Microbes-myeloid-tumor: a colonic triad against T cells

Immune checkpoint blockade has dramatically improved cancer therapy but remains ineffective for most colorectal tumors. In this issue of Immunity, Peuker et al. describe a microbiota-myeloid-tumor cell crosstalk that inhibits CD8+ T cells and promotes colorectal cancer progression. Immune checkpoint blockade has dramatically improved cancer therapy but remains ineffective for most colorectal tumors. In this issue of Immunity, Peuker et al. describe a microbiota-myeloid-tumor cell crosstalk that inhibits CD8+ T cells and promotes colorectal cancer progression. Therapies based on immune checkpoint blockade (ICB), such as targeting CTLA-4 or the PD-1/PD-L1 axis, have resulted in unparalleled clinical outcomes in numerous cancer types. Although ICB is effective for a subset of colorectal cancers (CRCs) characterized by DNA-mismatch repair deficiency and microsatellite instability (MSI), most CRC patients (85%) have microsatellite stable (MSS) tumors that do not respond. Hence, understanding the mechanisms of resistance to ICB and uncovering new therapeutic targets for MSS CRC remains an important unmet medical need. The susceptibility of MSI tumors to ICB is attributed to their high mutational load, resulting in a high neoantigen burden. However, MSI and MSS tumors also differ in their immune tumor microenvironment (TME) (Picard et al., 2020Picard E. Verschoor C.P. Ma G.W. Pawelec G. Relationships Between Immune Landscapes, Genetic Subtypes and Responses to Immunotherapy in Colorectal Cancer.Front. Immunol. 2020; 11: 369Crossref PubMed Scopus (195) Google Scholar). MSI tumors are characterized by high lymphocyte infiltration, particularly CD8+ T cells, which are a positive prognostic factor for CRC and response to ICB (Bruni et al., 2020Bruni D. Angell H.K. Galon J. The immune contexture and Immunoscore in cancer prognosis and therapeutic efficacy.Nat. Rev. Cancer. 2020; 20: 662-680Crossref PubMed Scopus (539) Google Scholar). In contrast, MSS tumors are poorly infiltrated by T cells but are enriched in immunosuppressive myeloid cells (Picard et al., 2020Picard E. Verschoor C.P. Ma G.W. Pawelec G. Relationships Between Immune Landscapes, Genetic Subtypes and Responses to Immunotherapy in Colorectal Cancer.Front. Immunol. 2020; 11: 369Crossref PubMed Scopus (195) Google Scholar). Therefore, targeting myeloid cells represents an attractive therapeutic approach for MSS CRC. In this issue of Immunity, Peuker et al. describe a microbiota-induced calcineurin-, nuclear factor of activated T cells (NFAT)-, and STAT3-dependent crosstalk among myeloid cells, T cells, and tumor cells that promotes CRC progression and metastasis via upregulation of B7H3 and B7H4 (Peuker et al., 2022Peuker K. Strigli A. Tauriello D.V.F. Hendricks A. Integration of microbial signals by myeloid calcineurin controls anti-tumor immunity in colorectal cancer.Immunity. 2022; 55: 701-717Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The contribution of microbiota to CRC development and progression is well established (Drewes et al., 2016Drewes J.L. Housseau F. Sears C.L. Sporadic colorectal cancer: microbial contributors to disease prevention, development and therapy.Br. J. Cancer. 2016; 115: 273-280Crossref PubMed Scopus (86) Google Scholar). Previous work from Zeissig and colleagues showed that this is in part through toll-like receptor (TLR)-dependent activation of the calcineurin-NFAT axis in intestinal epithelial cells, which supports tumor stem cell function (Peuker et al., 2016Peuker K. Muff S. Wang J. Künzel S. Bosse E. Zeissig Y. Luzzi G. Basic M. Strigli A. Ulbricht A. et al.Epithelial calcineurin controls microbiota-dependent intestinal tumor development.Nat. Med. 2016; 22: 506-515Crossref PubMed Scopus (80) Google Scholar). TLR agonists also activate the calcineurin-NFAT axis in myeloid cells. Thus, given the abundance of these cells in the TME of MSS CRC, Peuker at al. performed a series of elegant experiments to determine the role of myeloid calcineurin-NFAT in intestinal tumor development. They first generated mice with myeloid-specific calcineurin deficiency (LysM-Cre-Ppp3r1fl/fl) and crossed them with ApcMin/+ mice, which are a popular model for human CRC (hCRC). LysM-Cre-Ppp3r1fl/flApcMin/+ mice showed reduced number and size of intestinal tumors (Peuker et al., 2022Peuker K. Strigli A. Tauriello D.V.F. Hendricks A. Integration of microbial signals by myeloid calcineurin controls anti-tumor immunity in colorectal cancer.Immunity. 2022; 55: 701-717Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Similar results were noticed by using a more aggressive CRC model in LysM-Cre-Ppp3r1fl/fl mice. In line with previous reports (Drewes et al., 2016Drewes J.L. Housseau F. Sears C.L. Sporadic colorectal cancer: microbial contributors to disease prevention, development and therapy.Br. J. Cancer. 2016; 115: 273-280Crossref PubMed Scopus (86) Google Scholar), microbiota depletion with antibiotics (Abx) in calcineurin-proficient mice resulted in reduced intestinal tumor burden in both CRC models. However, Abx treatment provided no additional benefit to LysM-Cre-Ppp3r1fl/flApcMin/+ mice. Therefore, the authors concluded that microbiota activate myeloid cell calcineurin to promote CRC. The authors then used a similar genetic approach to delete NFATc1, the main NFAT transcription factor expressed by intestinal tumor-infiltrating immune cells (Peuker et al., 2016Peuker K. Muff S. Wang J. Künzel S. Bosse E. Zeissig Y. Luzzi G. Basic M. Strigli A. Ulbricht A. et al.Epithelial calcineurin controls microbiota-dependent intestinal tumor development.Nat. Med. 2016; 22: 506-515Crossref PubMed Scopus (80) Google Scholar). Mice with NFATc1-specific ablation in myeloid cells (LysM-CreNfatc1fl/flApcMin/+) phenocopied LysM-CrePpp3r1fl/flApcMin/+ animals. Additionally, selective inhibition of calcineurin-NFAT interaction in myeloid cells reduced tumor burden similarly to myeloid-specific calcineurin and NFATc1 deficiencies, suggesting that the pro-tumorigenic role of myeloid calcineurin is indeed mediated by NFAT activation. Interestingly, the antitumor effect of myeloid calcineurin deletion was lost in mice lacking B and T lymphocytes, indicating that activation of myeloid calcineurin-NFAT promotes CRC via regulation of adaptive immune responses. Further analysis of tumor-infiltrating immune cell populations revealed an increase in CD8+ T cell content, higher interferon gamma (IFNγ) production, and reduced expression of exhaustion markers in tumors from LysM-CrePpp3r1fl/flApcMin/+ mice or calcineurin-NFAT inhibition. Specific depletion of CD8+ T cells abolished the protective effect of myeloid calcineurin deficiency. These data indicate that, in addition to its tumor-cell-intrinsic role (Peuker et al., 2016Peuker K. Muff S. Wang J. Künzel S. Bosse E. Zeissig Y. Luzzi G. Basic M. Strigli A. Ulbricht A. et al.Epithelial calcineurin controls microbiota-dependent intestinal tumor development.Nat. Med. 2016; 22: 506-515Crossref PubMed Scopus (80) Google Scholar), calcineurin-NFAT activation supports CRC development via myeloid cell inhibition of antitumor CD8+ T cell responses. To further elucidate how myeloid calcineurin regulates CD8+ T cell immunity in their CRC models, the authors analyzed the expression of co-inhibitory and co-stimulatory molecules in tumors and normal intestinal mucosa from myeloid calcineurin-proficient or -deficient mice. Among the molecules analyzed, only expression of B7H3 and B7H4 was increased in tumors compared to what was seen in healthy tissue, and it was downmodulated by disruption of myeloid calcineurin-NFAT. B7H3 and B7H4, two members of the B7 family of immune checkpoint molecules, are overexpressed in several cancer types and associated with poor prognosis (Ni and Dong, 2017Ni L. Dong C. New B7 family checkpoints in human cancers.Mol. Cancer Ther. 2017; 16: 1203-1211Crossref PubMed Scopus (145) Google Scholar). B7H3 and B7H4 interact with unknown receptors on T cells and inhibit antitumor T cell responses. Peuker et al. found these molecules almost exclusively expressed on epithelial tumor cells and used loss- and gain-of-function approaches to investigate their role in the myeloid calcineurin pro-tumorigenic effect. Antibody-mediated blockade of B7H3 in control but not LysM-CrePpp3r1fl/flApcMin/+ mice resulted in increased CD8+ T cell infiltration, higher IFNγ production, and concomitant inhibition of tumor growth. Selective deletion of B7H3 or B7H4 in intestinal epithelial cells had a similar protective effect but only in the presence of adaptive immune cells. Finally, restoring intestinal epithelial expression of either of these molecules eliminated the protective effect of myeloid calcineurin deficiency. Together, these findings support the notion that activation of myeloid calcineurin-NFAT promotes tumorigenesis via epithelial cell B7H3/H4 inhibition of CD8+ T cell responses (Figure 1). Mechanistically, calcineurin-NFAT-driven production of interleukin (IL)-6 induced STAT3-dependent expression of B7H3 and B7H4 on tumor cells. Activation of epithelial STAT3 is a known mechanism of microbiota-induced colon tumorigenesis (Chung et al., 2018Chung L. Thiele Orberg E. Geis A.L. Chan J.L. Fu K. DeStefano Shields C.E. Dejea C.M. Fathi P. Chen J. Finard B.B. et al.Bacteroides fragilis toxin coordinates a pro-carcinogenic inflammatory cascade via targeting of colonic epithelial cells.Cell Host Microbe. 2018; 23: 203-214.e5Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar; Grivennikov et al., 2012Grivennikov S.I. Wang K. Mucida D. Stewart C.A. Schnabl B. Jauch D. Taniguchi K. Yu G.-Y. Österreicher C.H. Hung K.E. et al.Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.Nature. 2012; 491: 254-258Crossref PubMed Scopus (942) Google Scholar). Accordingly, expression of B7H3 and B7H4 was reduced in Abx-treated animals and restored by oral administration of the TLR4 agonist LPS. Several cytokines have been shown to facilitate microbial-induced colon carcinogenesis via epithelial STAT3 activation. For instance, colonization with enterotoxigenic Bacteroides fragilis, which is associated with hCRC, can activate epithelial STAT3 and promote tumorigenesis in an IL-6- and IL-23-independent manner in the ApcMin/+ model (Chung et al., 2018Chung L. Thiele Orberg E. Geis A.L. Chan J.L. Fu K. DeStefano Shields C.E. Dejea C.M. Fathi P. Chen J. Finard B.B. et al.Bacteroides fragilis toxin coordinates a pro-carcinogenic inflammatory cascade via targeting of colonic epithelial cells.Cell Host Microbe. 2018; 23: 203-214.e5Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Others have highlighted the importance of the IL-23-STAT3 pathway in microbial-induced colonic tumorigenesis (Grivennikov et al., 2012Grivennikov S.I. Wang K. Mucida D. Stewart C.A. Schnabl B. Jauch D. Taniguchi K. Yu G.-Y. Österreicher C.H. Hung K.E. et al.Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.Nature. 2012; 491: 254-258Crossref PubMed Scopus (942) Google Scholar). It remains to be determined whether particular bacterial taxa, such as those associated with CRC, regulate myeloid calcineurin or whether other mediators of STAT3 activation regulate B7H3/4 expression. The authors identified a population of Ly6G+ granulocytes as responsible for the pro-tumorigenic effect of myeloid calcineurin. Myeloid calcineurin deficiency affected IL-6 expression exclusively on tumor-infiltrating Ly6G+ cells, and depletion of this population resulted in reduced tumor burden and decreased tumor-associated IL-6 and B7H3 expression in calcineurin-proficient mice. Tumor Ly6G+ cells from myeloid calcineurin-proficient and -deficient mice had a similar immunosuppressive effect on T cells ex vivo. Interestingly, depletion of Ly6G+ cells did not impact tumor burden in LysM-CrePpp3r1fl/flApcMin/+ mice. This observation is somewhat surprising because previous studies showed a negative impact of direct myeloid-T cell crosstalk on CRCs (Picard et al., 2020Picard E. Verschoor C.P. Ma G.W. Pawelec G. Relationships Between Immune Landscapes, Genetic Subtypes and Responses to Immunotherapy in Colorectal Cancer.Front. Immunol. 2020; 11: 369Crossref PubMed Scopus (195) Google Scholar). Nevertheless, the data from Peuker et al. support their hypothesis that calcineurin activation in Ly6G+ myeloid cells hinders antitumor CD8+ T cell responses indirectly via induction of B7H3/4 on tumor cells. Tumor-infiltrating Ly6G+ cells have been previously implicated in the tumorigenic effect of CRC-associated bacteria (Chung et al., 2018Chung L. Thiele Orberg E. Geis A.L. Chan J.L. Fu K. DeStefano Shields C.E. Dejea C.M. Fathi P. Chen J. Finard B.B. et al.Bacteroides fragilis toxin coordinates a pro-carcinogenic inflammatory cascade via targeting of colonic epithelial cells.Cell Host Microbe. 2018; 23: 203-214.e5Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar; Lopès et al., 2020Lopès A. Billard E. Casse A.H. Villéger R. Veziant J. Roche G. Carrier G. Sauvanet P. Briat A. Pagès F. et al.Colibactin-positive Escherichia coli induce a procarcinogenic immune environment leading to immunotherapy resistance in colorectal cancer.Int. J. Cancer. 2020; 146: 3147-3159Crossref PubMed Scopus (43) Google Scholar). In line with this, and with the role of microbial-derived signals in myeloid calcineurin activation, in vitro assays showed that TLR agonists induced IL-6 production by splenic granulocytes in a calcineurin-NFAT-dependent manner. Finally, the authors studied B7H3 and B7H4 expression in hCRC. Expression of these molecules was elevated in hCRC mucosa compared with what was seen in healthy colonic tissue. Tumors with high B7H3/4 expression had increased infiltration with myeloid cells with nuclear NFATc1, suggestive of calcineurin-NFAT activation. Like their mouse models, B7H3/4 protein expression was restricted to tumor cells, increased with tumor stage, associated with reduced CD8+ T cell infiltration, and negatively correlated with patient survival. B7H3 and B7H4 were also expressed on most hCRC metastatic lesions and inversely correlated with CD8+ T cell infiltration. Antibody blockade of B7H3 in a mouse model of CRC liver metastasis increased CD8+ T cells and reduced metastatic burden. Combined, these findings support the proposed mechanism in which tumor cell expression of B7H3/4 induced by myeloid calcineurin-NFAT activation dampens antitumor CD8+ T cell immunity and promote CRC progression. Previous studies showed a negative association between CRC-associated bacteria and tumor-infiltrating lymphocytes in CRC patients (Lopès et al., 2020Lopès A. Billard E. Casse A.H. Villéger R. Veziant J. Roche G. Carrier G. Sauvanet P. Briat A. Pagès F. et al.Colibactin-positive Escherichia coli induce a procarcinogenic immune environment leading to immunotherapy resistance in colorectal cancer.Int. J. Cancer. 2020; 146: 3147-3159Crossref PubMed Scopus (43) Google Scholar; Mima et al., 2015Mima K. Sukawa Y. Nishihara R. Qian Z.R. Yamauchi M. Inamura K. Kim S.A. Masuda A. Nowak J.A. Nosho K. et al.Fusobacterium nucleatum and T Cells in Colorectal Carcinoma.JAMA Oncol. 2015; 1: 653-661Crossref PubMed Scopus (392) Google Scholar). Although the authors found an inverse correlation between B7H3/4 and infiltrating lymphocytes, they did not find associations between B7H3/4 expression and specific bacterial taxa. More effective therapies are needed to improve MSS CRC clinical outcomes. The study by Peuker et al. describes a microbiota-myeloid-tumor crosstalk that inhibits CD8+ T cell responses in CRCs (Figure 1). It remains to be elucidated whether specific CRC-associated bacteria or changes in microbiota composition engage the myeloid calcineurin-NFAT-B7H3/4 pathway and how microbiota regulate B7H3/4 in distant metastatic lesions. Nevertheless, interfering with this pathway offers a potential therapeutic avenue to increase CD8+ T cell recruitment and sensitize MSS CRC tumors to ICB. This research was supported by the Intramural Research Program of the NIH (NCI, Center for Cancer Research). The authors declare no competing interests. Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancerPeuker et al.ImmunityMarch 31, 2022In BriefThe mechanisms through which microsatellite-stable colorectal cancer inhibits anti-tumor immunity and resists immune checkpoint blockade are incompletely understood. Peuker et al. describe a microbiota-dependent pathway of crosstalk between myeloid cells, T cells, and tumor cells that inhibits CD8+ T cell-dependent anti-tumor immunity through the co-inhibitory proteins B7H3 and B7H4 and that is amenable to therapeutic targeting. Full-Text PDF Open Archive