Abstract
The gut microbiota is a crucial regulator of anti-tumour immunity during immune checkpoint inhibitor therapy. Several bacteria that promote an anti-tumour response to immune checkpoint inhibitors have been identified in mice1,2,3,4,5,6. Moreover, transplantation of faecal specimens from responders can improve the efficacy of anti-PD-1 therapy in patients with melanoma7,8. However, the increased efficacy from faecal transplants is variable and how gut bacteria promote anti-tumour immunity remains unclear. Here we show that the gut microbiome downregulates PD-L2 expression and its binding partner repulsive guidance molecule b (RGMb) to promote anti-tumour immunity and identify bacterial species that mediate this effect. PD-L1 and PD-L2 share PD-1 as a binding partner, but PD-L2 can also bind RGMb. We demonstrate that blockade of PD-L2–RGMb interactions can overcome microbiome-dependent resistance to PD-1 pathway inhibitors. Antibody-mediated blockade of the PD-L2–RGMb pathway or conditional deletion of RGMb in T cells combined with an anti-PD-1 or anti-PD-L1 antibody promotes anti-tumour responses in multiple mouse tumour models that do not respond to anti-PD-1 or anti-PD-L1 alone (germ-free mice, antibiotic-treated mice and even mice colonized with stool samples from a patient who did not respond to treatment). These studies identify downregulation of the PD-L2–RGMb pathway as a specific mechanism by which the gut microbiota can promote responses to PD-1 checkpoint blockade. The results also define a potentially effective immunological strategy for treating patients who do not respond to PD-1 cancer immunotherapy.
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Data availability
The 16S sequencing data have been deposited to National Institutes of Health BioProject database with the identifier PRJNA936792. Source data are provided with this paper.
Change history
01 June 2023
A Correction to this paper has been published: https://doi.org/10.1038/s41586-023-06237-8
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Acknowledgements
Figure 4a was created using BioRender (https://www.biorender.com). We would like to thank T. Yanortsang and J. Ramos for their assistance with GF mice; and members of the Sharpe Laboratory and the Kasper Laboratory for insightful discussions. The following institutions are acknowledged for funding: Quark Ventures (A31696 to J.S.P., F.S.G., D.L.K. and A.H.S.); the National Institutes of Health/National Institute of Childhood Health and Human Disease (T32 5T32HD55148-10 to F.S.G.); the National Institutes of Health/National Cancer Institute (1 K22 CA258960-01 to F.S.G.; 5F32CA247072-02 to J.S.P.; P50CA206963 and P50CA101942 to G.J.F.; P01 AI56299 to A.H.S. and G.J.F.; and 1F32CA260769-01 to G.M.); and the Cancer Prevention and Research Institute of Texas (CPRIT Training Award RP210028 to E.M.P.).
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Contributions
J.S.P., F.S.G., G.J.F., D.L.K. and A.H.S. designed the experiments. J.S.P. and F.S.G. performed the experiments. M.W. analysed the 16S sequencing data. A.K.L., J.G., M.W.L. and W.Z. assisted with the experiments. S.B.J., G.M., E.M.P., Y.Z., S.S.W. and J.A.W. obtained the patient stool samples. J.S.P. and F.S.G. analysed the data. G.J.F. generated the PD-L1, PD-L2 and RGMb antibodies. J.S.P., F.S.G., G.J.F., D.L.K. and A.H.S. wrote the manuscript.
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J.S.P., F.S.G., D.L.K. and A.H.S. are listed as inventors on US Utility application (17/311,587) covering identification of gut bacteria that promote an anti-tumour response to immunotherapy filed by President and Fellows of Harvard College. J.S.P., F.S.G., D.L.K. and A.H.S. are listed as inventors on US Utility application (17/473,083) covering methods and compositions for treating cancer or a tumour in a subject by administering to the subject a first agent that disrupts the interaction between PDL2/RGMb and a second agent that disrupts the interaction between PD-1/PD-L1 filed by President and Fellows of Harvard College. J.S.P., F.S.G., G.J.F., D.L.K. and A.H.S. are listed as inventors on PCT patent applications (PCT/US21/50674) covering methods of treating an individual that has failed an anti-PD-1/PD-L1 therapy filed by President and Fellows of Harvard College and Dana-Farber Cancer Institute. J.S.P., F.S.G., G.J.F., D.L.K. and A.H.S. are listed as inventors on PCT patent applications (PCT/US23/12139) covering methods and compositions for treating cancer or a tumour in a subject by administering to the subject T cells with reduced RGMb expression or activity and an immune checkpoint inhibitor such as a PD-1 or PD-Ll inhibitor filed by President and Fellows of Harvard College and Dana-Farber Cancer Institute. J.S.P., F.S.G., G.J.F., D.L.K. and A.H.S. are listed as inventors on provisional patent application covering PD-L2 modulated dendritic cell therapy filed by President and Fellows of Harvard College and Dana-Farber Cancer Institute. G.J.F. is listed as an inventor on US patent US11220545 on combination RGMb and PD-1 blockade for cancer immunotherapy assigned to Dana-Farber Cancer Institute. G.J.F. and A.H.S. have patents/pending royalties on the PD-1–PD-L1 pathway from Roche, Merck MSD, Bristol-Myers Squibb, Merck KGA, Boehringer-Ingelheim, AstraZeneca, Dako, Leica, Mayo Clinic, and Novartis as listed in Supplementary Table 1. G.J.F. has served on advisory boards for Roche, Bristol-Myers-Squibb, Xios, Origimed, Triursus, iTeos, NextPoint, IgM, Jubilant, Trillium, GV20, IOME, and Geode. G.J.F. has equity in Nextpoint, Triursus, Xios, iTeos, IgM, Trillium, Invaria, GV20, and Geode. A.H.S. has patents/pending royalties on the PD-1 pathway from Roche and Novartis. A.H.S. is on advisory boards for Surface Oncology, SQZ Biotechnologies, Elpiscience, Selecta, Bicara and Monopteros, Bicara, Fibrogen, IOME and Alixia. She also is on scientific advisory boards for the Massachusetts General Cancer Center, Program in Cellular and Molecular Medicine at Boston Children’s Hospital, the Human Oncology and Pathogenesis Program at Memorial Sloan Kettering Cancer Center, Bloomberg-Kimmel Institute for Cancer Immunotherapy, GlaxoSmithKline, Janssen and Amgen. She is an academic editor for the Journal of Experimental Medicine. A.H.S. has received research funding from Novartis, Roche, UCB, Ipsen, Merck, AbbVie, Moderna, Vertex and Erasca unrelated to this project. D.L.K. is on a scientific advisory board of IOME. S.S.W. is on the advisory board for Asylia Therapeutics and reports compensation from Ridgeline Therapeutics. J.A.W. reports compensation for speaker’s bureau and honoraria from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, PeerView, Physician Education Resource, MedImmune, and Bristol-Myers Squibb and serves as a consultant and advisory board member for Roche/Genentech, Novartis, AstraZeneca, GlaxoSmithKline, Bristol-Myers Squibb, Merck, Micronoma, and Biothera Pharmaceuticals, with stock options for Micronoma.
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Extended data figures and tables
Extended Data Fig. 1 SPF, MMB, and HMB promote anti-tumor responses to PD-1/PD-L1 blockade.
MC38 tumor growth with or without anti-PD-L1 in (a) Taconic SPF n = 10 mice per group. Significance indicated on graph and measured by Two Way ANOVA and Sidak’s multiple comparisons test or (b) GF n = 5 mice per group, performed at the same time as (a), representative experiment of 14 individual experiments. MC38 tumor cells were implanted subcutaneously in (c) GF mice n = 4 mice for isotype group and n = 5 mice for anti-PD-1 group. (d) GF mice were orally gavaged with MMB one week before implantation of MC38 tumor cells. N = 3 mice for no treatment group and n = 6 mice for anti-PD-L1 group. Significance indicated on graph and measured by Two Way ANOVA and Sidak’s multiple comparisons test. (e) MC38 tumor cells were implanted subcutaneously in ABX or ABX/HMB mice and treated with anti-PD-1 according to Fig. 1a, and monitored for tumor growth. N = 3 mice for ABX + isotype group and n = 4 mice for ABX + anti-PD-1, ABX/HMB + Isotype, and ABX/HMB + anti-PD-1 groups. Significance indicated on graph and measured by Two-Way ANOVA and Tukey’s multiple comparisons test. (a–e) Error bars show mean and s.e.m.
Extended Data Fig. 2 Immune cell responses in ABX, ABX/HMB, GF, and SPF mice.
MC38 tumor cells were implanted subcutaneously in ABX and ABX/HMB mice. Mice were treated with isotype or anti-PD-L1 on days 7, 10, 13, and 16, followed by sacrificed on day 24 after tumor implantation. For detection of cytokines, tumor-infiltrating lymphocytes were stimulated with PMA/Ionomycin for 5 h in the presence of Golgi inhibitors. (a) Ratio of CD8+ T cells to Treg cells n = 10 mice per group (b) Percent of Granzyme B+ CD8+ T cells n = 10 mice per group (c) Frequency of PD-1+TIM-3+ n = 10 mice per group or (d) CXCR5+ TIM-3− among CD8+ T cells in tumors n = 5 mice per group for ABX groups and ABX/HMB+ Isotype group and n = 7 for ABX/HMB + anti-PD-L1 group. Percent of (e)TNF-α+ n = 10 mice per group (f) IFNγ+n = 10 mice per group, and (g) TNF-α+IFNγ+ CD8+ T cells n = 10 mice per group for ABX groups and ABX/HMB + isotype and n = 9 mice for ABX/HMB + anti-PD-L1 (h) Percent of IFNγ+ CD4+ T cells n = 10 mice per group for ABX groups and ABX/HMB + anti-PD-L1 and n = 12 mice per group for ABX/HMB + isotype. Mice were analyzed on day 24 after tumor implantation, representative of two independent experiments. Significance measured by non-parametric one-way ANOVA and Dunn’s multiple comparisons and P values are indicated on graphs, error bars show mean and s.d. (i–l) MC38 tumor cells were implanted subcutaneously in ABX and ABX/HMB mice. Mice were treated with isotype or anti-PD-L1 on days 7 and 10 and sacrificed on day 13 after tumor implantation. (i) Percent of PD-1+ CD8+ T cells in tumors, dLNs, and mesenteric lymph nodes (MLNs) n = 5 mice per group (j) Percent TIM-3+ among PD-1+ CD8+ T cells in Tumors, dLNs, and MLNs n = 5 mice per group for all except MLN ABX/HMB + anti-PD-L1 n = 4 mice per group (k) Percent CD44+ expression on PD-1+ CD8+ T cells in tumors, dLNs, and MLNs n = 4 mice per group (l) Percent IFNγ+ CD8+ T cells in tumors, dLNs, and MLNs n = 5 mice per group. (i–l) Significance determined by non-parametric one-way ANOVA with Dunn’s multiple comparisons test, error bars show mean and s.d. Expression of (m) PD-L1, (n) CD80, (o) CD86, and (p) ICOSL on CD11c+ MHCII+ and CD11b+ MHCII+ cells in draining lymph nodes of ABX and ABX/HMB mice implanted with MC38 tumor cells subcutaneously and treated with isotype control mAb as in Fig. 1a. Mice were analyzed on day 13 after tumor implantation. N = 4 mice in ABX group and n = 5 mice in ABX/HMB group. Significance measured by unpaired two- tailed, Mann-Whitney test. Expression of PD-L2 on CD11c+ MHCII+, CD11b+ MHCII+ cells and CD8+ T cells in draining lymph nodes of (q) ABX vs. ABX/HMB mice n n = 4 mice per group for ABX and n = 5 mice per group for ABX/HMB or (r) GF vs. Taconic SPF mice n = 4 mice per group, implanted with MC38 tumor cells subcutaneously and treated with isotype control mAb as in Fig. 1a. Mice were analyzed on day 10 after tumor implantation. Significance measured by unpaired, two-tailed Mann-Whitney test, and significant P values indicated on graphs. ABX and ABX/HMB mice were sacrificed 24 days after implantation with MC38 tumor cells. PD-L2 was measured on MHCII+ CD11c+, MHCII+ CD11b+, and CD8+ T-cells in (s) draining lymph nodes n = 5 mice per group, (t) MLN n = 5 mice for ABX and n = 4 mice for ABX/HMB, (u) tumors n = 5 mice per group and (v) spleen n = 5 mice per group. Significance measured by Two-Way ANOVA and Sidak’s multiple comparison’s test. Significant P values indicated on graph. Representative experiment of two different experiments. Error bars show mean and s.d.
Extended Data Fig. 3 Combination therapy with anti-PD-1 or anti-PD-L1 plus anti-PD-L2 or anti-RGMb promotes an anti-tumor response in multiple, but not all, tumor types in SPF mice.
MC38, B16-OVA, MB49, Py8119-OVA, LLC-OVA cells were implanted subcutaneously, and E0771 cells were injected into mammary fat pad of C57BL/6 mice with the indicated microbiota. The mice were treated with four doses of anti-PD-L1 or anti-PD-1 with/without anti-PD-L2 3.2 or anti-PD-L2 2C9 or anti-RGMb 9D1 one week after tumor implantation as indicated. (a) Growth of MB49 tumors in ABX mice given isotype, anti-PD-1 alone or combined with anti-PD-L2 3.2 n = 10 mice per group for Isotype and anti-PD-1 + anti PD-L2 3.2 groups, n = 9 mice for anti-PD-1 group (b) Growth of MB49 tumors in ABX mice given isotype, anti-PD-L1 alone or combined anti-PD-L2 3.2 n = 10 mice per group (c) Growth of B16-OVA tumors in Taconic SPF mice given isotype, anti-PD-L1, anti-PD-L2 3.2 or anti-PD-L1 + anti-PD-L2. n = 5 mice per group for isotype and anti-PD-L2 3.2 groups and n = 10 mice per group for anti-PD-L1 and anti-PD-L1 + anti-PD-L2 3.2 groups. (d) Growth of Py8119-OVA in Taconic SPF mice given isotype or anti-PD-L1 and/or anti-PD-L2 3.2. n = 5 mice per group for all except n = 4 mice for anti-PD-L2 3.2 group (e) Growth of MC38 tumors in Taconic SPF mice given isotype, anti-PD-L1, anti-PD-L2 3.2, or anti-PD-L1 + anti-PD-L2 n = 10 mice per group for all except n = 9 mice for anti-PD-L2 3.2 group. (f–g) Growth of E0771 tumors in Taconic SPF mice (f) given isotype, or anti-PD-1 combined with anti-PD-L2 3.2, anti-PD-L2 2C9 or anti-RGMb 9D1 n = 10 mice per group or (g) given isotype n = 10 mice, anti-PD-L1 alone n = 9 mice or combined with anti-PD-L2 n = 5 mice. (h) Growth of LLC-OVA tumors in Taconic SPF mice given isotype, anti-PD-L1, or anti-PD-L1 and either anti-PD-L2 3.2 or anti-RGMb 9D1. n = 10 mice per group. Significance measured by two-way ANOVA and Tukey’s multiple comparisons test. Significant P vales are designated on graphs. Error bars show mean and s.e.m.
Extended Data Fig. 4 16S sequencing of fecal samples, tumor growth, and survival of mice colonized with melanoma patient stool.
(a) Patient characteristics for stool samples. *Samples were characterized as complete responder (CR) or non-responder (NR) based on RECIST 1.1 response criteria. Mean age is 56.3 years, mean BMI is 29.9 kg/m2. **Stage at start of ICB treatment. (b–h) Bacterial community configuration and distances in fecal samples collected from three different sets of mice colonized with melanoma patient stool: complete responder (CR), Non-responder 1 (NR1) and Non-responder 2 (NR2) as well as input. (b) Principal coordinates analysis (PCoA) of unweighted UniFrac distance measurements based on the 16S sequencing analysis of the composition of bacterial communities at 7 day after gavage. Each coloured circle represented a fecal community sampled from a mouse belonging to the indicated donor group or input gavage sample. Pairwise unweighted UniFrac distances of the composition of bacterial communities in fecal samples within (c) CR and across NR1 and NR2 groups compared to CR (d) NR1 and across CR and NR2 groups compared to NR1 and (e) NR2 and across CR and NR1 groups compared to NR2. Pairwise weighted UniFrac distances of the composition of bacterial communities in fecal samples within (f) CR and across NR1 and NR2 groups compared to CR (g) NR1 and across CR and NR2 groups compared to NR1 and (h) NR2 and across CR and NR1 groups compared to NR2. n= number of pairwise distances calculated between all samples in each group compared to the comparator group. *** q-values = 0.001, PERMANOVA. Minima, maxima, center, bounds, and percentiles of boxplots shown in source data. GF mice were colonized with stool from Responder (R) or Non-Responder (NR) melanoma patients treated with anti-PD-1, implanted with MC38 tumors, and given anti-PD-L1, anti-PD-L2 or anti-PD-L1 plus anti-PD-L2 mAbs. (i) Tumor growth in mice treated with anti-PD-L1 monotherapy. N = 6 mice for CR and NR1 groups and n = 5 mice for NR2 group. Significance determined by 2 way ANOVA and Tukey’s multiple comparisons test and significant p values are indicated on graph. Error bars show standard error of the mean. (j) Survival of mice given anti-PD-L1, anti-PD-L2 or anti-PD-L1 plus anti-PD-L2 mAbs. Survival defined as number of live mice with tumors <2 cm3 or <50% ulcerated. N = 6 for CR + anti-PD-L1, CR+ anti-PD-L1 + anti-PD-L2, NR1 + anti-PD-L1, NR1 + anti-PD-L1 and anti-PD-L2, NR2 + anti-PD-L1 and anti-PD-L2, n = 5 for CR + anti-PD-L2, NR2 anti-PD-L1, NR2 anti-PD-L2, n = 3 for NR1 + anti-PD-L2. Significance of anti-PD-L1 monotherapy versus combination therapy for each group of mice shown and significance indicated on graph.
Extended Data Fig. 5 Individual antibiotic treatments and 16S sequencing show Gram-positive species are associated with an anti-tumor response.
All mice were treated with Vancomycin, Neomycin, Metronidazole, and Ampicillin (VNMA) in the drinking water 4 days before tumor implantation until day 7 pi. VNMA mice continued with the antibiotic cocktail for the duration of the experiment. VNMA + HMB mice stopped VNMA at day 7 and were orally gavaged with HMB stock. Mice given individual antibiotics stopped VNMA at day 7 pi and water was replaced with an individual antibiotic and mice were orally gavaged with HMB stock. MC38 tumor growth curves in mice receiving HMB + (a) Vancomycin (Vanco) n = 5 mice per group (b) Metronidazole (Met) n = 5 mice per group (c) Ampicillin (AMP) n = 5 mice per group and (d) Neomycin (Neo) n = 5 mice for no treatment and n = 3 mice for anti-PD-L1 group with or without anti-PD-L1. N = 4 for VNMA groups and n = 5 for HMB groups. Significance determined by two-way ANOVA and Tukey’s multiple comparisons test and significant p values between individual antibiotic treatments versus HMB or VNMA are shown. Error bars show mean and s.e.m. (e–f) Pairwise weighted and unweighted UniFrac distances of the composition of bacterial communities in fecal samples within HMB and across treatment groups compared to HMB. Each dot represented the weighted or unweighted UniFrac distance between the configuration of bacterial populations as judged from the relative abundances of its members, determined by 16S sequencing, in fecal samples collected from members of the indicated treatment group. q-values were indicated for each group comparisons as determined by permutational analysis of variance (PERMANOVA) for beta diversity group significance. Minima, maxima, center, bounds, and percentiles of boxplots shown in source data. (g) Principal coordinates analysis (PCoA) of weighted UniFrac distance measurements based on the 16S sequencing data of the composition of bacterial communities in the fecal samples of mice with different treatment at day 13 and 23 pi. Each colored circle represented a fecal community sampled from a mouse belonging to the indicated antibiotic treatment group or input gavage sample. (h) The relative abundance of the taxa in the order of Clostridiales in each treatment group at day 23 pi after anti-PD-L1 treatment. These identified taxa were significantly (W-statistic = 39, 35 and 34 respectively, p-value < 0.05) associated with response to anti-PD-L1 by differential abundance testing of taxa between the responder group (HMB n = 5, Neomycin n = 3) to the non-responder group (ABX n = 3, Ampicillin n = 4, Vancomycin n = 3, and Metronidazole n = 2) using ANCOM (Analysis of Composition of Microbiomes).
Extended Data Fig. 6 Narrowing down bacteria isolated from HMB stocks that promote anti-tumor immunity and suppress PD-L1.
MC38 tumor growth in gnotobiotic mice colonized one week prior to tumor implantation with (a) Mix of overnight cultures of Longicatena caecimuris, Blautia hydrogenotrophica, Clostridium orbiscindens, Clostridium innocuum, Phocaeicola dorei, Coprobacillus cateniformis, and Ersyipelatoclostridium ramosum n = 4 mice for GF + Isotype, n = 5 mice per group for GF + anti-PD-L1, 7 mix + isotype, and 7 mix + anti-PD-L1 (b) mix of overnight cultures of C. innocuum, C. cateniformis, E. ramosum n = 4 mice for GF + isotype, n = 5 mice for GF + anti-PD-L1, n = 10 mice for 3 mix + Isotype, n = 9 mice for 3 mix + anti-PD-L1 (c) C. cateniformis n = 5 mice per group (d) E. ramosum n = 10 mice per group (e) C. innocuum n = 5 mice for isotype and n = 4 mice for anti-PD-L1. Significance at day 23 is shown and was determined by two-way ANOVA with Tukey’s multiple comparisons test for a and b. Significance is shown and was measured by two-way ANOVA with Sidak’s multiple comparisons test for all days was shown for c-e. Error bars for all tumor graphs show mean and s.e.m. (f) C.cateniformis-specific primers for the 16S gene were used to detect C. cateniformis in C. cateniformis stock, HMB stocks, Taconic feces, and E. ramosum stocks. C. cateniformis gene expression was normalized using the 16S universal primers in each group. N = 3 technical replicates for C. cateniformis and E. ramosum pure stocks, n = 5 biological replicates from 5 HMB stocks, n = 2 biological replicates of stool samples from two different Taconic mice. To show sequence specificity, data are shown as log fold change over the normalized C. cateniformis-specific 16S levels in E .ramosum stock. Error bars represent standard deviation. GF mice or GF mice monocolonized with E. ramosum one week prior to tumor implantation were sacrificed at day 10 p.i. PD-L2 was measured on MHCII+ CD11c+, MHCII+ CD11b+, and CD8+ T cells in (g) draining lymph nodes, n = 9 mice for GF and 7 mice for E. ramosum (h) MLN, n = 10 mice per group. Significance measured by Mann-Whitney test and error bars show mean and s.d.
Extended Data Fig. 7 C. cateniformis and its surface extracts impact immune function.
(a) BMDCs from WT, TLR2 KO, Dectin-1 KO, and MyD88 KO mice were treated with surface extracts isolated from C. cateniformis pellets. PD-L2 expression was measured by flow cytometry. Percent reduction (compared to vehicle treatment) of PD-L2 expressing dendritic cells is shown. Significance measured by Kruskal-Wallis test and P values compared to WT are indicated on graph. Error bars show mean and s.d., n = 3 wells of BMDCs per group. (b–f) Isotype treated-GF versus GF mice monocolonized with C. cateniformis one week prior to tumor implantation were sacrificed at day 13 p.i. and dLNs were analyzed by flow cytometry. Frequencies of (b) CD45+ cells, (c) CD8+ T cells (d) CD4+ T cells (e) MHCII+ CD11b+ cells and (f) MHCII+CD11c+ cells. (b–f) n = 10 mice for GF and n = 9 mice for C. cateniformis, significance measured by unpaired, two-tailed Mann-Whitney test and P values are indicated on graphs, error bars show mean and s.d. (g–j) GF mice were monocolonized one week prior to tumor implantation and treated with either isotype or anti-PD-L1. Mice were sacrificed at day 18 p.i. and tumors were harvested for analysis. Frequencies of CD8+ T cells expressing (g) Granzyme B (h) IFNγ (i) TIM-3 and PD-1 and (j) TNFα. For (g–j) n = 5 mice for isotype and n = 3 mice for anti-PD-L1, significance determined by unpaired, two-tailed Mann-Whitney tests and P values are indicated on graphs, error bars show mean and s.d. (k) Histograms of PD-L2 expression on BMDCs measured by flow cytometry. Red= BMDCs transduced with PD-L2 GPF lentivirus, Blue = BMDCs transduced with control GFP lentivirus, Gray = BMDCs from PD-L2 KO mice. (l–p) BMDCs transduced with GFP lentivirus (GFP) or lentivirus expressing GFP and PD-L2 (PD-L2-GFP) were treated with C. cateniformis extract 24 h before co-culture with CD8+ T cells. Expression measured by flow cytometry. (l) Example of flow cytometry plots of Granzyme B and CD107a expression on CD8+ T cells. Quantification of mean fluorescence intensity of (m) CD107a and (n) Granzyme B expression on CD8+ T cells displayed in (l). Quantification of mean fluorescence intensity of (o) CD44 and (p) CD25 on CD8+ T cells. (m–p) Significance determined by unpaired, two-tailed t tests, with Welch correction and P values indicated on graph, n = 4 wells of BMDC- CD8+ T cell co-culture per group, error bars show mean and s.d.
Extended Data Fig. 8 C. cateniformis treatment and PD-L2 blockade impacts anti-tumor immunity.
(a) Taconic mice were treated with broad spectrum antibiotics one week prior to tumor implantation. On days 7, 10, 13, 16 C. cateniformis or PBS was orally gavaged and antibodies were administered i.p. Significance between anti-PD-L1 + C. cateniformis treatment and the other groups at day 27 is shown. Significance measured by Two way ANOVA and Tukey’s multiple comparisons test, n = 10 mice per group, error bars show mean and s.e.m. (b) B16-OVA tumor growth in SPF mice that received GFP or PD-L2-GFP BMDCs at tumor site 3 days after tumor implantation. Significance indicated on graph and measured by 2 way ANOVA and Sidak’s multiple comparisons test, n = 10 mice per group, error bars show mean and s.e.m. (c–e) BMDCs were cultured for 7 (blue) or 10 (red) days and MHCII expression was measured by flow cytometry. (c) Expression of CD11c and MHCII gated on live cells from BMDC culture. (d) Histogram of MHCII expression gate on live cells from BMDC culture. (e) Histogram of MHCII expression gated on CD11c+ MHCII+ cells. (f) Growth of MC38 tumor cells implanted subcutaneously in β2m−/− mice (B2M KO), β2m+/− (Het), and WT littermate controls. n = 5 mice per group for WT/het + anti-PD-L1 and B2M KO + anti-PD-L1 and n = 3 mice for B2M KO + anti-PD-L1 and anti-PD-L2. Significance indicated on graph measured by one-way ANOVA and Tukey’s multiple comparisons, error bars show mean and s.e.m. Tumor growth of (g) MC38 tumor cells in GF mice treated with Isotype (n = 5 mice), anti-PD-L1 (n = 5 mice), anti-PD-L2 clone 3.2 (n = 5 mice), anti-RGMb (n = 4 mice), anti-PD-L1 + anti-PD-L2 3.2 (n = 4 mice), or anti-PD-L1 + anti-RGMb (n = 5 mice) and (h) B16-OVA tumor cells in Taconic mice treated with Isotype (n = 5 mice), anti-PD-L1 (n = 10 mice), anti-PD-L2 clone 3.2 (n = 5 mice), anti-PD-L2 clone 2C9 (n = 5 mice), anti-RGMb (n = 5 mice), anti-PD-L1 + anti-PD-L2 3.2 (n = 10 mice), anti-PD-L1 + anti-PD-L2 2C9 (n = 10 mice), or anti-PD-L1 + anti-RGMb (n = 10 mice). (g,h) Significance measured by Two-way ANOVA and Tukey’s multiple comparisons, P values are shown on graph, error bars show mean and s.e.m.
Extended Data Fig. 9 RGMb expression is modulated by the gut microbiota.
Tumor draining lymph nodes from GF and SPF mice implanted with MC38 tumor cells subcutaneously, as in Fig. 1a, were analyzed on day 11 after implantation. (a) Relative mRNA expression of RGMb in tumor draining lymph nodes of indicated cells. The levels of rgmb transcripts were normalized to expression of an internal control gene 18S rRNA. N = 5 mice for GF and n = 4 mice for SPF and (b–d) cell surface expression of RGMb protein in CD4+ T cells, CD8+ T cells, CD11c+ MHCII+ and CD11b+ cells. (b) Frequencies of RGMb -expressing CD4+ T cells, CD8+ T cells, CD11c+ MHCII+ and CD11b+ cells were measured using 9D3 clone mAb in dLN, n = 5 mice per group. (c–d) Geometric Mean Fluorescent Intensity (gMFI) of RGMb was assessed in indicated populations from (c) tumor and (d) tumor draining lymph nodes using αRGMb polyclonal antibody, n = 5 mice per group. Significance measured by unpaired, two tailed Mann-Whitney test and significant p values indicated on graph, error bars show mean and s.d. (e–g) GF mice were colonized with stools from three patients who received anti-PD-1 therapy and responded or did not respond - Complete Responder (CR, n = 6) or Non-Responder 1(NR1, n = 5) or Non-Responder 2 (NR2, n = 5). The mice were injected subcutaneously with MC38 tumor cells and treated with rat IgG2b isotype control. Frequencies of RGMb-expressing tumor-infiltrating (e) CD8+ T cells, (f) CD11b+MHC II+ cells, and (g) CD11c+MHC II+ cells were examined on post-implantation day 29. Significance measured by non-parametric one-way ANOVA with Dunn’s multiple comparisons test and significant p values indicated on graph. Error bars show mean and s.d. (h) MC38 tumor cells expressing GFP were implanted in GF or SPF mice. The mice were treated with two doses of isotype control or anti-PD-L1 one week after tumor implantation, and tumors were harvested 13 days after tumor implantation. MC38-GFP cells were isolated and examined to measure PD-L2 (Upper), PD-L1 (Middle), and RGMb (Lower) expression by flow cytometry. Representative of 5 mice per group. (i) GF mice were implanted with MC38 tumor cells subcutaneously and treated with indicated antibodies as in Fig. 1a. Tumor-infiltrating CD4+ T cells were isolated on day 11 after tumor implantation and stimulated with PMA/Ionomycin for 5 h. Frequencies of TNF-α producing cells among CD4+ T cell population were measured by intracellular staining and flow cytometry. N = 4 mice for isotype and n = 5 mice for anti-PD-L1, anti-RGMb, and anti-PD-L1 + anti-RGMb groups. Significance measured by non-parametric one-way ANOVA with Dunn’s multiple comparisons and indicated on graph. Error bars show mean and s.d. (j–l) GF mice were implanted with MC38 tumor cells subcutaneously and treated with indicated antibodies as in Fig. 1a. Cells in tumor and tumor draining lymph node were analyzed on day 11 after tumor implantation. Frequencies of tumor infiltrating CD8+ T cells expressing (j) PD-1 (k) TIM-3 and (l) LAG-3. N = 4 mice for isotype and n = 5 mice for anti-PD-L1, anti-RGMb, anti-PD-L1 + anti-RGMb groups. Significance measured by non-parametric one-way ANOVA with Dunn’s multiple comparisons and significant p values indicated on graphs. Error bars show mean and s.d. (m) Strategy to generate RGMb conditional knockout mice (n) Validation of CD4-Cre mediated deletion of RGMb in peripheral naïve CD8 T cells by qPCR (o) Validation of LysM-Cre mediated deletion of RGMb in bone marrow derived macrophages by qPCR, n = 2 mice per group. Error bars show mean and s.d. (p–s) WT or RGMb KO CD8+ T-cells were co-cultured with WT BMDCs. CD8+ T cells were analyzed by flow cytometry for (p) Mean Fluorescence Intensity (MFI) of T-Bet (q) MFI of CD107a (r) MFI of Granzyme B and (s) proliferation measured by Cell Trace Violet. N = 3 per group. Representative experiment of 3 experiments. Significance determined by unpaired Mann-Whitney test and significant p values indicated on graphs. Error bars show mean and s.d.
Extended Data Fig. 10 Gating strategies for flow cytometric analysis.
The following gating schemes were used to define CD11c+ MHC II+, CD11b+ MHC II+ cells, CD8+ T cells, CD4+ T cells and Treg cells in (a) draining lymph nodes and mesenteric lymph nodes and (b) tumor-infiltrating immune cells. (c) Gating strategy to examine OT-I cells stimulated by OVA-loaded BMDCs. (d) Gating strategy used to analyze tumor cells from the mice implanted with MC38 tumor cells expressing GFP. Gating strategies used to sort CD4+ T cells, CD8+ T cells, CD11c+MHC II+ cells and CD11b+ cells from (e) tumor-infiltrating leukocytes and (f) tumor draining lymph nodes.
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Park, J.S., Gazzaniga, F.S., Wu, M. et al. Targeting PD-L2–RGMb overcomes microbiome-related immunotherapy resistance. Nature 617, 377–385 (2023). https://doi.org/10.1038/s41586-023-06026-3
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DOI: https://doi.org/10.1038/s41586-023-06026-3
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