Abstract
The antitumor efficacy of cancer immunotherapy has been correlated with specific species within the gut microbiota. However, molecular mechanisms by which these microbes affect host response to immunotherapy remain elusive. Here we show that specific members of the bacterial genus Enterococcus can promote anti-PD-L1 immunotherapy in mouse tumor models. The active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in non-protective E. faecalis was sufficient to promote antitumor activity of clinically approved checkpoint targets, and its activity required the peptidoglycan sensor Nod2. Notably, SagA-engineered probiotics or synthetic muropeptides also promoted checkpoint inhibitor antitumor activity. Our data suggest that microbiota species with unique peptidoglycan remodeling activity may enhance immunotherapy and could be leveraged for next-generation adjuvants.
One Sentence Summary A conserved family of secreted NlpC/p60 peptidoglycan hydrolases from Enterococcus promote antitumor activity of immune checkpoint inhibitors.
Main Text
Cancer immunotherapy harnesses host immune mechanisms to impede tumor growth and has demonstrated clinical success across a range of solid and liquid tumors (1–3). In particular, antibodies that target immune inhibitory pathways such as CTLA-4 and the PD-1/PD-L1 signaling axis have been approved to treat a wide array of human cancers (3). However, patient response to immune checkpoint inhibitors is variable (3). The success of checkpoint blockade relies on numerous factors including mutational burden of the malignancy (4), successful tumor antigen presentation (5), recruitment and infiltration of lymphocytes (6), and signaling cues within the tumor microenvironment (7). Recently, the gut microbiota has emerged as a potent new factor associated with the efficacy of anti–CTLA4, anti–PD-1, and anti–PD-L1 treatment (8–13). In animal and human cohorts, the presence of specific microbial species was correlated with responsiveness to checkpoint blockade agents. The antitumor activity of these microbes was recapitulated in preclinical mouse models upon co-housing, fecal transplant, or direct inoculation, suggesting that the correlated microorganisms are direct causative agents of improved therapeutic response. Nevertheless, little is known about the exact, molecular factors by which these immune modulatory microbes act.
Multiple analyses of the commensal microbiome from human cohorts treated with immunotherapies targeting PD-1 have revealed that the bacterial genus Enterococcus is enriched in responding patients (12, 13). Although antibiotic-resistant strains of E. faecium and E. faecalis can be pathogenic (14), commensal strains of these bacteria have been used employed as probiotics in animals and humans (15). Recent studies also suggested that Enterococcus species can trigger immune signaling pathways and modulate infection (16–18), autoimmunity (19), and graft-versus-host-disease (20). These observations prompted our inquiry into whether specific Enterococcus species and strains are sufficient to improve checkpoint blockade.
To evaluate specific enterococci and their mechanism of action, we utilized mouse tumor models and orally administered bacteria. Here, specific pathogen-free (SPF) C57BL/6 mice from The Jackson Laboratory were pretreated for two weeks with a broad-spectrum antibiotic cocktail to clear resident microbial species and subsequently provided water supplemented with bacteria. Supplemented animals were then subcutaneously implanted with B16-F10 melanoma cells and treated with anti–PD-L1 (Fig. 1A). We first focused on the two most common Enterococcus species in the human gut microbiota: E. faecium (Efm) and E. faecalis (Efs). We found that supplementation with the human commensal E. faecium strain Com15 without therapeutic intervention did not alter tumor growth (Fig. 1B). However, treatment of supplemented animals with anti–PD-L1 at both high and low dosages showed a significant decrease in tumor size compared to mice treated with anti–PD-L1 alone. Using the low anti–PD-L1 dose protocol going forward, we then compared the activity of E. faecium with E. faecalis across multiple strains of each species—human-isolated, type, and multi-drug resistant—to ascertain if the observed synergistic activity was unique to E. faecium Com15. Inhibition of tumor growth was observed across all three isolates of E. faecium and not for any strain of E. faecalis (Fig. 1C). To ensure that these effects were not due to differences in bacterial load, fecal samples were plated onto Enterococcus-selective medium and enumerated. All bacterial strains yielded similar amounts of colony forming units (CFU, Fig. 1D), indicating that the observed antitumor activity of E. faecium strains was not due to differences in bacterial amount in the gut. In addition to E. faecium, 16S rRNA analysis of gut microbiota from responsive human patients also uncovered the enrichment of other Enterococcus species (12). We found that antitumor activity was conserved across multiple species including E. durans (Eds), E. hirae (Ehe), and E. mundtii (Emi) (Fig. 1E and S1A). CFU analysis of the colonized animals again revealed that activity did not correlate with bacterial load (Fig. 1F and S1B).
To explain the species-specific differences we observed across the Enterococcus genus, we examined possible sources of their immunomodulatory activity. Notably, our previous work indicated that E. faecium has unique peptidoglycan composition and remodeling capabilities to enhance host tolerance to enteric pathogens (16–18). To compare peptidoglycan composition across the enterococci, we isolated sacculus of each species and analyzed the digested peptidoglycan fragments by high performance liquid chromatography–mass spectrometry (HPLC-MS). All four of the immunotherapy-active enterococci (E. faecium, E. durans, E. hirae, and E. mundtii) showed similar peptidoglycan fragment patterns compared to the nonactive species E. faecalis and E. gallinarum (Egm) (Fig. 2A and S2A). Additionally, all four immunotherapy-active species showed an abundance of smaller, non-crosslinked peptidoglycan fragments, suggesting higher levels of peptidoglycan remodeling and turnover. The modification of peptidoglycan stem peptides is catalyzed by conserved families of amidases and peptidases (21), so we examined Enterococcus genomic assemblies for unique expression of peptidoglycan remodeling enzymes (Fig. S3). Based on this analysis, we identified a cluster of NlpC/p60 hydrolases that were highly conserved through all active enterococci (Fig. 2B). This group of related enzymes contained the peptidoglycan hydrolase secreted antigen A (SagA) from E. faecium, which we previously demonstrated improves host immunity against enteric infections (16–18). Using primary sequence alignment, we found that the putative SagA orthologs from other active enterococci were highly homologous, with greater than 90% sequence identity within the C-terminal NlpC/p60 hydrolase domain (Fig. 2C). Conversely, the closest related protein in E. gallinarum showed only 67% sequence identity in the predicted catalytic domain (Fig. S2b). No direct orthologs were found in E. faecalis, and the two most similar conserved enzymes, sagA-like proteins A and B (SalA and SalB) (22), possessed distinct C-terminal domains (Fig. 2C). Structural modeling revealed that the putative SagA orthologs also shared similar secondary structural organization compared to a crystal structure of the NlpC/p60 hydrolase domain from E. faecium SagA (Fig. S4).
To directly detect the expression of SagA orthologs in enterococci, we performed Western blotting on both the secreted and cell-associated protein fractions from each species. Using antiserum raised against E. faecium SagA (18), strong signals were observed in the supernatant fractions of E. faecium, E. durans, E. hirae, and E. mundtii but not E. faecalis or E. gallinarum (Fig. 2D), suggesting that the similar proteins are both highly expressed and secreted by these species. The signal was confirmed to be SagA-dependent using a strain of E. faecalis OG1RF engineered with a chromosomal sagA insertion (Efs-sagA). As expected, all tested strains of E. faecium also showed similar expression and secretion patterns for SagA (Fig. S5). To determine if these SagA orthologs were functional, we analyzed the hydrolytic activity of the purified recombinant proteins on peptidoglycan in vitro (Fig. 2E and S6). Proteins from E. durans, E. hirae, and E. mundtii showed D,L-endopeptidase activity against a model crosslinked peptidoglycan fragment similar to E. faecium SagA (Fig. S7). The hydrolytic activity of SagA was confirmed using a mutant construct lacking the cysteine active site residue, which was conserved in all orthologs (Fig. S8). E. faecalis SalB did not hydrolyze peptidoglycan at detectable levels in our assay, whereas SalA cleaved the crosslinked fragment in the cross-bridge region rather than the peptide stem (Fig. 2E and S9). These results show the immunotherapy-active enterococci possess similar peptidoglycan composition and remodeling activity.
We then investigated whether SagA was sufficient to enhance the efficacy of checkpoint inhibitor therapy. Since sagA is an essential gene in E. faecium (23), we compared the inactive, parental E. faecalis OG1RF strain with the engineered, SagA-expressing strain E. faecalis–sagA (Fig. 2D). The peptidoglycan profile of E. faecalis–sagA showed changes consistent with increased D,L-endopeptidase activity (Fig. S10A), indicative of active SagA expression (Fig. 2D). Antibiotic-treated animals that were orally supplemented with E. faecalis–sagA showed a significant decrease in B16-F10 tumor growth upon anti–PD-L1 compared to the parental E. faecalis strain (Fig. 3A). This antitumor activity was similar to the phenotype observed in E. faecium-supplemented animals. Furthermore, fecal CFU analysis showed that E. faecalis–sagA exhibited similar bacterial load as the parental E. faecalis strain and E. faecium (Fig. S10B). We then tested the outcome of SagA expression on checkpoint inhibitor therapy in an animal model with an intact, complex microbiota. SPF C57BL/6 mice from Taconic Biosciences were chosen as previous reports have found that these mice do not respond strongly to anti–PD-L1 therapy similar to germ-free animals (9, 13). Here, these mice were directly supplemented with enterococci-containing water without antibiotic pre-treatment prior to B16-F10 implantation (Fig. S11A). As seen in the antibiotic-pretreated model, animals supplemented with E. faecium or E. faecalis–sagA showed a significant decrease in tumor growth compared to treatment with antibody alone or antibody and parental E. faecalis (Fig. S11B). We found much lower overall Enterococcus CFU counts that did not significantly differ between the supplemented mice, suggesting that this effect did not require enteric domination by the newly administered, active enterococci (Fig. S11C). We then asked whether the antitumor phenotype occurred through changes in endogenous microbiota populations caused by SagA-expressing enterococci. Fecal samples were collected prior to and during bacterial administration and compared by 16S rRNA sequencing. No significant differences in microbiota composition were observed between samples regardless of species administered or duration (Fig. S11, D-G).
Given the activity of specific enterococci during anti–PD-L1 treatment of a subcutaneous melanoma model, we subsequently interrogated whether SagA-expressing bacteria would also improve the efficacy of other checkpoint antibodies against different cancer cell types. Subcutaneous tumors were established with MCA205 fibrosarcoma or MC38 colorectal carcinoma cells in antibiotics-pretreated animals colonized by enterococci and then treated with anti–PD-1 or anti–CTLA-4, respectively. In both cases, we also observed a significant decrease in tumor growth when animals were cotreated with SagA-expressing enterococci and checkpoint inhibitor (Fig. 3, B and C). Given the broad efficacy of these enterococci with different targeted therapies, we then asked whether this effect was mediated by an adaptive immune response. Using our antibiotic-pretreated model, we subcutaneously implanted B16-OVA tumor cells into animals colonized with either parental E. faecalis or E. faecalis–sagA and then treated with anti–PD-L1. Tumors were harvested the day following the third antibody treatment (five days after the start of treatment), and tumor-infiltrating lymphocytes were quantified by flow cytometric analysis (Fig. S12). Animals colonized with E. faecalis–sagA showed an overall increase in the absolute amount of intratumoral CD45+ leukocytes as well as CD3+ lymphocytes (Fig. 3, D and E). The composition of tumor-infiltrating CD3+ lymphocytes showed an increase in the proportion of CD8+ T cells but no change in CD4+FoxP3+ regulatory T cells (Fig. 3, F and G). Tumors contained higher amounts of CD8+ T cells that expressed granzyme B (Fig. 3H), a marker for activated cytotoxic T lymphocytes (24). Tetramer staining also revealed a significant increase in the number of OVA-specific CD8+ T cells (Fig. 3I), consistent with enhanced priming of a tumor antigen-specific immune response (9, 13).
To characterize the microbial mechanism of immune activation, we first examined whether SagA contributed to bacterial dissemination, as Enterococcus translocation from the gut has been implicated during autoimmunity (19), chemotherapy treatment (25), and alcoholic hepatitis (26). CFU analysis of mesenteric lymph nodes and whole spleens of supplemented animals only showed low levels of live bacteria in proximal tissues (Fig. S10C). The bacterial load in mesenteric lymph nodes was independent of SagA expression, suggesting that SagA did not improve barrier transit for live bacteria in our studies. We then turned our attention to peptidoglycan remodeling by SagA and the generation of muropeptides as a potential mechanism of action (18). Indeed, we found that Nod2, a key pattern recognition receptor for muropeptides (27, 28), was required for the anti–PD-L1 antitumor activity in animals supplemented with E. faecalis–sagA (Fig. 4A). To evaluate the enzymatic activity and utility of SagA to improve checkpoint blockade, we investigated heterologous expression in probiotic bacteria. Lactococcus lactis (Lls) has been explored extensively as a live, oral probiotic to deliver bioactive proteins and enzymes (29). Therefore, we produced L. lactis strains that chromosomally expressed wild-type, catalytically inactive (C443A), or secretion-deficient (ΔSS) SagA. All three constructs expressed well, and the wild-type and C443A mutant SagA constructs showed higher signal in the secreted fraction as expected (Fig. 4B). Antibiotic-pretreated animals were orally supplemented with these strains as well as parental L. lactis or E. faecium and used to monitor B16-F10 responsiveness to anti–PD-L1. Animals supplemented with L. lactis expressing wild-type SagA showed similar tumor growth inhibition as E. faecium-treated animals (Fig. 4C). L. lactis expressing catalytically inactive SagA (C443A) were not able to recapitulate the antitumor phenotype, indicating that the enzymatic activity of SagA is required to impede tumor growth. We found that the SagA secretion-deficient strain (ΔSS) did slow tumor growth, which suggests that the low amount of SagA secreted by this strain may have been sufficient to partially inhibit tumor growth. Alternatively, active, non-secreted SagA may escape from L. lactis due to cell lysis in the gut. Because SagA NlpC/p60 hydrolase activity was required, we asked whether synthetic muropeptide analogs of SagA enzymatic products such as muramyl dipeptide (MDP) could also elicit an improved response to checkpoint therapy. For these experiments, the Nod2-active MDP-L,D isomer or the inactive MDP-L,L diastereomer (Fig. 4D) were co-administered by intraperitoneal injection with anti–PD-L1. Animals that received active MDP-L,D along with anti–PD-L1 showed a significant antitumor effect that was not observed upon co-administration of the MDP-L,L negative control, suggesting the Nod2-active muropeptide agonist was sufficient to improve checkpoint blockade (Fig. 4E).
Together, our data indicate that enterococci with unique NlpC/p60 peptidoglycan hydrolase activity can generate Nod2-active muropeptides and modulate the efficacy of checkpoint blockade immunotherapy in vivo (Fig. S13). Although bacteria enriched in responding patients do not correlate well by phylogeny (30), specific enterococci and other microbiota species with privileged cell wall composition and remodeling activity could provide functional indicators of therapeutic efficacy. As peptidoglycan fragments can disseminate into circulation and prime systemic immune responses (31, 32), NlpC/p60 hydrolases like SagA and Nod2-active muropeptides may be utilized as biomarkers to predict personalized clinical responses. Moreover, our results suggest that peptidoglycan remodeling enzymes may be utilized to reprogram probiotic bacteria and activate Nod2 during checkpoint blockade. Lastly, since MDP and Nod2 have been implicated in direct activation of macrophages for tumor clearance (33), epigenetic reprogramming of monocytes (34), generation of conventional type 1 dendritic cells (35), and priming of dendritic cells for cross-presentation to CD8+ T cells (36), our study highlights how microbes and small molecules that activate this pattern recognition receptor could be employed as adjuvants for cancer immunotherapy.
Funding
This work was supported by the National Institutes of Health (1R01CA245292-01, H.C.H.) and in part by the Melanoma Research Foundation (Career Development Award, M.E.G.). M.E.G. is a Hope Funds for Cancer Research Fellow supported by the Hope Funds for Cancer Research (HCFR-19-03-02). This work is also partially funded by an NIH research service award training grant (A1070084, J.E.).
Author contributions
Conceptualization: M.E.G. and H.C.H.; Methodology: M.E.G., J.E., and H.C.H.; Investigation: M.E.G., J.E., and J.L.B.; Supervision: H.C.H.; Writing – original draft: M.E.G. and H.C.H.; Writing – review and editing: M.E.G., J.E., J.L.B., J.J., G.R.F., and H.C.H.; Resources – J.J. and G.R.F.
Competing interests
M.E.G. and H.C.H. have filed a patent application (PCT/US2020/019038) for the commercial use of SagA-bacteria to improve checkpoint blockade immunotherapy. Rise Therapeutics (J.J. and G.R.F.) has licensed the patent to develop immunological-based biologics.
Data and materials availability
All data are available in the main text and supplementary materials. Enterococcus strains used in the research are available from H.C.H. under a material transfer agreement.
Acknowledgments
We thank S. Mazel, A. Keprova, M. Jaimes, D. Tran, and the Rockefeller Flow Cytometry Resource Center for assistance with flow cytometric experiments, C. Steckler and H. Molina of the Rockefeller Proteomics Resource Center for assistance with peptidoglycan analysis, K. Eckartt for assistance in homology modeling, and M. Huse, T. Merghoub, K. Cadwell, and D. Mucida for helpful discussions.