for 45 minutes at 37°C. Leukocytes were enriched by 40% (for flow cytometry) or 40%/80% (for sorting) Percoll (GE Healthcare) gradient centrifugation. mLN were chopped and incubated in RPMI containing 2% FBS and 1 mg/ml collagenase II (Sigma-Aldrich), 20 μg/ml DNase I (Sigma-Aldrich) with shaking at 200 rpm for 20 minutes at 37°C, and cells were then dissociated using a Pasteur pipette, and filtered through a 70-μm cell strainer in PBS containing 0.5 mM EDTA and 2% FBS.

CTLA4-Ig binding assay

Following preparation of single-cell suspensions from the large intestine and mLN, 106 cells per condition were seeded into individual wells of a 96-well plate and incubated with CTLA4-Ig (100 μg/ml) at 4°C for 30 minutes. Cells were then washed twice with PBS to remove unbound reagent and immediately processed for flow cytometric staining. To detect cell-bound CTLA4-Ig, cells were stained with a PE-conjugated AffiniPure F(ab′)2 fragment donkey anti-human IgG antibody (Jackson ImmunoResearch Laboratories), and the presence of CTLA4-Ig was quantified by flow cytometry. To control for nonspecific Fc-mediated binding, parallel samples were incubated with total human IgG and processed identically. PBS-treated cells were included as a negative control.

H. hepaticus-specific TCR transgenic T cells and H. hepaticus-induced colitis model

Recipient mice (CD45.2+) were colonized with H. hepaticus (ATCC 51449/Hh3B1) by oral gavage (p.o.) with 3 doses every other day within 7 days before T cell transfer, using a protocol adapted from previously described methods (Lyu et al., 2022; Xu et al., 2018). Naive CD4+ T cells of H. hepaticus (HH7–2) TCR transgenic mice (CD45.1+) were isolated from spleen and lymph nodes by a FACSAria cell sorter (BD Biosciences) and were gated as CD45.1+CD5+CD4+CD25−CD44lowCD62LhiTCR Vβ6+. Recipient mice received 100,000 cells per mouse of HH7–2 TCR transgenic CD4+ T cells retro-orbitally and were analyzed 2 weeks after transfer, unless otherwise indicated. To induce colitis, mice were orally gavaged with H. hepaticus (ATCC 51449/Hh3B1), followed by intraperitoneal (i.p.) administration of anti-IL-10R antibody (clone 1B1.3A, BioXCell; 800 μg/mouse) on day 0 and every 6 days thereafter. Where indicated, mice were administrated i.p. injection of anti-CD80 (clone 16–10A1, BioXcell; 1 mg/mouse) and anti-CD86 (clone GL-1, BioXcell; 1mg/mouse), with IgG2a/2b isotype antibodies (clone 2A3/clone LTF-2, BioXcell; 1mg/mouse) as control. In separate experiments, CTLA4-Ig fusion protein (clone CTLA-4-Ig (hum/hum), BioXcell; 200 μg/mouse) or anti-CTLA4 (clone 9D9, BioXcell; 200 μg/mouse) was administered i.p., with IgG1 Fc (clone human Fc-G1, BioXcell; 200 μg/mouse) or IgG2b (clone MPC-11, BioXcell; 200 μg/mouse) serving as the respective controls.

Histology

Distal large intestinal tissues were fixed in 4% paraformaldehyde and embedded in paraffin. 5 μm sections were stained with H&E. Images were taken using a Nikon Eclipse Ti microscope and NIS-Elements 4.30.02 software (Nikon).

Statistics

P values for datasets were determined by unpaired two-tailed Student’s t-test or ordinary one-way ANOVA with 95% confidence intervals. All statistical analyses were performed with GraphPad Prism version 10 (GraphPad Software Inc.). P values smaller than 0.05 were considered significant.

Supplementary Material

1

Online supplemental material

Fig. S1 shows the experimental designs for B7 co-stimulation blockade and CTLA4-Ig or anti-CTLA4 administration in a mouse model with H. hepaticus (Hh) colonization and the development of Hh-specific RORγt+ Treg cells in the mLN and LI. It also presents Hh-specific and endogenous T cell changes in the mLN and LI of these mice. Fig. S2 illustrates the flow cytometry gating strategy developed in this study to identify major APC subsets and to quantify CTLA4-Ig binding across populations. It also demonstrates the CTLA4-Ig induced changes in the frequency and absolute number of multiple APC subsets in mLN and LI of C57BL/6J WT mice. Fig. S3 demonstrates the immune profiling in the Hh-induced colitis model, including c-Maf expression in Hh-specific RORγt+ T cells and phenotypic and inflammatory cytokine changes in endogenous RORγt+ T cells following IgG1 Fc or CTLA4-Ig treatment. It also shows the experimental designs for colitis induction by Hh colonization plus anti-IL-10R blockade, and for CTLA4-Ig treatment in RORγtCre H2-Ab1fl/fl mice and associated littermate controls.

Acknowledgements

We acknowledge members of the Sonnenberg Laboratory for discussions and critical reading of the manuscript. The illustrations in this manuscript were created in BioRender (https://BioRender.com). Research in the Sonnenberg Laboratory is supported by the National Institutes of Health (R01AI143842, R01AI123368, R01AI145989, U01AI095608, R01AI162936, R37AI174468, and R01CA274534), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, the Meyer Cancer Center Collaborative Research Initiative, the Dalton Family Foundation, and Linda and Glenn Greenberg. G.F.S. is a CRI Lloyd J. Old STAR. M.L. is supported by the National Institutes of Health (K99CA290052).

Non-standard abbreviations:

RORγt

retinoic acid receptor-related orphan receptor-γt

Treg

regulatory T cell

TH17

T helper 17

APC

antigen-presenting cell

cDC

conventional dendritic cell

ILC3

group 3 innate lymphoid cell

eTAC

extrathymic AIRE-expressing cell

CTLA4-Ig

cytotoxic T lymphocyte associated antigen-4 immunoglobulin

Hh

Helicobacter hepaticus

IBD

inflammatory bowel disease

LI

large intestine

mLN

mesenteric lymph node

Data availability

All data in the article and its supplementary materials are available upon request from the corresponding authors. The sequencing data used to generate Fig. S2 A are publicly available from the Gene Expression Omnibus (GEO) under accession number GSE184175.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2190463-supplement-1.pdf (15.4MB, pdf)

Data Availability Statement

All data in the article and its supplementary materials are available upon request from the corresponding authors. The sequencing data used to generate Fig. S2 A are publicly available from the Gene Expression Omnibus (GEO) under accession number GSE184175.

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