included from the following trials: Mel-lpi-Rx(NCTO1557114), and 2 Phase II trials at NYU combining ipilimumab with radiotherapy in either MM (NCTo1689974)or NSCLC (NCTO2221739). For memory T cell responses, blood samples were drawn from patients before and after 3 or 4 cycles of ipilimumab. High-throughput sequencing analyses (MiSeq Technology) of 16s rRNA gene amplicons in patients' feces pre-and post-injections of ilimumab (vo, VI, V2, V3)were performed according to n SC12-018; ID RCB: 2012 A01496-37 pilot study endpoints by gatC Biotech AG(Konstanz, Germany) Clinical studies GOLD: Prospective immunomonitoring study of patients with MM receiving four injections of ipilimumab every three weeks. Feces were collected before each ipilimumab injection. Feces samples were frozen at-80oC and sent for 16S rRNA gene sequencing analysis at GATC Biotech AG(Konstanz, Germany) MEL-IPI-RX: Phase I trial that combines ipilimumab and radiation therapy to assess the synergy between the two modalities. Dose escalation radiotherapy was administered with ipilimumab on week 1, 4, 7 and 10 weeks at 10mg/kg. Subsequently, a maintenance dose of ipilimumab was given every 12 weeks as long as the patient had a positive clinical response. Study code NCT01557114 MELANOMA ABSCOPAL TRIAL: Phase II randomized trial of ipilimumab versus ipilimumab plus radiotherapy in MM. Eligible patients had MM with at least 2 measurable sites of disease All patients were randomly assigned to receive ipilimumab 3mg/kg i.v. versus ipilimumab 3 mg/kg i v. plus fractionated radiotherapy to one of their measurable lesions. For patients assigned to the ipilimumab plus radiotherapy arm, ipilimumab treatment started after radiotherapy with a dose given on day 4 from the first radiotherapy fraction and repeated on days 25, 46, and 67 Response to treatment was evaluated at week 12 to assess clinical and radiographic responses in the non-irradiated measurable metastatic sites. Study code: NCTO1689974 IG CANCI BSCOPAL TRIAL: Phase II study of combined ipilimumab and radiotherapy in metastatic non-small cell lung cancer(NSCLC). Eligible patients had chemo-refractory metastatic NSCLC with at least 2 measurable sites of disease. Patients received ipilimumab 3mg/kg i.v., within 24 hrs of starting fractionated radiotherapy. Ipilimumab
3 included from the following trials: Mel-Ipi-Rx (NCT01557114), and 2 Phase II trials at NYU combining ipilimumab with radiotherapy in either MM (NCT01689974) or NSCLC (NCT02221739). For memory T cell responses, blood samples were drawn from patients before and after 3 or 4 cycles of ipilimumab. High-throughput sequencing analyses (MiSeq Technology) of 16S rRNA gene amplicons in patients’ feces pre-and post-injections of ipilimumab (V0, V1, V2, V3) were performed according to n° SC12-018; ID RCB: 2012- A01496-37 pilot study endpoints by GATC Biotech AG (Konstanz, Germany). Clinical studies. GOLD: Prospective immunomonitoring study of patients with MM receiving four injections of ipilimumab every three weeks. Feces were collected before each ipilimumab injection. Feces samples were frozen at -80°C and sent for 16S rRNA gene sequencing analysis at GATC Biotech AG (Konstanz, Germany). MEL-IPI-RX: Phase I trial that combines ipilimumab and radiation therapy to assess the synergy between the two modalities. Dose escalation radiotherapy was administered with ipilimumab on week 1, 4, 7 and 10 weeks at 10mg/kg. Subsequently, a maintenance dose of ipilimumab was given every 12 weeks as long as the patient had a positive clinical response. Study code: NCT01557114. MELANOMA ABSCOPAL TRIAL: Phase II randomized trial of ipilimumab versus ipilimumab plus radiotherapy in MM. Eligible patients had MM with at least 2 measurable sites of disease. All patients were randomly assigned to receive ipilimumab 3mg/kg i.v. versus ipilimumab 3 mg/kg i.v. plus fractionated radiotherapy to one of their measurable lesions. For patients assigned to the ipilimumab plus radiotherapy arm, ipilimumab treatment started after radiotherapy with a dose given on day 4 from the first radiotherapy fraction and repeated on days 25, 46, and 67. Response to treatment was evaluated at week 12 to assess clinical and radiographic responses in the non-irradiated measurable metastatic sites. Study code: NCT01689974. NON-SMALL CELL LUNG CANCER ABSCOPAL TRIAL: Phase II study of combined ipilimumab and radiotherapy in metastatic non-small cell lung cancer (NSCLC). Eligible patients had chemo-refractory metastatic NSCLC with at least 2 measurable sites of disease. Patients received ipilimumab 3mg/kg i.v., within 24 hrs of starting fractionated radiotherapy. Ipilimumab
was repeated on days 22, 43, and 64. Patients were re-imaged between days 81-88 and evaluated for responses in the non-irradiated measurable metastatic sites. Study code: NCT02221739 Mice. All animal experiments were carried out in compliance with French and European laws and regulations. Mice were used between 7 and 14 weeks of age. WT specific pathogen-free (SPF) C57BL/6J and BALB/c mice were obtained from Harlan(France)and Janvier(France) respectively, and were kept in SPF conditions at Gustave Roussy. C57BL/6 GF mice were obtained from Institut Pasteur and maintained in sterile isolators. 1-10- C57BL/6 mice and WT C57BL/6 control animals were kindly provided by Anne O Garra(National Institute for Medical Research, UK). NOD2, Il10,and NOD2-1110 mice(BALB/c background) were obtained from Institut Pasteur Lille C57BL/6 TIr2 mice were provided by Ivo Gompers Boneca(Institut Pasteur, Paris, France), TIr4 mice were bred and maintained in the animal facility of Gustave Roussy, Villejuif, France. Cell culture and reagents oVA-expressing mouse fibrosarcoma MCA205 cells, murine colon carcinoma MC38 cells, RET melanoma model (a transgene-enforced expression of the Ret protooncogene under the control of the metallothionein-1 promoter driving spontaneou melanomagenesis, kindly provided by Viktor Umansky)(class I MHC H-2, syngeneic for C57BL/6 mice)and mouse colon carcinoma CT26 cells(class I MHC H-2d syngeneic for BALB/c mice)were cultured at 37 C under 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum(FBS), 100 units/ml penicillin G sodium, 100 ug/ml streptomycin sulfate, 2mM L-glutamine, ImM sodium pyruvate and non-essential amino acid (from this point on referred to as complete RPMI-1640; all reagents from Gibco-Invitrogen Carlsbad, CA, USA). OVA-expressing MCA205 cells were selected in complete RPMI-1640 medium(as above) though supplemented with 50 ug/ml hygromycin B(Invitrogen, Life Technologies) Tumor challenge and treatment Mice were subcutaneously injected into the right flank with 1 x 10 MCA205-OVA or MC38 with 0.5 eT or with 0.2 x 10 CT26 tumor cell lines When tumors reached a size of 20 to 40 mm(day 0), mice were injected intraperitoneally(i p with 100ug of anti-CTLA-4 mAb(clone 9D9)or isotype control(clone MPCI1) Mice were
4 was repeated on days 22, 43, and 64. Patients were re-imaged between days 81-88 and evaluated for responses in the non-irradiated measurable metastatic sites. Study code: NCT02221739. Mice. All animal experiments were carried out in compliance with French and European laws and regulations. Mice were used between 7 and 14 weeks of age. WT specific pathogen-free (SPF) C57BL/6J and BALB/c mice were obtained from Harlan (France) and Janvier (France), respectively, and were kept in SPF conditions at Gustave Roussy. C57BL/6 GF mice were obtained from Institut Pasteur and maintained in sterile isolators. Il-10-/- C57BL/6 mice and WT C57BL/6 control animals were kindly provided by Anne O’Garra (National Institute for Medical Research, UK). NOD2-/-, Il10-/-, and NOD2-/-Il10-/- mice (BALB/c background) were obtained from Institut Pasteur Lille. C57BL/6 Tlr2-/- mice were provided by Ivo Gompers Boneca (Institut Pasteur, Paris, France), Tlr4-/- mice were bred and maintained in the animal facility of Gustave Roussy, Villejuif, France. Cell culture and reagents. OVA-expressing mouse fibrosarcoma MCA205 cells, murine colon carcinoma MC38 cells, RET melanoma model (a transgene-enforced expression of the Ret protooncogene under the control of the metallothionein-1 promoter driving spontaneous melanomagenesis, kindly provided by Viktor Umansky) (class I MHC H-2b , syngeneic for C57BL/6 mice) and mouse colon carcinoma CT26 cells (class I MHC H-2d , syngeneic for BALB/c mice) were cultured at 37°C under 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 2mM L-glutamine, 1mM sodium pyruvate and non-essential amino acids (from this point on referred to as complete RPMI-1640; all reagents from Gibco-Invitrogen, Carlsbad, CA, USA). OVA-expressing MCA205 cells were selected in complete RPMI-1640 medium (as above) though supplemented with 50 μg/ml hygromycin B (Invitrogen, Life TechnologiesTM). Tumor challenge and treatment. Mice were subcutaneously injected into the right flank with 1 × 106 MCA205-OVA or MC38, with 0.5 x 106 RET or with 0.2 × 106 CT26 tumor cell lines. When tumors reached a size of 20 to 40 mm2 (day 0), mice were injected intraperitoneally (i.p) with 100μg of anti-CTLA-4 mAb (clone 9D9) or isotype control (clone MPC11). Mice were
injected 5 times at 3-day intervals with 9D9, and tumor size was routinely monitored by means of a caliper. In order to evaluate the synergistic effect of vancomycin and anti-CTLA4, a prophylactic setting was established. In figure S21, anti-CTLA4 treatment started two days after umor inoculation. In experiments using anti-IL-12p40 mAb(clone C17.8, 500ug per mouse)or anti-Ly6G mAb(clone 1A8, 200ug per mouse), mAbs(or their isotype controls, clone 2A3 in both cases) were injected i.p. every 2 days starting from day 0 until the final anti-CTLA-4 injection. Anti-ICOS mAb(clone 17G9)or isotype control mAb(clone LTF-2)were injected at 250ug per mouse, at the same times as for anti-CTLA4 mAb. In order to deplete CD4+ and CD8+ cells, clone GK1.5 and clone 53.72. 1. or isotype control mAb were injected ip at 200ug/mice 4 days before anti-CTLA4 treatment. All mAbs for in vivo use were obtained from BioXcell(West Lebanon, NH, USA), using the recommended isotype control mAbs. The inhibitor of iNOS, N-monomethyl-L-arginine (L-NMMA; Sigma), was administered in PBS daily to mice i.p. at a dose of 2mg per mouse from the start of anti-CTLA4 treatment Antibiotic treatments. Mice were treated with antibiotics 2-3 weeks before tumor implantation and continued on antibiotics until the end of the experiment. A mix of ampicillin(1 mg/ml) streptomycin (5 mg/ml), and colistin(1 mg/ml)(Sigma-Aldrich), or vancomycin alone(0.25 mg/ml), or imipenem alone(0.25 mg/ml), or colistin alone(2.10U/ml)were added in sterile drinking water. Solutions and bottles were changed 2-3 times a week, or daily for experiments with imipenem. Antibiotic activity was confirmed by macroscopic changes observed at the level of caecum(dilatation) and by cultivating the fecal pellets resuspended in BHI+15% glycerol at 0.1g/ml on blood agar plates for 48h at 37C with in aerobic or anaerobic conditions Flow cytometry. Tumors, spleens and lymph nodes were harvested two days after the third injection of anti-CTLA-4 for antibiotics experiments or at the end of tumor growth(around day 20)for germ-free experiments. Excised tumors were cut into small pieces and digested in RPMI medium containing Liberase at ml(roche) and DNasel at 150UI/ml(Roche)for minutes at 37C. The mixture was uently passaged through a 100um cell strainer. 2 x 10 splenocytes(after red blood cells lysis), lymph nodes or tumor cells were preincubated with purified anti-mouse CD16/CD32(clone 93; eBioscience) for 15 minutes at 4C, before membrane staining. For intracellular staining, the FoxP3 staining kit(eBioscience) was used
5 injected 5 times at 3-day intervals with 9D9, and tumor size was routinely monitored by means of a caliper. In order to evaluate the synergistic effect of vancomycin and anti-CTLA4, a prophylactic setting was established. In figure S21, anti-CTLA4 treatment started two days after tumor inoculation. In experiments using anti-IL-12p40 mAb (clone C17.8, 500μg per mouse) or anti-Ly6G mAb (clone 1A8, 200μg per mouse), mAbs (or their isotype controls, clone 2A3 in both cases) were injected i.p. every 2 days starting from day 0 until the final anti-CTLA-4 injection. Anti-ICOS mAb (clone 17G9) or isotype control mAb (clone LTF-2) were injected at 250μg per mouse, at the same times as for anti-CTLA4 mAb. In order to deplete CD4+ and CD8+ cells, clone GK1.5 and clone 53.72.1. or isotype control mAb were injected ip at 200μg/mice 4 days before anti-CTLA4 treatment. All mAbs for in vivo use were obtained from BioXcell (West Lebanon, NH, USA), using the recommended isotype control mAbs. The inhibitor of iNOS, NG-monomethyl-L-arginine (L-NMMA; Sigma), was administered in PBS daily to mice i.p. at a dose of 2mg per mouse from the start of anti-CTLA4 treatment. Antibiotic treatments. Mice were treated with antibiotics 2-3 weeks before tumor implantation and continued on antibiotics until the end of the experiment. A mix of ampicillin (1 mg/ml), streptomycin (5 mg/ml), and colistin (1 mg/ml) (Sigma-Aldrich), or vancomycin alone (0.25 mg/ml), or imipenem alone (0.25 mg/ml), or colistin alone (2.103 U/ml) were added in sterile drinking water. Solutions and bottles were changed 2-3 times a week, or daily for experiments with imipenem. Antibiotic activity was confirmed by macroscopic changes observed at the level of caecum (dilatation) and by cultivating the fecal pellets resuspended in BHI+15% glycerol at 0.1g/ml on blood agar plates for 48h at 37°C with in aerobic or anaerobic conditions. Flow cytometry. Tumors, spleens and lymph nodes were harvested two days after the third injection of anti-CTLA-4 for antibiotics experiments or at the end of tumor growth (around day 20) for germ-free experiments. Excised tumors were cut into small pieces and digested in RPMI medium containing LiberaseTM at 25μg/ml (Roche) and DNase1 at 150UI/ml (Roche) for 30 minutes at 37°C. The mixture was subsequently passaged through a 100μm cell strainer. 2 × 106 splenocytes (after red blood cells lysis), lymph nodes or tumor cells were preincubated with purified anti-mouse CD16/CD32 (clone 93; eBioscience) for 15 minutes at 4°C, before membrane staining. For intracellular staining, the FoxP3 staining kit (eBioscience) was used
Dead cells were excluded using the Live/Dead Fixable Yellow dead cell stain kit(Life TechnologiesM) Stained samples were run on a Canto II(BD Bioscience, San Jose, CA, USA) cytometer, and analyses were performed with Flow Jo software(Tree Star, Ashland, OR, USA) For cytokine staining, cells were stimulated for 4 hrs at 37C with 50ng/ml of phorbol 12 myristate 13-acetate(PMA; Calbiochem), lug/ml of ionomycin( Sigma), and BD Golgi STOP (BD Biosciences). Anti-CD45.2(104), anti-FoxP3(FJK-16s), anti-ICOS(7E17G9), anti-IFN-Y (XMG1. 2), anti-TNF-a(MP6-XT22), anti-CXCR3( CXCR3-173)and isotype controls rat IgGl (eBrGl), IgG2a(eBRG2a), IgG2b(eBRG2b) were purchased from eBioscience. Anti-CD3 (145-2C11), anti-CD25(PC61.5.3), KI67(FITC mouse anti-human KI67 set), rat IgGlK were obtained from BD Bioscience. Anti-CD4(GK1.5), anti-CD8B (YTS1567.7), Rat IgG2a (RTK2758)were purchased from Biolegend(San Diego, CA, USA). Anti-CCR6(140706)was obtained from R&D Systems, Minneapolis, MN. Eight-color flow cytometry analysis was performed with antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, phycoerythrin cyanin 7, peridinin chlorophyll protein cyanin 5.5, allophycocyanin cyanin 7, pacific blue, or allophycocyanin. All cells were analyzed on a FACS CANTO II(BD) flow cytometer with FlowJo(Tree Star) software Microbial DNA extraction, 454 pyrosequencing and bacteria identification. Fecal samples used in this study were collected before or after one injection of anti-CTLA4 (or isotype control) from mice under vancomycin regimen or water, and were kept at -80oC until further analysis Library preparation and sequencing were conducted at GATC Biotech AG(Konstanz, Germany) Bacterial isolation, culture, and identification. Fecal pellet contents were harvested and suspended in BHI+15% glycerol at 0.1g/ml. Serial dilutions of feces were plated onto sheep's blood agar plates and incubated for 48h at 37C with 5%CO2 in aerobic or anaerobic conditions After 48h, single colonies were isolated and Gram staining was performed. The identification of specific bacteria was accomplished through the combination of morphological tests and analysis by means of an Andromas MALDI-TOF mass spectrometer(Andromas, France) Gut colonization with dedicated bacterial species. For inoculation of gF mice or mice treated bad-spectrum antibiotics, colonization was performed the day following the first anti- CTLA4 injection by oral gavage with 100ul of suspension containing 1 x 10 bacteria. Efficient
6 Dead cells were excluded using the Live/Dead Fixable Yellow dead cell stain kit (Life TechnologiesTM). Stained samples were run on a Canto II (BD Bioscience, San Jose, CA, USA) cytometer, and analyses were performed with FlowJo software (Tree Star, Ashland, OR, USA). For cytokine staining, cells were stimulated for 4 hrs at 37°C with 50ng/ml of phorbol 12- myristate 13-acetate (PMA; Calbiochem), 1μg/ml of ionomycin (Sigma), and BD Golgi STOP TM (BD Biosciences). Anti-CD45.2 (104), anti-FoxP3 (FJK-16s), anti-ICOS (7E17G9), anti-IFN-Ȗ (XMG1.2), anti-TNF-Į (MP6-XT22), anti-CXCR3 (CXCR3-173) and isotype controls rat IgG1 (eBRG1), IgG2a (eBRG2a), IgG2b (eBRG2b) were purchased from eBioscience. Anti-CD3 (145-2C11), anti-CD25 (PC61.5.3), KI67 (FITC mouse anti-human KI67 set), rat IgG1ț were obtained from BD Bioscience. Anti-CD4 (GK1.5), anti-CD8ȕ (YTS1567.7), Rat IgG2a (RTK2758) were purchased from Biolegend (San Diego, CA, USA). Anti-CCR6 (140706) was obtained from R&D Systems, Minneapolis, MN. Eight-color flow cytometry analysis was performed with antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, phycoerythrin cyanin 7, peridinin chlorophyll protein cyanin 5.5, allophycocyanin cyanin 7, pacific blue, or allophycocyanin. All cells were analyzed on a FACS CANTO II (BD) flow cytometer with FlowJo (Tree Star) software. Microbial DNA extraction, 454 pyrosequencing and bacteria identification. Fecal samples used in this study were collected before or after one injection of anti-CTLA4 (or isotype control) from mice under vancomycin regimen or water, and were kept at −80°C until further analysis. Library preparation and sequencing were conducted at GATC Biotech AG (Konstanz, Germany). Bacterial isolation, culture, and identification. Fecal pellet contents were harvested and resuspended in BHI+15% glycerol at 0.1g/ml. Serial dilutions of feces were plated onto sheep’s blood agar plates and incubated for 48h at 37°C with 5% CO2 in aerobic or anaerobic conditions. After 48h, single colonies were isolated and Gram staining was performed. The identification of specific bacteria was accomplished through the combination of morphological tests and analysis by means of an Andromas MALDI-TOF mass spectrometer (Andromas, France). Gut colonization with dedicated bacterial species. For inoculation of GF mice or mice treated with broad-spectrum antibiotics, colonization was performed the day following the first antiCTLA4 injection by oral gavage with 100μl of suspension containing 1 × 109 bacteria. Efficient
colonization was checked by culture of feces 48h post oral gavage. Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides distasonis, Bacteroides umiformis, Lactobacillus plantarum and Enterococcus hirae were grown on blood agar plates(Biomerieux) for 48h at 37C in anaerobic conditions. E. coli and Burkholderia cepacia were grown on blood agar plates for 24h at 37C in aerobic conditions. Bacteria were harvested from the agar plates, suspended in sterile PBS, centrifuged and washed once with PBS, then resuspended in sterile PBS at an optical density(600nm) of 1, which corresponds approximately to 1 x 10 colony-forming units (CFU)ml In cases where more than one bacteria was administered, an equal volume of each bacteria suspension was mixed to give a suspension of equal proportion of each type of bacteria to a total 1 x 10 bacteria/ml. For bacteria reconstitution experiments using mice previously treated with antibiotics, antibiotics treatment was stopped after 2-3 weeks at the first anti-CTLA4 injection, and mice were orally gavaged with 1 x 10 CFU the following day. B. distasonis, B niformis, E. hirae, and E coli isolates used in the experiments were originally isolated from feces or mesenteric lymph nodes of SPF mice treated with anti-CTLA4 and identified as described above. L. plantarum, B. fragilis and B. thetaiotaomicron were provided by the Biobank of the Institut Pasteur, Paris, France. Burkholderia cepacia was kindly provided by the IUH Mediterranee Infection, Marseille, France( Supplemental Table 2). LPS-EK and LTA-SA (Invivogen)were administrated by oral gavage, at a dose of 500ug per mouse TCR cross-linking assays. For cross-linking experiments, total cells isolated from draining or contralateral lymph nodes(after red blood cell lysis)were incubated in MaxiSorp plates(Nunc; 2 x 10 cells per well), precoated with anti-CD3 mAb(145-2Cll)(0.5 g per well; eBioscience The supernatants were assayed at 48h by ELISa for mouse IFN-Y(BD) Cytokine and antimicrobial peptide quantification. IL-12p70, IFN-Y(BD Biosciences)and IL-10(R&D Systems, Minneapolis, MN), were measured by ELisa following the manufacturers instructions. For quantification of lipocalin-2 from feces and caecum contents individual(not pooled) samples were reconstituted in PBS containing 0. 1% Tween 20(at 100 g/ml) and vortexed .20 min to get a homogenous fecal suspension. These samples were then centrifuged for 10 min at 10,000 rpm. Clear supernatants were collected and stored at -20C
7 colonization was checked by culture of feces 48h post oral gavage. Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides distasonis, Bacteroides uniformis, Lactobacillus plantarum and Enterococcus hirae were grown on blood agar plates (Biomerieux) for 48h at 37°C in anaerobic conditions. E. coli and Burkholderia cepacia were grown on blood agar plates for 24h at 37°C in aerobic conditions. Bacteria were harvested from the agar plates, suspended in sterile PBS, centrifuged and washed once with PBS, then resuspended in sterile PBS at an optical density (600nm) of 1, which corresponds approximately to 1 × 109 colony-forming units (CFU)/ml. In cases where more than one bacteria was administered, an equal volume of each bacteria suspension was mixed to give a suspension of equal proportion of each type of bacteria, to a total 1 × 109 bacteria/ml. For bacteria reconstitution experiments using mice previously treated with antibiotics, antibiotics treatment was stopped after 2-3 weeks at the first anti-CTLA4 injection, and mice were orally gavaged with 1 × 109 CFU the following day. B. distasonis, B. uniformis, E. hirae, and E.coli isolates used in the experiments were originally isolated from feces or mesenteric lymph nodes of SPF mice treated with anti-CTLA4 and identified as described above. L. plantarum, B. fragilis and B. thetaiotaomicron were provided by the Biobank of the Institut Pasteur, Paris, France. Burkholderia cepacia was kindly provided by the IUH Méditerranée Infection, Marseille, France (Supplemental Table 2). LPS-EK and LTA-SA (Invivogen) were administrated by oral gavage, at a dose of 500μg per mouse. TCR cross-linking assays. For cross-linking experiments, total cells isolated from draining or contralateral lymph nodes (after red blood cell lysis) were incubated in MaxiSorp plates (Nunc; 2 × 105 cells per well), precoated with anti-CD3 mAb (145-2C11) (0.5 g per well; eBioscience). The supernatants were assayed at 48h by ELISA for mouse IFN-γ (BD). Cytokine and antimicrobial peptide quantification. IL-12p70, IFN-γ (BD Biosciences) and IL-10 (R&D Systems, Minneapolis, MN), were measured by ELISA following the manufacturer’s instructions. For quantification of lipocalin-2 from feces and caecum contents, individual (not pooled) samples were reconstituted in PBS containing 0.1% Tween 20 (at 100 mg/ml) and vortexed for 10-20 min to get a homogenous fecal suspension. These samples were then centrifuged for 10 min at 10,000 rpm. Clear supernatants were collected and stored at -20°C