The evolution and popularization of next-generation sequencer led to technological innovations in the studies of enteric microbiota, and uncovered that enteric microbiota profoundly affects host immune system [
44‐
48]. This prompted to conduct investigations focusing on the relationship between efficacy of ICI and intestinal flora, and some intriguing findings have been reported mainly in melanoma. The finding that intestinal flora affected the efficacy of a therapy targeting PD-1/PD-L1 axis was initially reported with an experimental murine melanoma model [
49]. It was demonstrated that oral administration of
Bifidobacterium species including
B. breve and
B. longum resulted in an improved tumor control without additional treatments, and that the improvement was further augmented in combination with anti-PD-L1 antibody treatment. Tumor-specific T cells increased in the tumor tissue as well as the periphery in those mice, and the depletion of CD8
+ T cells canceled the therapeutic effects. Furthermore, the
Bifidobacterium feedings enhanced the capacity of dendritic cells to stimulate CD8
+ T cells. These suggested that the colonization of
Bifidobacterium species modulated dendritic cell activation, leading to the exertion of anti-tumor effects via evoking T cell immunity [
49]. Previous studies demonstrated that some species of
Bifidobacterium had a potential to modulate DC activation directly and to influence T-cell responses [
50‐
55]. Although innate immune systems including Toll-like receptors should be involved [
56‐
61], heat inactivation of those bacteria wiped out the anti-tumor effects after their oral administration [
49]. This suggested that live bacteria were indispensable and that bacterial components alone were insufficient.
Based on the results described above, analyses using feces of metastatic melanoma patients who had received anti-PD-1 antibody therapies were conducted by the same group at University of Chicago [
62]. In that cohort study, stool specimens were collected from 42 metastatic melanoma patients prior to the anti-PD-1 antibody treatment, and the correlations between the compositions of intestinal flora and the therapeutic efficacies were examined. It was demonstrated that eight bacterial species, including
Bifidobacterium longum,
Collinsella aerofaciens, and
Enterococcus faecium, were enriched in the responders to the PD-1-based therapy as compared to the non-responders. On the other hand, two bacterial species,
Ruminococcus obeum and
Roseburia intestinalis, were found to be more abundant in the non-responders than the responders [
62]. Fecal microbiome transplantation into germ-free mice revealed that feces of the responders, but not that of the non-responders, had a capacity to control tumor growth. Fecal transplantation from the responder increased tumor-specific T cells not only in the spleen but also in the tumor tissue of the mice as compared to that from the non-responder, indicating that colonization of the beneficial bacteria primed tumor antigen-specific immunity locally as well as systemically [
62]. Furthermore, the experimental fecal transplantation from the responders into mice augmented the therapeutic effects of anti-PD-L1 antibody, whereas that from the non-responders abrogated the effects. Collectively, colonization of several bacterial species including
Bifidobacterium longum, which was found also in the murine study described above, was associated with anti-tumor efficacies of PD-1-based therapy in the cohort of this study [
62].
In another cohort study conducted at The University of Texas MD Anderson Cancer Center, fecal samples were collected from 112 metastatic melanoma patients before and after the anti-PD-1 antibody treatment, and the correlations between the diversity and compositions of the intestinal flora and the clinical responses were analyzed [
63]. The diversity of microbiota of the responders of the therapy was profoundly higher than that of the non-responders, resulting that the patients with high diversity had significantly longer progression-free survival (PFS) than those with intermediate or low diversity [
63]. It was also uncovered that Ruminococcaceae family and
Faecalibacterium genus were enriched in fecal microbiota of the responders, whereas Bacteroidales was abundant in those of the non-responders. Consistent with this finding, patients with high
Faecalibacterium abundance displayed longer PFS than those with lower abundance, while patients with high Bacteroidales abundance had shorter PFS than those with lower abundance [
63]. Immunohistochemical analyses of the tumor tissues revealed that the infiltration of CD8
+ T cells into the tumor and the abundance of the
Faecalibacterium, the Ruminococcaceae, and the Clostridiales in the gut were positively correlated [
63]. Moreover, in the systemic circulation, the patients with the high abundance of the
Faecalibacterium, the Ruminococcaceae, and the Clostridiales displayed high frequencies of CD8
+ T cells and effector CD4
+ T cells. In contrast, the patients with the high abundance of the Bacteroidales exhibited high frequencies of immune-suppressive cell populations such as regulatory T cells (T
reg) and myeloid-derived suppressor cells in the systemic circulation [
63]. Fecal microbiome transplantation into germ-free mice revealed that feces of the responders significantly suppressed tumor growth as compared to that of the non-responders. Moreover, the experimental fecal transplantation from the responders into mice improved the therapeutic efficacy of anti-PD-L1 antibody treatment, although that from the non-responders worsened [
63]. Tumor tissues of the mice receiving the feces from the responders exhibited higher levels of CD8
+ T cell infiltration and of PD-L1 expression than those form the non-responders, suggesting that colonization of the beneficial bacteria in the gut would generate the immunologically “hot” microenvironment in the tumor tissues. Moreover, high frequency of innate immune effector cells expressing CD45, CD11b, and Ly6G [
64], and low frequency of myeloid suppressor cells expressing CD11b and CD11c [
65] were observed in the tumor tissues of mice receiving the feces of the responders as compared to those of non-responders. In contrast, the mice receiving the feces of the non-responders displayed higher frequencies of T
reg in their spleens than those of the responders [
63]. Consistent with the aforementioned results in another cohort, these results indicated that colonization of specific bacteria in the gut would influence anti-tumor immunity not only systemically but also locally. Altogether, in PD-1-based therapy on the cohort of this study, enrichment of Ruminococcaceae family and
Faecalibacterium genus was correlated with the effectiveness, whereas that of Bacteroidales was correlated with the ineffectiveness [
63].