immunology.sciencemag.org/cgi/content/full/5/46/eaax0062/DC1

Supplementary Materials for

Gut-resident CX3CR1hi macrophages induce tertiary lymphoid structures and IgA

response in situ

Balázs Koscsó, Sravya Kurapati, Richard R. Rodrigues, Jelena Nedjic, Kavitha Gowda, Changsik Shin, Chetna Soni, Azree Zaffran Ashraf, Indira Purushothaman, Maryknoll Palisoc, Sulei Xu, Haoyu Sun, Sathi Babu Chodisetti, Eugene Lin,

Matthias Mack, Yuka Imamura Kawasawa, Pingnian He, Ziaur S. M. Rahman, Iannis Aifantis, Natalia Shulzhenko, Andrey Morgun, Milena Bogunovic*

*Corresponding author. Email: milena.bogunovic@umassmed.edu

Published 10 April 2020, Sci. Immunol. 5, eaax0062 (2020)

DOI: 10.1126/sciimmunol.aax0062

The PDF file includes:

Methods Fig. S1. Model of streptomycin-pretreated Salmonella-induced colitis in mice on a Salmonella-resistant background. Fig. S2. Flow cytometry gating strategy to identify MP populations in infected LB. Fig. S3. Response to Salmonella infection in mice lacking Mϕs or cDC2. Fig. S4. Gene expression profiles of inflammatory MP subsets. Fig. S5. Intestinal GC B cells express genes related to IgA class switch. Fig. S6. Response to Salmonella infection in mice after CD4+ T cell depletion. Fig. S7. Development strategy and characterization of Ccr7fl/fl and Lyz2:Ccr7–/– mice. Fig. S8. Role of CX3CR1+ Mϕ subsets in the induction of Salmonella-specific T cell responses. Fig. S9. Response to Salmonella infection in Ccr7–/– mice. References (99–104)

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/5/46/eaax0062/DC1)

Table S1. Parametric P values and geometric means of expression levels of cDC and Mϕ genes analyzed in fig. S1B (separate Excel file). Table S2. List of genes and pathways included in pathway analysis (separate Excel file). Table S3. Parametric P values and geometric means of expression levels of each pathway (separate Excel file). Table S4. Parametric P values and geometric means of expression levels of all genes in the analyzed pathways (separate Excel file). Table S5. Raw data file with P values (separate Excel file).

Methods

In vivo cell depletion experiments.

We previously established that mucosal Ms in the normal intestine express CSF1R, require

CSF1R signaling for their development, and are selectively depleted by CSF1R blocking

monoclonal antibody (mAb) with no significant effect on mucosal cDCs, granulocytes or

mononuclear phagocyte (MP) populations of the mesenteric lymph nodes (MLN) (38). Batf3–/–

mice were used as an established model lacking cDC1 (40). ItgaxCre

Notch2fl/fl

mice were used as

an established model deficient in cDC2 (36, 37).

To deplete monocytes mice were injected intraperitoneally with 20 μg of MC-21 CCR2 mAb

(97) or with control rat IgG on days 1, 2 and 3 of infection with Salmonella. Monocyte depletion

was confirmed in the blood.

To deplete CD4+ T cells, mice were injected intraperitoneally with 250 μg of CD4 mAb (clone

GK1.5, BioXCell, West Lebanon, NH) or with control rat IgG 5 days before infection, and the

injection was repeated every two days until infection with Salmonella. The injections were

repeated with the same dose twice every week during the course of the experiment. T cell

depletion was confirmed in the blood.

Tamoxifen treatment

To induce Cre expression, Cx3cr1CreER/WT

H2-Ab1WT/fl

or Cx3cr1CreER/WT

H2-Ab1fl/fl

mice were

treated with 0.125 mg/g of body weight of tamoxifen (Sigma) dissolved in corn oil by oral

gavage 7 days before infection. The treatment was repeated with the same dose on day 3 post-

infection (Fig. S9J). The model was adapted from previously published studies (66, 98) by

titrating down tamoxifen dose and treatment regimen to target only the CX3CR1hi

M

population.

Adoptive OT-II T cell transfer

For adoptive transfer experiments, OT-II mice were backcrossed to CD45.1+ background to

create congenic OT-II mice and bred with 129S1 mice. OT-II T cells were purified from spleens

and lymph nodes of mice from F1 generation of congenic B6 CD45.1+ OT-II mice crossed to

129S1 mice by negative selection using biotinylated CD11c, F4/80, CD19, NK1.1 and MHCII

mAbs (Thermo Fisher Scientific) and Streptavidin MicroBeads (Miltenyi Biotec, Auburn, CA).

Mouse strains backcrossed to Salmonella-resistant background as described above were used as

recipients. Recipient mice were anesthetized by ketamine/xylazine, and 4-8x106 CFSE-labeled

(Thermo Fisher Scientific) OT-II T cells were transferred i.v. via retro-orbital injection on the

day of infection with SL-OVA.

Tissue preparation

Single cell suspensions were prepared from MLNs, SB after removing Peyer’s patches and LB

(cecum and colon) after removing cecal and colonic patches as described previously (72). As cell

surface CXCR5 expression is undetectable after collagenase treatment we used an alternative

tissue homogenization approach to detect Tfh cells. Briefly, LB was processed and epithelial

cells were removed as described (72) followed by cutting the tissue to 2 mm pieces and

incubation for an additional 45 min in HBSS containing 2% fetal bovine serum (FBS) and EDTA

in a shaking incubator at 37°C. Tissue pieces were homogenized by pipetting and passed through

70 μm nylon mesh filters.

Immunohistochemistry

Colons were harvested from mice at days 0, 7, or 10 after infection with SL1344, rinsed in PBS,

and rolled around a sterile tooth pick before freezing in OCT compound (Sakura Finetek,

Torrance, CA) on dry ice. 8 μm cross-sections were cut using a cryostat (Leica Biosystems,

Buffalo Grove, IL). Tissues sections were fixed with 4% PFA or acetone and blocked with goat

or donkey serum and stained with the following antibodies: anti-mouse CD45R (B220, clone

RA3-6B2), anti-human/mouse GL7 (clone GL-7), anti-mouse CD4 (clone GK1.5), anti-mouse

CD45.1 (clone A20), anti-mouse MAdCAM-1 (rabbit polyclonal, R&D Systems, Minneapolis,

MN) and BacTrace FITC-labeled goat polyclonal anti-Salmonella CSA-1 antibody (Kirkegaard

& Perry Laboratories, now in Seracare) followed by the secondary reagents. For CXCL13

staining infected Cx3cr1GFP/WT

mice were injected i.p. with 0.25 mg brefeldin A (Enzo Life

Sciences, Farmingdale, NY) 6 hours before euthanasia. Colons were removed and washed in

HBSS containing 10 μg/ml brefeldin A followed by fixation in 4% PFA and 2% sucrose. Fixed

colons were rolled around a sterile toothpick before freezing in OCT compound. 8 μm cross-

sections were cut using a cryostat blocked with donkey serum and stained using anti-mouse

B220 (clone RA3-6B2) and goat polyclonal anti-mouse CXCL13 Ab (R&D Systems) (64).

Tissue sections were counterstained with DAPI. Slides were mounted on Prolong Diamond

(Thermo Fisher Scientific) and imaged using Leica DM4000 (Leica Microsystems, Buffalo

Grove, IL), Zeiss Axio Observer.Z1 fluorescent microscopes (Carl Zeiss Microscopy, White

Plains, NY) or Nikon Eclipse Ti confocal microscope. Images were analyzed using Las-X

(Leica), Zeiss ZEN 2.3 pro or Volocity (PerkinElmer, Waltham, MA) software packages. For

quantification, tertiary lymphoid structures (TLS) were identified as diffuse non-encapsulated

aggregates of B220+ cells (follicles) with an area greater than 10

-3 mm

2. Follicular area was

determined using the tools in the image analyzer software packages listed above. Follicle

numbers for each colon were determined by calculating the average of follicle numbers counted

on at least four sections cut 100 μm apart from each other.

Flow cytometry

The following primary antibodies were used for flow cytometry: anti-mouse CD45 (clone 30-

F11), anti-mouse CD11c (clone N418), anti-mouse CD11b (clone M1/70), anti-mouse CD103

(clone 2E7), anti-mouse MHCII (I-A/I-E) (clone MS/114.15.2), anti-mouse CD115

(cloneAFS98), anti-mouse F4/80 (clone BM8), anti-mouse Ly6C (clone HK1.4), anti-mouse

Ly6G (clone 1A8-Ly6g), anti-mouse CD45R (B220) (clone RA3-6B2), anti-mouse CD19 (clone

eBio1D3), anti-human/mouse GL7 (clone GL-7), anti-mouse CD4 (clone GK1.5), anti-mouse

CD4 (clone RM4-5), anti-mouse TCRβ (clone H57-597), anti-mouse CD45.1 (clone A20), anti-

mouse CD25 (clone PC61.5), anti-mouse CD199 (CCR9) (clone eBioCW-1.2), anti-mouse V

alpha 2 TCR (clone B20.1), anti-mouse CD101 (clone Moushi101) from Thermo Fisher

Scientific; anti-mouse CD95 (clone Jo2), anti-mouse CXCR5 (clone 2G8) from BD Pharmingen

(San Diego, CA); anti-mouse CD279 (PD-1) (clone 29F.1A12), anti-mouse CD16/32 (clone 93),

anti-mouse CD64 (clone X54-5/7.1), anti-mouse XCR1 (clone ZET) from Biolegend (San Diego,

CA). Samples were acquired on LSRII flow cytometers (BD). For cell sorting a FACSAria III

instruments were used (101). The purity of sorted populations was confirmed by running the

collected cells on LSRII flow cytometer (Fig. S2C). Data analysis was performed using FlowJo

software (FlowJo LLC, Ashland, OR). Gating strategies to identified MP populations, GC B

cells and Tfh cells are shown on Fig. S2A-B, Fig. S5A and Fig. S6D, respectively. Total cell

numbers in LB were determined as described previously (72).

To compare MP populations in steady state and inflammation with unbiased analysis, control

(day 0) and infected (day 3) LB samples were analyzed by flow cytometry and 30000 MP events

from each (see MP subset gate on Fig. S2A) were concatenated. Unsupervised t-SNE analysis

was performed with FlowJo using the t-SNE plugin set to 10 phenotypic markers (MHCII,

CD11b, CD16/32, CD64, CX3CR1-GFP, CD11c, CD103, CD101, XCR1, Ly6G), 2500

iterations, perplexity of 100, and Eta at 200.

RNA isolation and RT-PCR

Total RNA was isolated from sorted cells using Trizol as described previously (72). RNA was

reverse transcribed using RNA to cDNA Ecodry premix (Takara Bio, Kusatsu, Japan). Gene

expression was assessed using Power SYBR Green PCR Master mix (Life Technologies) on a

StepOnePlus qPCR instrument. Il10, Tgfb1, Aicda, Ccr7, Cxcl13, Tnf, Tnfsf13, Tnfsf13b and

Actb PrimeTime qPCR primers were purchased from Integrated DNA Technologies (Skokie, IL).

Additional primers used: IaF (5’-CCAGGCATGGTTGAGATAGAGATAG-3’), CaR (5’-

GAGCTGGTGGGAGTGTCAGTG-3’), ImF (5’-CTCTGGCCCTGCTTATTGTTG-3’) (49).

The primers IaF and CaR were used to detect IgA germline transcript (aGT), and ImF and CaR

were used to detect rearranged, post switch IgA transcript (PST). Gene expression levels were

calculated by normalization to Actb, and shown as fold change over the population described in

figure legends.

RNA sequencing

Total RNA was isolated from cell populations sorted from LB of control mice and mice infected

with SL1344 (day 5). Samples for each population were prepared by pooling cells sorted from 5

mice. In total, 5 samples were prepared, representing 5 independent cell sorting experiment. The

cDNA libraries were prepared using the QuantSeq 3’mRNA-Seq Library Prep Kit FWD for

Illumina (Lexogen, Vienna, Austria) as per the manufacturer’s instructions. Briefly, total RNA

was reverse transcribed using oligo (dT) primers. The second cDNA strand was synthesized by

random priming in a manner that DNA polymerase is efficiently stopped when reaching the next

hybridized random primer, and only the fragment close to the 3’ end gets captured for later

indexed adapter ligation and PCR amplification. The processed libraries were assessed for size

distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent

Technologies, Santa Clara, CA). Pooled libraries were diluted to 2 nM in EB buffer (Qiagen,

Germantown, MD) and then denatured using the Illumina protocol. The denatured libraries were

diluted to 10 pM in pre-chilled hybridization buffer, loaded onto a TruSeq v2 Rapid flow cell on

an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe according to the

manufacturer's instructions. De-multiplexed sequencing reads were generated using Illumina

bcl2fastq (released version 2.18.0.12) allowing no mismatches in the index read.

Sequences were processed to remove adapter, poly A and low-quality bases by BBTools

(https://jgi.doe.gov/data-and-tools/bbtools/) using bbduk parameters of k=13, ktrim=r,

forcetrimleft=12, useshortkmers=t, mink=5, qtrim=r, trimq=15, minlength=20. Reads were

aligned to mouse genome and transcriptome (ENSEMBL NCBIM37) using Tophat (v2.1.1)

(102) with default parameters. Number of reads per million for mouse genes were counted using

HTSeq (v 0.6.0) (103) and quantile normalized.

In order to better understand differences between M populations and cDCs we focused our

analyses on specific pathways known to be associated with MP. Genes and pathways for analysis

were selected based on their described roles in physiological processes linked to MP and enteric

https://jgi.doe.gov/data-and-tools/bbtools/

infections using NCBI BioSystems and Kyoto Encyclopedia of Genes and Genomes (KEGG)

databases. The list of genes and pathways is shown in Table S2.

The expression of pathways was compared between cell populations using a previously

described approach with minor modifications (104). Specifically, the gene expression per sample

was normalized by the gene’s median expression across all samples. Then for each sample, the

geometric mean of all genes in a pathway was calculated to represent the expression of the

pathway in the sample (Table S3, S4). These values (per pathway per sample) were used for

class comparison analysis in BRBArrayTools (https://brb.nci.nih.gov/BRB-ArrayTools/) to

identify differentially expressed pathways between cell populations. The values of geometric

means of the pathway per cell populations were used to generate heat maps in R using heatmap.2

(gplots), row scaling, and the pathways were manually organized.

Individual gene expression was compared between cell populations using BRBArrayTools

software. Adjustment for multiple hypotheses was performed by calculating False Discovery

Rate using Benjamini-Hochberg Procedure. The expression levels of differentially expressed

genes (FDR < 1%) obtained by comparing (day 0) SB cDC2 vs Per. MFs was used for

hierarchical clustering of samples in BRB with centered correlation and average linkage.

Individual heat maps per pathway illustrate the geometric mean of gene expression per cell

population. The heat maps were generated in R using heatmap.2 (gplots), row scaling, and the

genes were organized as per decreasing (CX3CR1hi

D.5 SL / CX3CR1lo

D.5 SL) fold change.

In vitro B cell cultures

MP populations were sorted from the LB of Cx3cr1GFP/WT

mice harvested at day 6 of infection. B

cells were purified from spleens of naïve CD45.1+ congenic mice using EasySep mouse B cell

isolation kit (STEMCELL Technologies, Vancouver, Canada). B cells were cultured with MP

subsets in RPMI-1640 medium supplemented with 10% FBS, 5.5x10-5

M 2-ME, 10 mM HEPES,

1 mM sodium pyruvate (RPMI), 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml

amphotericin B and 100 μg/ml gentamicin in presence or absence of SL1344. At day 4 of

culture, supernatants were analyzed to assess IgG and IgA levels with ELISA and cells were

collected and analyzed by flow cytometry to detect GC B cells.

In vitro antigen presentation assays

MP subsets were sorted from the LB of Cx3cr1GFP/WT

mice harvested at day 6 after infection. T

cells were purified from MLNs and spleens of congenic OT-II mice using EasySep mouse CD4+

T cell isolation kit (STEMCELL). MP subsets were incubated with HK SL-OVA in RPMI in

presence of rGM-CSF/rM-CSF (Ms) or rFlt3L (DCs) for 2 hours. Isolated OT-II T cells labeled

with CFSE were added to the wells at an APC:T cell ratio of 1:1 in RPMI with 100 units/ml

penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B and 100 μg/ml gentamicin. T cell

activation and proliferation were assessed by flow cytometry after culturing for 4 days at 37°C

and 5% CO2. Bone marrow derived (BM) DCs and Ms were prepared as described previously

(105). BM Ms or BM DCs cells were cultured in RPMI with HK SL-OVA for 18 h at 37C and

5% CO2. Thereafter, OT-II T cells labeled with CFSE were added to SL-OVA loaded Ms or

DCs at a 1:1 ratio. After 72 hours of co-culture, OT-II T cell activation and proliferation was

assessed by flow cytometry.

Ex vivo Transwell assay

For B cell migration assays 96-well Transwell support system was used (Corning, NY, USA)

with 5 um pore size. CX3CR1hi

Ms were sorted from the LB of Cx3cr1GFP/WT

mice harvested at

day 12 of infection cultured for 3 hours in the lower chamber of Transwell plate in presence or

absence of CXCL13 Ab (50 g/ml) (64). Spleens were collected from naïve CD45.1+ congenic

mice and after homogenization splenocytes were re-suspended in RPMI-1640 containing 10 mM

HEPES, 0.5% fatty acid-free BSA (Sigma), 100 units/ml penicillin, 100 μg/ml streptomycin,

0.25 μg/ml amphotericin B and 100 μg/ml gentamicin and incubated in 37C water bath for 30

min. Splenocytes were added to the upper chamber of the Transwell plate at 1:1 ratio and

incubated for 3 hours at 37C and 5% CO2. For input wells, splenocytes were added to the lower

well without CX3CR1hi

Ms. After the incubation, cells from the lower well were collected and

the numbers of migrated B cells were analyzed using flow cytometry.

Fig. S1. Model of streptomycin-pretreated Salmonella-induced colitis in mice on a

Salmonella-resistant background. WT mice were infected with SL1344 as described in

Methods.

(A) Salmonella CFU in the lumen, intestinal mucosa and isolated organized gut associated

lymphoid tissue (GALT) from various intestinal regions (D.1 p.i.) (n=5).

(B) Body weight loss during Salmonella-induced colitis (n=8).

(C) Lipocalin-2 concentrations in fecal pellets of WT mice with Salmonella-induced colitis

(n=4).

(D) Neutrophil counts in various intestinal regions shown as % of total viable cells (D.14 p.i.)

(n=3-10).

(E) Counts of myeloid cell subsets in the small bowel (SB) and large bowel (LB) (n=5-8).

Graphs show mean±SEM, data is representative or combination of 2 independent experiments.

Statistical analysis: two-way ANOVA. *p

Fig. S2. Flow cytometry gating strategy to identify MP populations in infected LB.

Cx3cr1GFP/WT

, Notch2fl/fl

(Cont.), ItgaxCre

Notch2fl/fl

(Itgax:Notch2–/–

) and

ItgaxCre

Notch2fl/fl

Cx3cr1GFP/WT

mice were infected with SL1344.

(A) Flow cytometry dot plots show gating strategy to identify myeloid cells in the infected (D.3

p.i.) large bowel (LB) of Cx3cr1GFP/WT

mice.

(B) Flow cytometry dot plots show gating strategy to identify mononuclear phagocyte (MP)

populations in normal (D.0) and infected (D.10 p.i.) large bowel (LB) of Cx3cr1GFP/WT

mice.

(C) Representative flow cytometry dot plots show the purity of MP populations sorted from

normal (D.0) LB, SB and peritoneal lavage and from infected LB (D.5 p.i.).

(D) Flow cytometry dot plots show percentage (%) of CCR2+ cells (identified as RFP

+ cells)

among blood monocyte and LB MP populations in Ccr2RFP/WT

Cx3cr1GFP/WT

mice infected with

Salmonella (D.8 p.i.).

(E) Graphs show myeloid cell counts in the LB before and after infection with SL1344 in

Notch2fl/fl

(Cont.) and ItgaxCre

Notch2fl/fl

(Itgax:Notch2–/–

) mice (n=2-4).

(F) Flow cytometry dot plots show MP populations in the LB and SB of Notch2fl/fl

Cx3cr1GFP/WT

(Cont.) and ItgaxCre

Notch2fl/fl

Cx3cr1GFP/WT

(Itgax:Notch2–/–

) mice infected with Salmonella (D.3

p.i.).

Graphs show mean±SEM, data is representative of 2 independent experiments. Statistical

analysis: two-way ANOVA. *p

Fig. S3. Response to Salmonella infection in mice lacking Mϕs or cDC2. WT or Cx3cr1GFP/WT

mice were injected with CSF1R mAb to deplete Ms (CSF1R Ab) or control IgG before

infection with SL1344 (A). ItgaxCre

Notch2fl/fl

(Itgax:Notch2–/–

) mice were used as a cDC2-

deficient model, their Notch2fl/fl

(Cont.) littermates were used as control (D-J). Mice were

rescued with ampicillin (G-H).

(A) Graphs show myeloid cell counts in the LB before and after infection with SL1344 in

Cx3cr1GFP/WT

mice injected with CSF1 mAb to deplete Ms (∆M) or control IgG (Cont.) (n=5-

8).

(B) Salmonella CFU in the mesenteric lymph nodes (MLNs) and spleen of WT mice at indicated

time points p.i (n=7-14).

(C) Numbers of Salmonella (SL)-specific IgA-producing cells in the LB and MLNs of WT mice

before and at indicated time points after infection as measured by ELISpot (n=2-8).

(D) Survival curves of control and Itgax:Notch2–/–

mice (n=8-9).

(E) Weight loss as percent of body weight at D.0 by control and Itgax:Notch2–/–

mice (n=8-9).

(F) Salmonella CFU in fecal pellets in control and Itgax:Notch2–/–

mice (D.1 p.i.) (n=6-7).

(G) Lipocalin-2 concentrations in fecal pellets of infected control and Itgax:Notch2–/–

mice (n=8-

9).

(H) Salmonella (SL)-specific antibody titers in fecal pellets (mucosal IgA) and blood (serum IgA

and IgG) of control and Itgax:Notch2–/–

mice (n=8-9).

(I) Numbers of TLS follicles (TLF) per colonic section in control and Itgax:Notch2–/–

mice (D.10

p.i.) (n=3).

(J) Area of follicles in colonic sections of control and Itgax:Notch2–/–

mice (D.10 p.i.) (n=3).

Graphs show mean±SEM and are representative or combination of at least 2 independent

experiments. Statistical analysis: Student’s t-test or two-way ANOVA. *p

Fig. S4. Gene expression profiles of inflammatory MP subsets. Cx3cr1GFP/WT

mice were

infected with SL1344.

(A) RNAseq heat maps show expression levels of genes related to the indicated pathways in MP

subsets isolated by FACS from Salmonella-infected LB (D.5 p.i.). Because intestinal MP subsets

were sorted based on their expression of the antigen-presenting molecule MHCII, to highlight the

differences in the expression of genes from the Antigen presentation/MHCI and Antigen

presentation/MHCII pathways among LB MP subsets, they are compared to MHCII– peritoneal

Ms isolated from normal mice on the same genetic background. Each sample represents the

geometric mean of 5 independent samples.

Fig. S5. Intestinal GC B cells express genes related to IgA class switch. Mice were infected

with WT Salmonella SL1344.

(A) Gating strategy to identify GC B cells in normal and infected (D.10 p.i.) LB of WT mice as

CD19+B220

+GL7

+CD95

+ cells. Gated on viable DAPI

–CD45

+ singlets.

(B) Expression of Aicda, IgA germline transcript (aGT) and mature rearranged IgA (Iµ-Cα)

transcript (PST) by GL7+ and GL7

– CD19

+B220

+ B cells (Fig. S5A) purified by FACS from

infected LB (D.10 p.i.) as measured by qPCR (n=3-4).

(C) Flow cytometry dot plots show proportions of B cells (thick gates) and CD4+ T cells among

total lymphocytes in tertiary lymphoid structures (follicles) (TLFs), mucosa, and colonic and

cecal patches of infected LB isolated from a WT mouse (D.14 p.i.). Gated on CD45+CD11b

–

CD11c– cells.

Graphs show mean±SEM and are representative of at least 2 independent experiments. Statistical

analysis: Student’s t test or two-way ANOVA. *p

Fig. S6. Response to Salmonella infection in mice after CD4+ T cell depletion. H2-Ab1

fl/fl

(Cont.), Lyz2Cre/WT

H2-Ab1fl/fl

(Lyz2:MHCII–/–

), WT mice treated with control IgG (Cont.) or CD4

Ab to deplete CD4+ T cells were infected with SL1344.

(A) Statistical summary of flow cytometry data show proportions of MHCII+ cells among MP

populations and B220+ B cells in the LB and MLNs (for B cells only) isolated from infected

Cont. and Lyz2:MHCII

–/– mice (D.7 p.i.) (n=5).

(B) Bone marrow derived (BM) Ms and DCs from Cont. and Lyz2:MHCII–/–

mice were co-

cultured with OT-II cells in the presence or absence of 2x106 CFU of heat-killed (HK) SL1344-

OVA (SL-OVA). Flow cytometry dot plots show percentages of proliferating CFSE low cells

among OT-II Vα2+CD4

+ T cells on D.4 of co-culture.

(C) Summary of B. Data is representative of 2 independent experiments (n=2).

(D) Gating strategy to identify Tfh cells as TCRβ+CD4

+CXCR5

+ PD1

+ cells in the LB from

SL1344-infected WT mice. Gated on viable DAPI–CD45

+ singlets.

(E) Survival curves of mice treated with IgG control or CD4 Ab (n=8).

(F) Weight loss as percent of D.0 in mice treated with IgG control or CD4 Ab (n=8).

(G) Lipocalin-2 concentrations in fecal pellets collected from mice treated with IgG control or

CD4 Ab (n=8).

(H) Salmonella-specific antibody titers in fecal pellets (mucosal IgA) and blood (serum IgA and

IgG) of mice treated with IgG control or CD4 Ab (n=8).

(I) Numbers of Salmonella-specific IgA-producing cells in the LB of mice treated with IgG

control or CD4 Ab (D.7 p.i.) as measured by ELISpot (n=5-6).

(J) Number of follicles per colonic section in mice treated with IgG control or CD4 Ab (D.10

p.i.) (n=8-9).

(K) Area of follicles in colonic sections of mice treated with IgG control or CD4 Ab (D.10 p.i.)

(n=8-9).

Graphs show mean±SEM and are representative of at least 2 independent experiments. Statistical

analysis: Student’s t-test or two-way ANOVA. *p

Fig. S7. Development strategy and characterization of Ccr7fl/fl

and Lyz2:Ccr7–/–

mice.

(A, B) Gene targeting strategy for generation of Ccr7fl/fl

animals. A Pgk-NEO cassette flanked by

Frt sites was inserted in the intronic region between exon II and exon III. LoxP sites are flanking

exon III, which allows for Cre recombinase mediated deletion of the targeted locus. Primer sets

P1/P2 and the outlined probe were used for screening of ES colonies by PCR and Southern Blot.

Grey bars depict the end of 5’ and 3’ homology arms.

(C) PCR screen with primer combination P1/P2. Recombination frequency is 3.5% (12/346

screened clones).

(D) Ccr7 expression by cDC1 and Ms isolated by FACS from LB of SL1344-infected Ccr7fl/fl

(Cont.) and Lyz2Cre

Ccr7fl/fl

(Lyz2:Ccr7–/–

) mice (D.5 p.i.) as measured by qPCR. Data shows fold

change over control (n=4-8).

(E) Absolute numbers of MP subsets in LB of infected Ccr7fl/fl

Cx3cr1GFP/WT

(Cont.) and

Lyz2Cre

Ccr7fl/fl

Cx3cr1GFP/WT

(Lyz2:Ccr7–/–

) mice (D.5 p.i.) (n=4-7).

Bar graphs show mean±SEM (n=3-7) and are combination of 2 independent experiments.

Statistical analysis: Student’s t test. *p

Fig. S8. Role of CX3CR1+ Mϕ subsets in the induction of Salmonella-specific T cell

responses. WT, H2-Ab1fl/fl

(Cont.) and Lyz2:MHCII–/–

mice were infected with SL1344 or

SL1344-OVA as indicated.

(A) Gating strategy to identify adoptively transferred CD45.1+ OT-II cells in LB of WT recipient

mice infected with either WT SL1344 (SL) or SL1344-OVA (SL-OVA).

(B) Control and Lyz2:MHCII–/–

mice were infected with SL1344-OVA one day prior to receiving

CFSE-labeled congenic CD45.1+ OT-II T cells. Flow cytometry dot plots show percentages of

proliferating CFSE low cells among total CD45.1+Vα2

+CD4

+CCR9

– (LB) and

CD45.1+Vα2

+CD4

+ (MLNs) T cells in the LB and MLNs on D.5 p.i.

(C) Statistical summary of B.

Bar graphs show mean±SEM (n=4-10) and are combination of 2 independent experiments.

Statistical analysis: Student’s t test or two-way ANOVA. ***p

Fig. S9. Response to Salmonella infection in Ccr7–/–

mice. Cx3cr1GFP/WT

control (Cont.) and

Cx3cr1GFP/WT

Ccr7–/–

mice, WT (Cont.) and Ccr7–/–

mice or Cx3cr1CreER/WT

H2-ab1WT/fl

and

Cx3cr1CreER/WT

H2-ab1fl/fl

mice were infected with SL1344. Mice were rescued with ampicillin

(B, C).

(A) Absolute numbers of MP subsets in LB of infected Cx3cr1GFP/WT

control and Cx3cr1GFP/WT

Ccr7–/–

mice (D.5 p.i.) (n=4).

(B) Lipocalin-2 concentrations in fecal pellets of Cont. and Ccr7–/–

mice (n=5-6).

(C) Salmonella-specific antibody titers in fecal pellets (mucosal IgA) and blood (serum IgA and

IgG) of Cont. and Ccr7–/–

mice (n=7-8).

(D) Numbers of follicles per colonic section in Cont. and Ccr7–/–

mice before and after infection

(n=3-6).

(E) Area of follicles in colonic sections of control and Ccr7–/–

mice after infection (D.10 p.i.)

(n=3-6).

(F) Kinetics of Tnfsf13a expression by MP subsets FACS-isolated from the normal (D.0) and

infected LB as measured by qPCR. Data shows fold change over CX3CR1hi

Ms (D.0) (n=2-3).

(G) Gating strategy to identify a B220+ subset among CX3CR1

hi Ms in the LB of SL1344-

infected Cx3cr1GFP/WT

mice. Gated on viable CD45+CD11b

lo/hiCD11c

lo/hiMHCII

+CX3CR1

hi

population.

(H) CX3CR1hi

Ms and cDC1 isolated from LB of infected Cx3cr1GFP/WT

mice (D.5 p.i.) were co-

cultured in vitro with CFSE-labeled OT-II CD4+ T cells in the presence of 2x10

6 CFU of heat-

killed (HK) SL1344-OVA (SL-OVA) or OVA protein. Flow cytometry dot plots show

percentages of proliferating CFSE low cells among OT-II Vα2+CD4

+ T cells on day 4 of co-

culture.

(I) Summary of H (n=1-3).

(J) Graphical summary of the experiment to selectively deplete CX3CR1hi

Ms in

Cx3cr1CreER

H2-ab1fl/fl

mice. Mice were treated with tamoxifen (Tam) 7 days before and 3 days

after infection with SL1344.

(K) Statistical summary of flow cytometry data show proportions of MHCII+ cells among B220

+

B cells in the infected LB isolated from tamoxifen-treated Cx3cr1CreER/WT

H2-ab1WT/fl

(Cre:WT/fl)

or Cx3cr1CreER/WT

H2-ab1fl/fl

(fl/fl) mice (Cre:fl/fl) (D.7 p.i.) (n=3-6).

Bar graphs show mean±SEM and are representative or combination of at least 2 independent

experiments. Statistical analysis: Student’s t-test or two-way ANOVA. *p