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Abstract
Background Disbalance in the intestinal microbiota composition (i.e. dysbiosis) alters the antitumoral immune response and is associated with primary resistance to immune checkpoint blockade (ICB).1–4Fecal microbiota transplantation (FMT) is one of effective microbiota centered interventions to circumvent ICB resistance.5–7 The understanding of the mechanisms underlying the success or failure of FMT engraftment in circumventing ICB resistance remains an open conundrum. Here, we describe how engraftment of patient donor microbiota influences peripheral and antitumoral immune and metabolic tonus in recipient mice.
Methods To study the impact of FMT on immune tonus, we generated avatar mice with humanized microbiota by colonization with a ‘dysbiotic’ human microbiota, enriched in Enterocloster species.8 Subsequently, the mice received fecal transplant from another donor (FMT 2A or FMT 2B) twice. We evaluated the impact of these different FMTs, in tumor bearing mice (MCA-205 murine fibrosarcoma model), on the response to anti-PD-1. Twenty days after tumor inoculation, tumors, tumor lymph nodes draining (tdLN) and spleens were collected for flow cytometry analyses. Cytokine assays in plasma at different time points were carried out (Olink®, 48-plex Panel). Furthermore, metabolic analysis on plasma and 16S-rRNA sequencing on feces samples were performed.
Results We found that depending on the FMT engraftment, the immune infiltration of the tumor environment (TME) and the systemic tonus were different. Five days after the second FMT, FM2B mice showed a lower α-diversity (p=0.00963). Lactobacillus and Bacteroides species were increased in FMT2A mice, and associated with metabolic modifications, such as an increase of hexoses (p=0.0031), polyols (p=0.0358), and inositol (p=0.0037) metabolites. Compared to the FMT2A, FMT2B induced imunosuppressive RORγt+ Treg (Tr17) lymphocytes in the tdLN of anti-PD-1 treated mice (p= 0.084), as well as macrophages (p=0.0111) in the spleen. These populations were associated with an increase of the tumor size. In addition, this cellular infiltration correlated with higher circulatory plasma protein concentration of Treg and TH17 cytokines (IL-17α, p=0.0317 ; IL-7 p=0.0317 ; IL-10 p=0.0079), TH2 cytokines (IL-4 p=0.0159) and also Cfs3 (p=0.0159).
Conclusions We have shown that depending on the FMT engraftment, the impact on peripheral and anti-tumor immune tonus is different. The populations of RORγt+ T lymphocytes and macrophages seem to be particularly influenced by the FMT performed. Furthermore, the hexoses and polyols pathways were perturbated by the microbiota engrafted. However, it remains necessary to understand by what mechanisms FMT acts on immune tonus.
Acknowledgements ST is supported by Paris Saclay University. MF, LZ and GK are supported by the SEERAVE Foundation.
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Ethics Approval Feces collection for metagenomics analysis were performed under the study ONCOBIOTICS* (Sponsor Protocol N: CSET 2017/2619, ID-RCB N: 2017-A02010-53) according to the ethical guidelines and approval of the local ethical committee (Comite Consultatif de Protection des Personnes dans la Recherche Biomedicale (CCPPRB) of the Kremlin Bicetre Hospital). All animal experiments were performed in compliance with French and European laws and regulations. The local institutional animal ethics board and French Ministère de la Recherche approved all mouse experiments (permission numbers: 2020_037_25421).
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