Microbiota-induced T cell plasticity enables immune-mediated tumour control

Although specific bacterial taxa have been associated with favourable clinical responses to immune checkpoint blockade (ICB) in cancer patients^{12,13,18,19,20,21,22}, the mechanisms by which the intestinal microbiota influences anti-tumour immune responses remain poorly defined. Products of the microbiota, including metabolites^23,24,25 and innate receptor ligands²⁶, may reprogramme myeloid cells²⁷, lowering the activation threshold for antigen presentation and thereby facilitating priming and activation of tumour-reactive T cells. Alternatively, T cells that recognize antigens shared between commensals (microbial-associated antigens (MAAs)) and tumours (tumour-associated antigens (TAAs)) may become activated in the setting of ICB, thus enhancing anti-tumour immune responses. Because the gut microbiome encodes an enormous antigenic repertoire, commensal-derived antigens can elicit T cell responses that, in some cases, cross-react with tumour epitopes—a plausible mechanism for commensal-driven tumour control²⁸. Despite correlative clinical data²⁹, the causality of such antigenic mimicry has not yet been demonstrated definitively in vivo. It is possible, however, to test this premise, particularly the relationship of microbe-specific T cells and intratumoural T cells, in animal models. An immunization model with skin-associated Staphylococcus epidermidis engineered to express antigens shared with implanted tumours was shown to elicit effective anti-tumour responses³⁰, but the ability of gut commensals that elicit stereotyped T cell responses to program anti-tumour immunity has not been explored. Here we studied how a small intestine-resident commensal microbe, SFB, which induces a regulatory-like T helper 17 (T_H17) cell response that enhances intestinal barrier integrity^31,32, influences efficacy of ICB in controlling growth of distal tumours that share antigen with the bacterium. We found that tumour-specific T_H17 cells, primed by SFB in the gut, infiltrate the tumour as trans-differentiated pro-inflammatory T helper 1 (T_H1)-like cells following ICB. These cells remodel the tumour microenvironment (TME), promoting recruitment, expansion and maturation of CD8⁺ effector T cells that contribute critically to anti-tumour immunity. Our results indicate that defined constituents of the intestinal microbiota can be harnessed to elicit desired effector T cell programs that restrain tumour growth.

To explore how gut commensal microbiota influence immune-mediated tumour control, we developed a synthetic neoantigen mimicry tumour model in mice by engineering B16-F10 (B16-3340) melanoma cells to express an immunodominant protein fragment of the gut-colonizing commensal microbe SFB (Fig. 1a,b). We chose SFB because it reliably colonizes specific-pathogen-free (SPF) mice and elicits a robust, well-characterized CD4⁺ T cell response that can be tracked with peptide-MHCII tetramers and TCR-transgenic mice^17,33,34. Lysates from B16-3340, but not from empty vector controls (B16-EV), robustly activated TCR-transgenic T cells (TCR^7B8) ex vivo, demonstrating effective processing and presentation of the SFB-derived epitope (Fig. 1c).

a, Amino acid sequence of the SFB-3340 protein fragment containing the CD4⁺ T cell epitope recognized by TCR^7B8 (red). The codon-optimized gene was fused to an EF-1α promoter (5′) and c-Myc tag (blue, 3′) by a flexible linker (L). s.c., subcutaneous. b, Immunoblot showing expression of the SFB-3340 fragment in transfected B16-F10 (B16-3340) cells, detected with anti-c-Myc antibody. Empty vector transfected cells (B16-EV) served as control. Size markers (kDa) are shown on the left. c, Ex vivo activation of naive SFB-specific CD4⁺ T cells from TCR^7B8 transgenic mice co-cultured with syngeneic splenocytes plus lysates from B16-3340 or B16-EV cells. Surface expression of activation markers CD69 and CD25 was analysed 24 h later by flow cytometry. d, Experimental design comparing SFB-colonized (SFB⁺) and SFB-free (SFB⁻) C57BL/6J mice implanted with B16-3340 or B16-EV tumours. e, Tumour growth curves from caliper measurements (n = 10 mice per group). Mice received anti-PD-1 antibody (250 µg per mouse, i.p.) on days 4, 7 and 10 post-implantation. Data represent mean ± s.d.; significance determined by two-way analysis of variance (ANOVA) with Sidak’s correction. f, Representative B16-3340 tumours excised from SFB⁺ and SFB⁻ mice on day 14 post tumour implantation. g, Excised tumour weights from SFB⁺ and SFB⁻ mice on day 14 (n = 8 mice per group); mean ± s.d., unpaired two-sided Mann–Whitney test. h, Kaplan–Meier survival curves of SFB⁺ and SFB⁻ mice (n = 10 mice per group) bearing B16-3340 tumours, with or without anti-PD-1 therapy. Following the initial challenge, surviving mice were re-challenged with the same tumour cells, and monitored without further anti-PD-1 antibody treatment. P values were determined by log-rank (Mantel-Cox) test. All experiments in e–h were repeated independently at least twice with similar results. In panel a, the ribbon-helix model was generated using AlphaFold2 to illustrate the predicted structure of the SFB-3340 protein fragment containing the TCR^7B8 epitope. This fragment was used to engineer cancer cell lines (B16-F10, MC-38 and LLC1) to stably express the SFB-3340 antigen, resulting in the generation of B16-3340, MC-3340 and LLC1-3340 cell lines. Schematics in a and d were created using BioRender (https://biorender.com).

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Next, we examined the effect of SFB colonization on tumour growth in SPF mice (Jackson Laboratories) bearing subcutaneously implanted B16-3340 and B16-EV tumours, with (Fig. 1d) or without (Extended Data Fig. 1a) anti-programmed cell death protein 1 (PD-1) treatment. In the absence of PD-1 blockade, there was no notable difference in tumour growth between mice implanted with either B16-3340 or B16-EV, regardless of SFB colonization status (SFB⁺ or SFB⁻) (Extended Data Fig. 1b,c). However, when animals were treated with anti-PD-1 antibody, the growth of B16-3340 tumours was markedly reduced in SFB⁺ mice compared with SFB⁻ mice. There was no notable difference in the growth of control B16-EV tumours between SFB⁺ and SFB⁻ mice receiving anti-PD-1 treatment (Fig. 1e–g and Extended Data Fig. 1d). The combination of SFB colonization and anti-PD-1 treatment of B16-3340 tumours also conferred a survival advantage compared with the other groups (Fig. 1h). Mice that survived the primary B16-3340 challenge subsequently rejected tumour re-challenge even without additional anti-PD-1 treatment, demonstrating durable, memory-like protection mediated by SFB colonization in concert with ICB (Fig. 1h). This robust SFB-dependent enhancement of anti-tumour immunity prompted further investigation into the underlying mechanisms by which SFB modulates tumour-directed immune responses and potentiates the efficacy of ICB.

To test whether SFB can act therapeutically to enhance anti-PD-1 efficacy after tumour establishment, we performed staged post-implantation gavage experiments (Extended Data Fig. 1e). Mice bearing B16-3340 were treated with anti-PD-1 (days 4–10) and gavaged with SFB at defined intervals (days 8–12, 12–16 or 15–19) or left SFB-free. Early gavage (days 8–12, group 1) produced the largest reduction in tumour growth, with progressively diminished benefit for later administrations (groups 2 and 3) (Extended Data Fig. 1f), indicating a narrow post-implantation window in which microbial antigen exposure most effectively synergizes with PD-1 blockade. These data further show that tumour expression of the SFB-derived neoantigen is required for microbiota-dependent augmentation of anti-PD-1 and that therapeutic SFB colonization is most effective when delivered early.

We next determined whether antigenic mimicry promotes microbiota-mediated control of additional tumours, extending our study to Lewis lung carcinoma (LLC1-3340) and MC-38 colon adenocarcinoma (MC-3340). SFB-colonized (SFB⁺) or SFB-free (SFB⁻) C57BL/6J mice were implanted subcutaneously with these engineered tumour cells and treated with anti-PD-1 beginning at the earliest stage of palpable tumour growth (Extended Data Fig. 1g,i). In both tumour models, SFB⁺ mice exhibited substantially delayed tumour growth compared with SFB⁻ cohorts (Extended Data Fig. 1h,j).

Profiling of T cells from B16-3340 and B16-EV tumours in anti-PD-1 treated SFB⁺ and SFB⁻ mice showed that SFB colonization significantly increased the intratumoural CD8⁺ to regulatory T (T_reg) cell ratio compared with either SFB⁻ mice with B16-3340 tumours or SFB⁺ mice with B16-EV tumours subjected to anti-PD-1 treatment (Fig. 2a,b). In contrast, SFB colonization did not alter CD8⁺ to T_reg cell ratio in the small intestine lamina propria (SILP) of anti-PD-1 treated mice (Extended Data Fig. 2a). Concurrently, CD8⁺ tumour-infiltrating lymphocytes (TILs) from anti-PD-1 treated, B16-3340 tumours from SFB⁺ exhibited markedly enhanced effector functions, with higher frequencies of IFNγ⁺, TNF⁺IFNγ⁺ and Gzm-B⁺TNF⁺ CD8⁺ TILs compared with either CD8⁺ TILs in anti-PD-1 treated B16-3340 tumours in SFB⁻ or B16-EV tumours in SFB⁺ mice (Fig. 2c and Extended Data Fig. 2b). Similar results were demonstrated previously in the microbiota-mediated response to PD-1 blockade³⁵.

a, Schematic of the synthetic mimicry model used to evaluate how gut SFB colonization alters the distal immune TME. b, Top, representative flow cytometry plots of tumour-infiltrating CD8⁺ T cells and Foxp3⁺ CD4⁺ T_reg cells from B16-3340 (SFB⁻, n = 10 mice and SFB⁺, n = 9 mice) and B16-EV (SFB⁺; n = 6) following anti-PD-1 treatment. Bottom, quantification of absolute counts of T_reg cells, CD8⁺ T cells, and CD8:T_reg ratio per tumour. c, Top, representative cytokine flow cytometry plots (TNF⁺ IFNγ⁺) of CD8⁺ TILs from B16-3340 tumours in SFB⁻ and SFB⁺ mice, and B16-EV tumours in SFB⁺ mice. Bottom, frequencies of TNF⁺ IFNγ⁺ CD8⁺ TILs: B16-3340 (SFB⁻, n = 8), B16-3340 (SFB⁺, n = 9) and B16-EV (SFB⁺, n = 8). d, Left, SFB-peptide-specific MHCII tetramer staining of CD4⁺ TILs from B16-3340 tumours (SFB⁻ versus SFB⁺). Right, absolute counts of tetramer⁺ CD4⁺ TILs per tumour (n = 9 mice per group). e, Left, expression of RORγt and T-bet in tetramer⁺ CD4⁺ TILs from B16-3340 tumours of SFB⁻ and SFB⁺ mice. Right, frequencies of T-bet⁺RORγt⁺ and RORγt⁺ subsets (n = 9 mice per group). f, Left, IFNγ and IL-17A expression in tetramer⁺ CD4⁺ TILs from B16-3340 tumours in SFB⁺ mice. Right, frequencies of IFNγ⁺ and IL-17A⁺ tetramer⁺ CD4⁺ TILs (n = 9 mice per group). g, Left, RORγt and T-bet expression in SILP tetramer⁺ CD4⁺ T cells (SFB⁻ versus SFB⁺). Right, quantification of transcription factor expression in SILP tetramer⁺ CD4⁺ T cells from SFB⁻ (n = 6) and SFB⁺ (n = 7) mice. h, Left, IFNγ and IL-17A expression in SILP tetramer⁺ CD4⁺ T cells (SFB⁺). Right, frequencies of IFNγ⁺ and IL-17A⁺ SILP tetramer⁺ CD4⁺ T cells (n = 7 mice per group). Data are mean ± s.d., each data point representing an individual mouse. Statistical comparisons were determined by unpaired two-sided Mann–Whitney t-test, with P values indicated. All experiments shown were repeated at least twice with similar results. Schematic in a was created using BioRender (https://biorender.com).

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Next, because SFB colonization induces antigen-specific T_H17 cells in the ileal lamina propria, we compared the CD4⁺ T cell phenotypes in the remotely located B16-3340 tumours. First, using a panel of antibodies specific for TCR Vβs, we found a greater proportion of Vβ14⁺ CD4⁺ T cells in tumours from SFB⁺ compared with SFB⁻ mice (Extended Data Fig. 2c). This bias is consistent with the known preferential interaction of this subset of TCRs with immunodominant SFB peptides in the SILP of SFB-colonized mice³⁶ (Extended Data Fig. 2d). Second, using SFB-3340 peptide-loaded MHCII tetramers revealed that colonization with SFB caused increased infiltration of SFB-3340-specific CD4⁺ T cells into B16-3340 tumours (Fig. 2d). Remarkably, tumour-resident tetramer⁺ CD4⁺ T cells displayed an IFNγ producing T_H1-like phenotype, unlike SILP tetramer⁺ T cells that, as expected, were IL-17A producing T_H17 cells (Fig. 2e–h and Extended Data Fig. 2e–g). Although tumour-resident tetramer⁺ CD4⁺ T cells in both SFB⁺ and SFB⁻ mice were T-bet⁺, a minor fraction from SFB⁺ mice co-expressed RORγt, consistent with gut imprinting (Fig. 2e and Extended Data Fig. 2g). By contrast, the bulk of tetramer⁻ CD4⁺ T cells in both B16-3340 and B16-EV tumours, irrespective of SFB colonization, were regulatory-like T cells, expressing both T-bet and Foxp3 (Extended Data Fig. 2h,i), a phenotype associated with strong suppression of anti-tumour immune responses³⁷.

ELISpot assays confirmed significant enrichment of IFNγ-producing CD4⁺ TILs in SFB⁺ B16-3340 tumours compared with SFB⁻ cohorts (Extended Data Fig. 3a) and, accordingly, tetramer⁺ CD4⁺ TILs from those tumours exhibited a robust T_H1 cytokine (IFNγ and TNF) response following ex vivo stimulation (Extended Data Fig. 3b). Furthermore, whereas the frequencies of both T-bet⁺Foxp3⁻ and T-bet⁺Foxp3⁺ cells in the tetramer⁻ CD4⁺ T cell population were comparable across groups (Extended Data Fig. 2h,i), B16-3340 tumours in SFB⁺ mice contained a significantly higher fraction of tetramer⁻ CD4⁺ T cells that produced moderate amounts of IFNγ and TNF following ex vivo stimulation compared with either B16-3340 tumours in SFB⁻ mice or B16-EV tumours in SFB⁺ mice (Extended Data Fig. 3c,d). Together, these findings suggest that SFB-specific pro-inflammatory CD4⁺ T cells contribute to remodelling the TME, thereby increasing its responsiveness to PD-1 blockade.

The T cell composition in the MC-38 tumours expressing the SFB antigen (MC-3340) was similarly altered, with SFB colonization promoting accumulation of tetramer⁺ CD4⁺ T cells exhibiting a T-bet⁺IFNγ⁺ T_H1-like program, and an enrichment of IFNγ⁺Gzm-B⁺ CD8⁺ TILs (Extended Data Fig. 3e–h). These data show that SFB-induced antigen-specific CD4⁺ T cell priming and T_H1-like polarization, together with enhanced CD8⁺ T cell effector function, potentiate PD-1 blockade across melanoma, lung and colon tumour models when the tumour expresses the cognate microbial epitope.

Next, given that the effective anti-tumour immune response requires synergistic cooperation of CD4⁺ and CD8⁺ T cells in ICB-mediated tumour control^38,39,40, we aimed to investigate whether the combination of SFB-induced CD4⁺ T cells and tumour-infiltrating CD8⁺ T cells, together with anti-PD-1 therapy, is essential for controlling B16-3340 tumour growth in SFB⁺ mice. In vivo depletion of either CD4⁺ (Extended Data Fig. 4a,b) or CD8⁺ (Extended Data Fig. 4a,g) T cells in B16-3340 tumour-bearing mice significantly impaired the efficacy of anti-PD-1 treatment (Extended Data Fig. 4c and 4h). CD8⁺ TILs from CD4-depleted, SFB⁺ mice exhibited a marked functional impairment, with significant reductions in T-bet⁺IFNγ⁺, TNF⁺Gzm-B⁺ and IFNγ⁺TNF⁺ cells relative to controls (Extended Data Fig. 4d–f), indicating that microbiota-dependent CD4⁺ T cells are critical for the acquisition of full CD8⁺ TIL effector function in tumours. Conversely, depletion of CD8⁺ T cells modestly reduced the proportion of T-bet⁺IFNγ⁺ CD4⁺ TILs, consistent with reciprocal but asymmetric cross-talk between these T cell lineages (Extended Data Fig. 4i,j). Together, these results demonstrate that SFB colonization enhances the efficacy of PD-1 blockade through a coordinated CD4–CD8 T cell axis: microbiota-induced, pro-inflammatory CD4⁺ T cells provide critical help for CD8⁺ TIL maturation and cytotoxic function, whereas both T cell subsets are jointly required for durable, SFB-dependent tumour control under anti-PD-1 therapy.

To examine the relationship of intestinal and tumour-infiltrating T cells in SFB⁻ and SFB⁺ mice with B16-3340 tumours, we performed paired single-cell RNA sequencing (scRNA-seq) and TCR repertoire analysis (scTCR-seq) on sorted CD4⁺ T cells from SILP and B16-3340 tumours (Fig. 3a). Unsupervised clustering of the scRNA-seq data resolved transcriptionally distinct CD4⁺ T cell subsets in each tissue (nine clusters in SILP and ten in B16-3340 tumours), defined by canonical lineage markers (Extended Data Fig. 5a). As anticipated, SFB colonization selectively expanded an IL-17A⁺ T_H17 cluster in the SILP (cluster 2) of SFB⁺ mice (Fig. 3b). In contrast, tumours from SFB⁺ mice were enriched for an IFNγ⁺ T_H1-like subset (cluster 1), a population absent from tumours of SFB⁻ mice (Fig. 3c), highlighting the divergent, tissue-specific programs of antigen-specific CD4⁺ T cells.