Animals and tumor model

All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Sanford Burnham Prebys Medical Discovery Institute (approval # 13–130, 16–028, and 17–001) and complied with all relevant ethical regulations for animal testing and research. WT and Rnf5−/− (originally generated in ref. 43) were generated from common Rnf5+/− heterozygous, which were obtained from at least 10 generations backcross to WT C57BL/6 mice and were bred in house for several generations. All experiments used littermate controls, or their immediate descendants. MyD88−/− mice were obtained with permission as gift from Dr. Shizuo Akira44. Rnf5−/− and MyD88−/− (C57BL/6) were crossed to generate MyD88+/−Rnf5+/− heterozygous, and MyD88−/−Rnf5−/− double-knockout mice were generated from MyD88+/−Rnf5+/− heterozygous. In total, 6–14-week-old mice were used for all experiments12. Germ-free C57BL/6 mice and ASF-bearing C3H/HeN mice were bred and maintained at the University of Nebraska-Lincoln (UNL) Gnotobiotic Mouse Facility under gnotobiotic conditions in flexible film isolators. Experiments involving GF and gnotobiotic mice were approved by the Institutional Animal Care and Use Committee (IACUC) at UNL (protocol #1534). All mice were fed an autoclaved chow diet ad libitum (LabDiet 5K67, Purina Foods). Germ-free status was routinely checked as previously described45. Briefly, fresh feces were collected and analyzed by bacterial 16S rRNA gene-specific PCR (30 cycles, universal bacteria primers 8F and 1391R)46 in combination with aerobic and anaerobic culture of feces in Brain Heart Infusion, Wilkins-Chalgren and Yeast Mold broths, and on Tryptic Soy Agar plates (all media from Difco™ Becton Dickinson) at 37 °C for 7 days. ASF colonization status was verified by qPCR analysis of fecal samples as previously published45. Briefly, genomic DNA was extracted from fecal samples and ASF bacteria were quantified by qPCR with species-specific primers. Mouse selection for experiments was not formally randomized or blinded. For tumor growth experiments, mice were injected subcutaneously (s.c.) with 1 × 106 tumor cells. Tumor size was measured twice a week for calculation of tumor volume. Tumors were weighed at the time of excision.

Cell lines and gene silencing

Mouse melanoma cell lines used in this study include YUMM1.5, YUMM1.3, YUMM1.7, YUMM1.9 (Marcus Bosenberg, Yale University)12, B16F10 (ATCC), and B2905 (Glenn Merlino, NCI), and the mouse intestinal epithelial cell line MODE-K (Richard Blumberg Harvard Medical School), were cultured in the DMEM containing 10% FBS. All cell lines were free of mycoplasma and were authenticated. Cells were transfected with shRNAs using jetPRIME (PolyPlus). The RNAi Consortium lentiviral pLKO.1 control vector served as the shControl. The shRNAs were purchased from Sigma-Aldrich. Lentiviral particles were prepared using standard protocols. Briefly, shRNA plasmid and the second generation of packaging plasmids delta R8.2 and VSV-G (Addgene) were transfected into HEK293T cells. Viral supernatants were collected after 48 h of culture and used with polybrene (Sigma) for infection of indicated cell line.

Quantitative real-time PCR amplification of 16S rRNA gene sequences

Fecal samples were weighed and bacterial DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen). The abundance of specific bacterial genes was amplified by qPCR with genera-specific 16S rRNA gene primers (Supplementary Table 6). The qPCR program started with an initial step at 95 °C for 5 min, followed by 35 cycles of 30S at 95 °C and 45S at 55 °C. The qPCRs were done using SYBR green supermix (Bio-Rad). Bacterial numbers were determined using standard curves with reference bacteria E. coli (ATCC). Standard curves were generated by qPCR using V3 primer pairs together with undiluted and a 10-fold dilution series of genomic template. The Ct values of each dilution were used to generate a standard curve of absolute abundance. qPCR measures the number of 16S rRNA gene copies per sample, not actual bacterial numbers.

Bacterial strains and culture

The closest available bacterial strains matching those observed in Rnf5−/− mice were identified. The 12 strains selected for administration as a cocktail in GF mice are shown in Supplementary Table 2. Lyophilized bacteria were resuspended and cultured anaerobically in the respective media formulations suggested by the vendors. Equal concentrations of each species, as measured by OD600, were pooled to make the bacterial cocktail. The Altered Schaedler Flora consisted of the following eight isolates: ASF 356, Clostridium sp.; ASF 360, Lactobacillus intestinalis; ASF 361, Lactobacillus murinus; ASF 457, Mucispirillum schaedleri; ASF 492, Eubacterium plexicaudatum; ASF 500, Pseudoflavonifractor sp.; ASF 502, Clostridium sp.; and ASF 519, Parabacteroides goldsteinii.

Bacterial administration to germ-free mice

A cocktail of the bacterial species listed in Supplementary Table 2 or B. rodentium alone, was resuspended in PBS and administered via two oral gavages 1 day apart. Two weeks before tumor inoculation, each mouse was given 100 µl containing either a bacterial cocktail of 1.13 × 107 organisms or 2.25 × 107 B. rodentium alone along with 100 µl of cecal contents from ASF-bearing mice prepared as previously described. ASF cecal contents were harvested from ASF-bearing mice after euthanasia and homogenized in sterile 10% glycerol in phosphate-buffered saline (PBS) without calcium and magnesium, pH 7 (Corning Cellgro, Manassas, VA) at a ratio of 1-g cecal contents per 10 mL of 10% glycerol in PBS under rigorous vortexing. Inoculations were performed under sterile aerobic conditions in a biosafety cabinet.

Bacterial DNA extraction and 16S library preparation

Mouse microbiota displays a stable homeostatic state when they are around 8 weeks47. In following this protocol, we did not collect microbiota earlier than 8 weeks. Mouse fecal pellets were frozen on dry ice, and stored at −80 °C. Bacterial DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen). To ensure efficient cell lysis, a 5-min bead-beating step using a Mini-Beadbeater-16 was included (Biospec Products, OK, USA). Library preparation for the Illumina MiSeq platform was performed by amplification of the V3–V4 region of the bacterial 16S ribosomal DNA gene using Forward primer: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′ and reverse primer: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GACTACHVGGGTATCTAATCC-3′. Adapter and barcode sequences for dual indices were used as described by Illumina. PCR clean up steps were performed with QIAquick 96-PCR Clean up kit (Qiagen, Germany), and library quantification was performed using a KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, MA, USA). An Experion Automated Gel Electrophoresis System (Bio-Rad, CA, USA) was used to measure the DNA concentration and purity of the pooled libraries. The 16S libraries were sequenced at the Analytical Genomics Core of Sanford Burham Prebys Medical Discovery Institute (Lake Nona, FL, USA) and Beijing Genomics Institute (Beijing, China).

16S sequencing data processing

The original FASTQ files from Illumina 250 basepair paired-end sequencing of the 120 samples (30 mice per group) were processed using a novel 16S amplicon sequencing pipeline HiMap (http://github.com/taolonglab/himap; bioRxiv 565572). The output of HiMap is Operational Strain Unit (OSU) which contains one or more bacterial strains that best match the 16S sequence and can not be further distinguished by it. The percentage similarity between the 16S sequence and the aligned region of 16S rRNA genes of the strains in the OSU is indicated. OSUs mapped to the same strains are grouped together (adding read counts), if the percentage similarities are within 2%. Read counts are converted into relative abundance as described in HiMap. Log10-transformed relative abundances were used for comparisons between samples under different experimental conditions.

Taxa selection

Taxa that distinguished Rnf5−/− mice microbiota from WT mice were selected as the overlap of the following three sets: (1) for each time point at day 0 (before tumor injection) and at day 24 (before tumor collection), Welch two-tailed t test was performed on the log10-transformed relative abundances of all OSU groups at the specified time point, and p-values were adjusted with Benjamini–Hochberg correction for multiple comparisons. OSU groups with adjusted p-value < 0.05 at both day 0 and day 24 were selected in this first set; (2) Wilcox rank-sum test was performed on relative abundances of all OSU groups at day 0 and day 24. OSU groups with adjusted p-value less than 0.05 at both day 0 and day 24 were selected in the second set; (3) the third set of OSU groups were selected by calculating spearman correlation between each of the OSU groups and tumor size at day 24 and keeping OSU groups with adjusted p-value <0.05. Initial OSU groups used in the above three tests are the ones with median relative abundance greater than 10−4 in at least one of the four groups: WT at day 0, Rnf5−/− at day 0, WT at day 24, and Rnf5−/− at day 24. The final set consists of 38 OSU groups that distinguished Rnf5−/− mice microbiota from WT mice (Supplementary Table 1, Supplementary Figure 3C, D).

In vivo antibody treatments

CD4+ or CD8+ T cells were depleted by intraperitoneal (i.p.) injection of 200 μg of anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), rat IgG2b isotype control, or rat IgG2a isotype control on days 0, 3, 6, 11, and 16 following tumor inoculation. The efficacy of depletion was confirmed by FACS analysis of blood samples. For anti-CTLA-4 or anti-PD-1 antibody treatment, mice were injected i.p. with 200 μg of anti-PD-1 (clone RMP1–14), anti-CTLA-4 (clone 9H10), Syrian hamster IgG isotype control (clone SHG-1), or rat IgG2a isotype control on days 7, 10, 13, and 16 after tumor inoculation. All mAbs for in vivo use were GoInVivo™ grade from BioLegend (San Diego, CA, USA).

Mouse melanoma model subjected to anti-CTLA4 treatment

B2905 cells generated in Glenn Merlino Laborartory (NCI) were derived from a UV-induced melanoma in Hgf-tg mouse. Cells (1 × 106) were subcutaneously implanted in the right flank of C57BL/6 mice. When tumors reached 75 mm3 in average, mice were randomized and anti-CTLA-4 (BioXCell, BE0164) or isotype control (BioXcell, BE0086) antibodies were administered i.v (final dose of 10 mg/kg). Treatment was done twice per week for four doses. Tumors were measured twice per week and collected 39 days post implantation when growth kinetics distinguished “responders” and “non-responders” to anti-CTLA-4.

Bone marrow chimeras

WT or Rnf5−/− recipient mice were lethally irradiated (1000 rads) and reconstituted by intravenous (i.v.) injection of 1 × 107 bone marrow (BM) cells isolated from the femurs and tibias of donor WT or Rnf5−/− mice. The recipient mice were treated with antibiotics (trimethoprim 8 mg/ml and sulfamethoxazole 40 mg/ml in the drinking water) for 3 weeks after injection. Reconstitution was confirmed 6–8 weeks after BM transfer, and the chimeric mice were then injected subcutaneously with 1 × 106 YUMM1.5 melanoma cells.

Tumor digestion

Tumors were excised, minced, and digested with 1 mg/ml collagenase D (Roche) and 100 µg/ml DNase I (Sigma) at 37 °C for 1 h. Digests were then passed through a 70-μm cell strainer to generate a single-cell suspension. The cells were washed twice with PBS containing 2 mM EDTA, and then stained for flow cytometry.

Flow cytometry

Tumor-derived single-cell suspensions were washed twice with FACS staining buffer, fixed for 15 min with 1% formaldehyde in PBS, washed twice, and resuspended in FACS staining buffer.

For intracellular cytokine staining, cells were resuspended in complete RPMI-1640 (containing 10 mM HEPES, 1% nonessential amino acids and L-glutamine, 1 mM sodium pyruvate, 10% heat-inactivated fetal bovine serum (FBS), and antibiotics) supplemented with 50 U/mL IL-2 (NCI), 1 mg/mL brefeldin A (BFA, Sigma), and incubated with phorbol myristate acetate (10 ng/ml) and ionomycin (0.5 μg/ml) for 5 h at 37 °C. The cells were then fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences) before staining.

Antibodies to the following proteins were obtained: CD45.2 (104), CD8α (53–6.7), CD4 (GK1.5), CD44 (IM7), PD-1 (RMP1–30), LAG-3 (C9B7W), TIM-3 (RMT3–23), CD45.1 (A20), TNF-α (MP6-XT22), IFN-γ (XMG1.2), CD11c (N418), CD11b (M1/70), MHC class II (M5/114.15.2), CD86 (GL1), CD40 (1C10), CD80 (16–10A1), PDCA (129c1), and B220 (RA3–6B2) were from BioLegend; antibodies to IL-2 (JES6–5H4) and MHC class I (AF6–88.5.5.3) were from eBioscience. All data were collected on an LSRFortessa (BD Biosciences) and analyzed using FlowJo Software (Tree Star). Gating strategies for immune cells analysis and cell sorting were provided in Supplementary Figure 8A–E.

Histology and immunohistochemistry

Immediately after killing, the entire gastrointestinal tract was removed from the mice, split open lengthwise, rinsed, and rolled up from the proximal to distal end to form a “Swiss roll.” The tissues were fixed in 4% formalin overnight at 4 °C, washed with PBS, and embedded in paraffin. The embedded samples were sliced into 5-μm-thick sections and stained with H&E. For quantitative analysis, the villi length and the crypt depth were scored for each section. The score distributions between experimental groups were compared using proportional odds logistic regression using R software.

For immunohistochemistry, the tissue sections were deparaffinized, rehydrated, washed in PBS, subjected to antigen retrieval using Dako target retrieval solution, and incubated with 3% hydrogen peroxide for 30 min to quench endogenous peroxidase activity. The sections were then incubated overnight at 4 °C with an antibody to BiP (C50B12, Cell Signaling Technology) diluted 1:100 and BiP (ab21685, abcam) dilute 1:200 in Dako antibody diluent. Slides were then washed three times with PBS/Tween-20, incubated with Dako Labeled Polymer-HRP for 1 h at room temperature, washed again with PBS/Tween-20, developed with DAB, Bajoran Purple chromogen kit (BioCare) and with simple stain AEC solution (Histofine) and counterstained with hematoxylin. For mouse tissues, BiP staining was scored using a four tier intensity scale from 0 to 3 (low to high intensity) multiplied by the percentage of positively stained cells to generate an H score (maximum score of 300). For human melanoma tissues, specimens were scored as IHC 0, 1, 2, 3, or 4, if negative, ≥1% but <25%, ≥25% but <50%, ≥50% but <75%, or ≥75% of cells were BiP positive, respectively. All stained tissues were blindly evaluated by pathologists.

Antibiotic treatments

Mice were treated for 2 weeks before tumor inoculation with ampicillin (1 mg/ml), neomycin (1 mg/ml), vancomycin (0.5 mg/ml), and metronidazole (1 mg/ml) (all Sigma-Aldrich) added to sterile drinking water. Solutions and bottles were changed 2–3 times per week.

Western blotting

Cells were washed once with PBS at room temperature and resuspended in RIPA buffer (PBS containing 1% NP-40, 1% sodium deoxycholate, 1% SDS, 1 mM EDTA, and phosphatase and protease inhibitors). Lysates were sonicated on ice with a Microtip sonicator, centrifuged, and the supernatants were removed and subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Osmonics Inc., MN, USA). Membranes were blocked and incubated with the respective primary antibodies followed by Alexa Fluor-conjugated secondary antibodies. The blots were imaged with an Odyssey detection system (Amersham Bioscience, NJ, USA). All uncropped blots are shown in Supplementary Figure 9.

Antibodies and chemicals

The anti-RNF5 antibody was described previously48. Antibodies to IREα (3294) and sXBP1 (12782) were from Cell Signaling Technology, and antibodies to pIREα (ab48187) and XBP1 (ab28715) were from Abcam. Brefeldin A (BFA) and thapsigargin (TG) were purchased from Sigma-Aldrich. IRE1 inhibitor (MKC-4485) was kindly provided by Dr. John Patterson.

Bone marrow-derived macrophages

Mice were euthanized with CO 2 , the femurs were removed, and the BM cells were harvested and washed. For macrophage differentiation, BM cells were resuspended in the RPMI-1640 medium containing 10% FBS, penicillin/streptomycin, and 2 mM L-glutamine and placed in Petri dishes. The supernatant from L929 cells (a source of macrophage-colony stimulating factor, M-CSF) was added at 30% (vol/vol), and the cells were incubated for 7 days. Differentiated macrophages were then harvested and used in the experiments.

MODE-K cells stimulation with B. rodentium

MODEK-K cells were harvested, washed, and stimulated for 4 h with the growth medium alone or with medium containing rehydrated B. rodentium at a final ratio of 1:10 MODE-K cells to bacterial cells.

BMDC activation and in vitro CD8+ T cell stimulation assay

To prepare conditioned media, MODE-K-EV or MODE-K-shRNF5 cells were incubated in DMEM supplemented with 10% FBS for 24 h at 37 °C to allow cell attachment. The cells were then treated with medium or MKC-4485 for 48 h in advanced DMEM (Thermo Fisher) containing 2 mM L-glutamine without serum. The medium was then collected and centrifuged at 2000 × g for 10 min at 4 °C to remove cellular debris. The resulting conditioned medium was used for treatment of BMDCs.

BM cells were isolated from the tibiae and femurs of WT C57BL/6 mice and cultured in the DMEM medium containing 10% FBS, 1% penicillin/streptomycin, and recombinant mouse GM-CSF (20 ng/ml; BioLegend) for 8 days at 37 °C. BMDCs were incubated for 24 h with the MODE-K conditioned medium (20% v/v) prepared as described above. OT-1 or P14 CD8+ T cells were incubated with 5 μM CFSE for 10 min at 37 °C and washed. Cells were then mixed 1:1 with the BMDCs and incubated for 72 h pulsed with 2 μg/ml of OVA peptide (SINFEKL) (AnaSpec) or GP33 peptide (AnaSpec). T-cell proliferation was monitored by CFSE dilution using flow cytometry and the division index (a measure of the average number of divisions which includes the undivided cells) was determined using FlowJo software (Tree Star). For intracellular cytokine staining, the cells were fixed, permeabilized with Cytofix/ Cytoperm (BD Biosciences), and stained with anti-TNF-α and anti-IFN-γ.

Patient samples

Patients with melanoma provided written informed consent for the collection of tissue and blood samples for research and genomic profiling, as approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board (DF/HCC Protocol 11–181), and the Kantonal Ethics Committee of Zurich (EK.647/800 & ZH.Nr.2014–0425) and Rambam Health Care Campus Institutional Review Board (RMB-0634–16131539). Fresh primary tumor specimens were obtained from patients prior to immunotherapy administration (see Supplementary Tables 3–5). Formalin-fixed tissue was analyzed to confirm that viable tumor was present via hematoxylin and eosin (H&E) staining and was used for immunohistochemical analysis and purification of RNA. Additional fresh tissue was processed immediately for purification of RNA. Of the MGH/Dana-Farber Cancer Center cohort, 36 of the 40 patients received immunotherapy for metastatic stage IV melanoma, 4 patients were given adjuvant therapy after definitive surgical resection. All patients classified as responders (R) showed clear radiographic decrease in disease and maintained an ongoing response without progression through to last follow-up. Patients classified as nonresponders (NR) did not respond to treatment radiographically and/or had clear and rapid progression. In the case of the five patients who received adjuvant therapy, R was defined as lack of post-treatment recurrence through to last follow-up. From the Zurich hospital cohort of 25 patients, all received immunotherapy for metastatic stage IV melanoma. All patients classified as R showed a reduction of tumor lesions within the first 12 weeks of treatment; whereas, NR had no measureable response to treatment at the first clinical end-point (12 weeks), as in Krieg et al.49. From the Rambam Health Care Center a cohort of 21 patients, all with metastatic melanoma received anti-PD-1 immunotherapy as a single agent, first-line systemic therapy was included in the study. In eight patients, more than one biopsy was examined. Response was assessed by a board certified oncologist (G.BS) who was blinded to the immunohistochemical results. All patients classified as responders (R) showed radiographic decrease of disease and maintained an ongoing response without progression through to last follow-up. Patients classified as nonresponders (NR) did not respond to treatment radiographically and/or had clear and rapid progression. For survival evaluation, the medical files of the patients were reviewed and tumor assessmemts were conducted according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, before treatment and then every 11–14 weeks until disease progression or death.

RNA extraction and qRT-PCR analyses

The total RNA was extracted from mouse tumor samples individually using the RNeasy Fibrous Tissue Midi kit (QIAGEN), cells using GenElute (Sigma-Aldrich) or formalin-fixed paraffin-embedded (FFPE) patient samples using GenEluteTM (Sigma-Aldrich) FFPE RNA purification kit. Fresh patient tumor samples were homogenized and disrupted using a mortar and pestle followed by use of a QIAshredder. A QIAcube was used to harvest RNA from patient biopsies using the RNeasy Mini Protocol (Qiagen). The total RNA was reverse transcribed using high Capacity Reverse Transcriptase kits (Invitrogen) or the Superscript VILO cDNA Synthesis Kit (Invitrogen) according to the protocol by the manufacturer. Purity and concentration of extracted RNAs were checked and quantified by reading at 260 and 280 nm in a NanoDrop spectrophotometer (Thermo Fisher).

The qRT-PCR analyses were performed using Syber Green RT-PCR kits (Invitrogen) on a Bio-Rad CFX Connect Real-Time system or Roche LightCycler. Expression levels normalized to 18S or Tubb5 controls. Sequence-specific primers used in this study are shown in Supplementary Table 6.

RNF5 gene deletion by CRISPR/Cas9 technology

RNF5-deficient cells were created using the CRISPR/Cas9 system50. A transient strategy was used to avoid nonspecific effects due to stable Cas9/sgRNA genome integration. YUMM1.7 cells were transiently transfected with a Cas9 and single-guide RNA (sgRNA) expression plasmid encoding GFP (Addgene plasmid #44719). The guide sequence was designed using the Optimized CRISPR Design at http://crispr.mit.edu: 5′-CGCTCGCGATTTGGCCCTTC-3′. After transfection, GFP-positive cells were sorted by FACS, cloned, and screened for Rnf5 deletion by immunoblot analysis. Independent knockout clones and the control parental cells were analyzed as indicated.

Isolation of intestinal epithelial cells

A 10 -cm section of mouse small intestine was opened longitudinally, minced, washed in 150 mM NaCl containing 1 mM DTT, and then resuspended in dissociation buffer (130 mM NaCl, 10 mM EDTA, 10 mM Hepes [pH 7.4], 10% FCS, and 1 mM DTT). The sections were incubated at 37 °C for 30 min with vigorous shaking to release the epithelial cells from the lamina propria. The epithelial cell suspension was then carefully aspirated, centrifuged, and washed in ice-cold PBS.

Serum cytokine and chemokine detection

Cytokines and chemokines in the sera of tumor-bearing WT and Rnf5−/− mice were quantified using the LEGENDplexTM mouse inflammation panel and mouse proinflammatory chemokine panel (BioLegend), respectively. All data were collected on an LSRFortessa (BD Biosciences) and analyzed using LEGENDplexTM software (BioLegend).

NanoString nCounter assay

For each NanoString assay, an aliquot of 100 ng RNA was mixed with a NanoString code set mix and incubated at 65 °C overnight (16 h). The reaction mixes were loaded onto the NanoString nCounter Prep Station for binding and washing, and the resulting cartridge was transferred to the NanoString nCounter digital analyzer for scanning and data collection. Quantified expression data were analyzed using NanoString nSolver Analysis Software v2.0. After performing image quality control using a predefined cutoff value, we excluded the outlier samples using a normalized factor based on the sum of positive control counts greater than threefold. Data were normalized by scaling with the geometric mean of the built-in control gene probes for each sample.

Bioinformatics analysis of the NanoString nCounter assay

For gene expression data from the NanoString nCounter assay, filtering of samples using quality control criteria was performed according to the manufacturer’s recommendations. Raw counts of samples passing quality control were normalized using 20 reference genes as internal controls (Abcf1, Alas1, Edc3, Eef1g, Eif2b4, G6pdx, Gusb, Hdac3, Hprt, Nubp1, Oaz1, Polr1b, Polr2a, Ppia, Rpl19, Sap130, Sdha, Sf3a3, Tbp, and Tubb5). Data were log2-transformed and used for further analysis. Student’s t test was applied to compare normalized expression values between groups. Ingenuity pathway analysis was used to interpret data in the context of biological processes, pathways, and networks.

In vivo OT-1 T-cell proliferation assay

CD8+ T cells were isolated from the spleens of naive OT-1 CD45.1+ mice, labeled with CFSE, and injected i.v. into CD45.2+ WT or Rnf5−/− mice (C57BL/6 background). After 24 h, the mice were injected s.c. with 1 × 106 B16-OVA melanoma cells and the mice were left for 7 days. The spleen, tumor-draining lymph nodes, and non-draining lymph nodes were harvested and analyzed by flow cytometry. The proliferation of OT-1 CD8+ T cells was assessed by analysis of CFSE dilution within the population of gated CD45.1+ CD8+ T cells.

Small intestine organoid culture

For small intestine organoid culture, crypt number was counted after isolation from tumor-bearing mice. A total of 500 crypts were mixed with 50 µl of Matrigel and 500 μl of organoid culture medium (Advanced DMEM/F12 containing 10 mM HEPES, 1× Glutamax, 1× N2 supplement, 1× B27 supplement, 50 ng/ml EGF, 1000 ng/ml R-spondin1, and 100 ng/ml Noggin).

Statistical analysis

Unless otherwise noted, all data are shown as the mean ± s.e.m. Before statistical analysis, data were subjected to the Kolmogorov–Smirnov test to determine distribution. Variance similarity was tested using an F test for two groups and Bartlett’s test for multiple groups. Two groups were compared using the two-tailed t test for parametric data or the Mann–Whitney U test for nonparametric data. Multiple groups were compared using one-way ANOVA with Tukey’s, Dunnett’s, or Bonferroni’s correction for parametric data or using the Kruskal–Wallis test with Dunn’s correction for non-parametric data. Tumor growth curves were analyzed using two-way ANOVA with Sidak’s, Tukey’s, or Bonferroni’s correction for multiple comparisons. Kaplan–Meier estimates and the log-rank test were used to analyze statistical differences in overall and progression-free survival between melanoma patients treated with immunotherapy, whose pre-treatment tumor biopsies showed low versus high melanoma cell expression of BiP.

Reporting Summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.