Enhancing the clinical coverage and anticancer efficacy of immune checkpoint blockade through manipulation of the gut microbiota

The equilibrium linking the intestinal microbiota, the intestinal epithelium and the host immune system determines host health and homeostasis.Citation¹ Perturbation of the mutualistic symbiosis between the intestinal microbiota and the host can result in chronic inflammatory and autoimmune immunopathologies.Citation² Previous studies from our groupCitation³ and othersCitation⁴ have suggested the constitution of the gut microbiota can also influence the immunostimulatory capacity of cancer therapies, particularly those that harness the anti-tumor immune response to mediate their efficacy.

In our latest research published in Science,Citation⁵ we questioned whether the therapeutic efficacy of CTLA-4 blockade, as mediated by the FDA/EMEA-approved clinical immunotherapy ipilimumab,Citation⁶ might also be determined by components of the intestinal microbiota. By comparing the efficacy of anti-CTLA-4 Ab against various established tumors in mice maintained under specific pathogen-free (SPF) and germ-free (GF) conditions, we identified that tumor progression was controlled in SPF but not in GF animals. The antitumor effects of CTLA-4-specific Ab were similarly compromised in mice treated with a broad-spectrum antibiotic cocktail to eliminate gut microbiota. In addition, anti-CTLA-4-mediated activation of tumor-infiltrating lymphocytes (TILs) and splenic lymphocytes was significantly reduced in GF and antibiotics-treated mice.

We next investigated anti-CTLA-4-mediated effects at the gut–microbiota interface, given that this is where microbiota alteration is initially sensed by the immune system and equally where blockade of CTLA-4 by ipilimumab often results in irAEs.Citation⁷ We identified alteration of the mucosal barrier following administration of anti-CTLA-4 Ab in mice, consistent with a “subclinical colitis”. This was more prominent in SPF than in GF animals, suggesting a role for distinct commensals in this anti-CTLA-4 Ab-promoted pathology. Antibody treatment additionally increased the proliferation and the T cell-mediated apoptosis of intestinal epithelial cells (IECs). Using intestinal crypt-derived enteroid cultures, this apoptosis was found to be induced by intraepithelial lymphocytes (IELs) from anti-CTLA4-treated (but not isotype-treated) mice, and to be dependent on the presence of microbial products. Coinciding with these mucosal alterations, FISH analysis revealed an accumulation of distinct Bacteroides spp. in the inner part of the mucus layer, potentially within reach of mucosal dendritic cells (DCs). Taken together, these findings signified that CTLA-4 Ab dysregulates the equilibrium between IECs, IELs and the microbiota at intestinal barriers (Fig. 1).

To identify potential changes in bacterial species caused by this dysregulation, we performed high-throughput pyrosequencing of 16S ribosomal RNA gene amplicons from murine feces. CTLA-4 blockade induced underrepresentation of Bacteroidales and Burkholderiales, and a relative increase in Clostridiales. We recolonized antibiotics-treated or GF mice with bacterial species associated with these perturbations, and identified that Bacteroides fragilis, Bacteroides thetaiotaomicron, Burkholderia cepacia, or the combination of B. fragilis and B. cepacia could each restore the anti-CTLA-4 Ab-mediated anticancer responses. Notably, oral feeding with B. fragilis induced Th1 immune responses in tumor-draining lymph nodes and promoted DC maturation in tumor beds. The microbiota-dependent immunostimulatory effects produced by CTLA-4 blockade required IL-12, this cytokine likely produced by mobilized B. fragilis-stimulated CD11b⁺ DC from the lamina propria.

To test whether these findings translated to the clinical setting, we analyzed the gut microbiome composition in metastatic melanoma patients before and after ipilimumab treatment. Three distinct microbiome clusters (enterotypes) were revealed, for which segregation was determined by the Bacteroides and Prevotella genera (Alloprevotella/Prevotella driving cluster A, and distinct Bacteroides spp. driving clusters B and C). Fecal microbial transplantation of feces highlighted that the microbial composition of cluster C, rich in immunogenic Bacteroides species (e.g., B. fragilis), could restore anti-CTLA4 Ab efficacy, while cluster B enriched in tolerogenic Bacteroides species mediated complete resistance to the Ab. Taken together, this suggests ipilimumab adjusts the level of immunostimulatory Bacteroides spp. in the gut, to facilitate its antitumor efficacy.

Given the exceptional clinical outcomes and lengthened overall survival that can be induced by ipilimumab, it is unfortunate that many patients receiving this immunotherapy develop irAEs.Citation⁷ Interestingly, we identified that administration of the combination of B. fragilis and Burkholderia cepacia, mandatory to restore the efficacy of CTLA4 blockade in antibiotics-treated animals, failed to induce signs of subclinical colitis, rather inducing a protection against anti-CTLA4-induced intestinal lesions. This protection was associated with the capacity of B. fragilis to promote the proliferation of ICOS⁺ Treg in the lamina propria, possibly via mobilizing plasmacytoid DC seen to accumulate and mature in mesenteric lymph nodes after B. fragilis monocolonization of GF mice treated with anti-CTLA4 Ab.Citation^5,8 In support of this, blockade of ICOS or IL-10 plus anti-CTLA4 Ab treatment resulted in an overt and deadly colitis in tumor bearers reared in SPF conditions. Therefore, efficacy and toxicity following CTLA-4 blockade could be uncoupled in this model of B. fragilis and B. cepacia bicolonization.

We therefore show that the efficacy of CTLA-4 blockade is facilitated by constituents of the microbiota, especially certain Bacteroides spp. and Burkholderiales, which enhance tumor control via stimulation of Th1 immune responses during anti-CTLA-4 therapy. Our findings put forward the possibility to (re)establish a favorable enteric microbiota in patients with an ineffective pre-existing enteric microbial microflora that may be associated with a poor prognosis to ipilimumab therapy. Taking our findings,Citation⁵ and recent evidence that the anticancer actions of PDL-1 blockade are enhanced in the presence of Bifidobacterium spp.,Citation⁹ the search is on for components of the microbiota that enhance the action of other immunotherapies.

Disclosure of potential conflicts of interest

The authors have declared no conflicts of interest. Gustave Roussy has signed a collaborative contract agreement with Enterome.