Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal anti-tumor immunity

Gut microbiota, specifically gut bacteria, are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anti-cancer immune responses, however, are largely unknown. Here, we find that ICT induces the translocation of specific endogenous gut bacteria into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, ICT induces lymph node remodeling and dendritic cell (DC) activation, which facilitates the translocation of a selective subset of gut bacteria to extraintestinal tissues to promote optimal anti-tumor T cell responses in both the tumor-draining lymph nodes (TDLN) and the primary tumor. Antibiotic treatment results in decreased gut microbiota translocation into MLN and TDLN, diminished DC and effector CD8+ T cell responses, and attenuated response to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anti-cancer immunity.


16S rRNA Sequencing Library Preparation
We used the reverse primer 926R, 5'-CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3": the italicized sequence is the reverse MiSeq primer i7; NNNNNNNN designates the unique 8-base barcode used to tag each PCR product; the bold sequence is the broad-range 16S bacterial primer containing the pad-link-16SR. The forward primer used was 515F, 5'-AATGATACGGCGACCACCGAGA TCTACAC-NNNNNNNN-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3': the italicized sequence is MiSeq Primer i5; the NNNNNNNN designates the unique 8-base barcode used to tag each PCR product; and the bold sequence is the broad range 16S bacterial primer containing the pad-link-16SF. PCR reactions consisted of 17ul Accuprime Pfx Supermix, 1000 nM of each primer, and 20ng of template. Reaction conditions were 2 min at 95℃, followed by 30 cycles of 20 s at 95℃, 15 s at 55℃, 5 min at 72℃, then 10 min at 72℃, and a hold at 4℃ on an Eppendorf Mastercycler. For tissue and tumor microbiome profiling, two rounds of PCR amplification were performed. Products were verified on a 1% agarose gel, and normalized using the AmPure Normalization plate protocol using the KingFisher Flex platform. Each plate was then pooled into a single tube, and the PCR product size and library quality of each individual pooled plate was checked using Agilent Technologies D1000 ScreenTape electrophoresis. Additionally, KAPA Biosystems PCR Library Quantification kit was used to quantify each pooled plate. Illumina spikein (PhiX) was included at 4 pM at 10%, and the pooled sample library was included at 4pM at 90% yielding a final library concentration of 3.6 pM and PhiX concentration of 0.4 pM.

Gastrointestinal barrier function assays.
FITC-dextran permeability assay. Mice were fasted overnight. FITC-dextran (500 mg/kg; Sigma Aldrich; 4 kD) was administered via oral gavage. Mice were kept without food and water for 4 hours. Blood samples were obtained by terminal cardiac puncture and collected in BD Vacutainer SST tube (BD) 4 hours after FITC-dextran administration. Blood samples were centrifuged at 2,000xG at RT for 10 min. Serum was collected by taking the upper layer after centrifugation. The serum fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a spectrophotometer (Synergy HT, BioTek).  (87) and transcript abundances were normalized to 18S rRNA transcript abundance. Amplification of target genes was conducted using following primers.

(B)
Cultured bacterial levels in MLN. MLN tissue homogenates were serially diluted, plated on BHI/Blood, YCFA, and CME0151 agar media and incubated at 37°C under anaerobic conditions for 24-72 hours. Quantification of colony-forming unit (CFU) from each agar plate was normalized to the tissue weight. n=7-8 per group. Points represent values from individual animals. Bars represent the mean + SEM. Statistical analysis by Mann-Whitney test. *P<0.05, **P<0.01. ***, P<0.001.

(C)
Relative abundance of cultured bacteria. Representative colonies (10-20 colonies with similar morphology and/or color) on each agar plate were selected and subsequently cultured in corresponding liquid media. gDNA was extracted. Full-length 16s rRNA gene (V1-V9 region) was amplified, purified, and sequenced (Sanger sequencing). Sequences were entered into the NCBI standard nucleotide Basic Local Alignment Search (BLAST) tool utilizing the rRNA/ITS databases. Bacterial species identification was ascertained from BLASTN results with the highest Total Score, with percent identity score >95% and E value <0.01. Relative abundance of cultured bacteria from secondary lymphoid organs and tumor tissue recovered from C57BL/6J mice (n=6-8) bearing melanoma tumors and receiving anti-PD-1 and anti-CTLA-4 antibody treatment, as described in fig 1A. Tissue homogenates were serially diluted in reduced PBS and plated on YCFA agar and incubated for 24-72 hours at 37℃ under anaerobic conditions. Colony-forming units (CFUs) were counted. Representative colonies based on morphology (~10-20 for each morphology type) were picked and cultured in YCFA medium for additional 24-48 hours at 37℃ under anaerobic conditions. gDNA was isolated from bacterial cultures. Full-length 16s rRNA gene (V1-V9 region) was amplified, purified, and sequenced (Sanger sequencing). Sequences were entered into the NCBI standard nucleotide Basic Local Alignment Search (BLAST) tool utilizing the rRNA/ITS databases. Bacterial species identification was ascertained from BLASTN results with the highest Total Score, with percent identity score >95% and E value <0.01.

Figure S3. Principal coordinate analysis of tissue and gut microbiome of mice bearing melanoma tumor and receiving ICT
Principal coordinate analysis of tissue and gut 16S rRNA sequencing data (as detailed in fig. 1), weighted and normalized by Bray-Curtis distances. The proportion of variance accounted by each principal component is indicated. Vector analysis (as indicated by arrows) performed by singular value decomposition of 16S rRNA sequencing data interpreted visually as a linear biplot.

Figure S18. Comparison of dendritic cell microbiomes from mice bearing melanoma tumors and treated with or without anti-PD-1 and anti-CTLA-4 antibody treatment.
Microbiome composition determined by analysis of 16S rRNA sequencing (V4 region) of dendritic cells recovered from MLNs in C57BL/6J mice bearing melanoma tumors treated with or without anti-PD-1 and anti-CTLA-4 antibody treatment (ICT). Differential bacterial taxonomic abundance between groups was analyzed by linear discriminant analysis effect size (LEfSe) projected as histograms. All listed bacterial groups (phylum (p), class (c), order (o), family (f), or genus (g)) were significantly enriched (>2 log-fold increase in linear discriminate analysis, LDA, score and P<0.05, Kruskal-Wallis test).

Figure S19. Bacterial levels in tumors of wildtype and CD11c-dtr mice after intra-MLN bacterial injection.
CD11c-dtr mice (female, 6-8 wks, Jackson) were injected with 100 ng diphtheria toxin (DT) intraperitoneally on day 3 post tumor implantation to deplete CD11c+ DCs. Wild-type C57BL/6 and DT-treated CD11c-dtr mice were implanted with B16-F10 tumor. Mice with comparable tumor volumes were randomized before the ICT. 3 doses of ICT or isotype controls were injected intraperitoneally. 1x10 7 GFP+ E. coli was injected directly into the MLN. Tumor tissue was collected 24 hours post E. coli injection. Tumor homogenates were spread on TSA-Kanamycin Agar plates. GFP+ colonies were enumerated after 24 hours of incubation at 37℃. n=3 per group. Squares represent values from individual mice. Bars represent the mean + SEM. Statistical analysis by t-test. *P<0.05. Figure S20. Secreted cytokine profiling of MLN CD8+ T cells isolated from mice + antibiotics C57BL/6 mice (female, 6-8 wks, Jackson) were treated + antibiotics (ABX, 2 mg/ml streptomycin and 1500 U/ml penicillin G in drinking water) for 7d before B16-    C57BL/6 mice (female, 6-8 wks, Jackson) were treated + antibiotics (ABX, 2 mg/ml streptomycin and 1500 U/ml penicillin G in drinking water) for 7d before B16-F10 tumor inoculation. Mice were treated with 200µg anti-PD-1 and 200µg anti-CTLA-4 mAb intraperitoneally on days 4, 8, and 12 after tumor implantation. n=10 mice per group. Mice were sacrificed on day 13 and CD11c+ dendritic cells isolated from 5 mice were pulled into one sample for the downstream cytokine secretion profiling (two pooled samples per group). Isolated CD11c+ dendritic cells were then stimulated with PMA and Ionomycin for 6 hours and supernatants were collected for multiplex cytokine profiling (Isoplexis IsoSpark; 16-plex mouse inflammation Codeplex chip panel). Bars represent the mean + SEM. Statistical analysis by one-way ANOVA with Fisher's Least Significant Difference (LSD) test. Figure S24. Oral administration of live Enterococcus faecalis and Escherichia coli induces melanoma tumor volume reduction in mice treated with ICT C57BL/6 mice (female, 6-8 wks, Jackson) were treated with antibiotics (ABX, 2 mg/ml streptomycin and 1500 U/ml penicillin G in drinking water) for 7 days. Mice were then inoculated with 1 × 10 5 B16-F10 cells subcutaneously in the right flank and orally gavaged with a total of 2 x 10 8 CFU live bacteria every two days: