(A) Migratory cDCs present endogenously loaded Helicobacter antigens to T cells. Resident (MHCIIint CD11chi) or migratory (MHCIIhi CD11cint) cDCs from the distal mesenteric lymph node (dMLN) were …
(A) Representative post-sort analysis of resident (MHCIIint CD11chi) or migratory (MHCIIhi CD11cint) conventional dendritic cells (cDCs) from the distal mesenteric lymph node (dMLN) used in Figure …
All three main subsets of migratory cDCs present endogenous Helicobacter antigens to T cells ex vivo. Migratory CD103+ CD11b– cDC1s (CD103+ SP), CD103+ CD11b+ cDC2s (DP), or CD103– CD11b+ cDC2s …
(A) Antigen presenting cells (APCs) in the CD103– CD11b+ cDC2 (CD11b+ SP) gate are mostly conventional dendritic cells (cDCs) by Zbtb46GFP expression.
Representative fluorescence-activated cell sorting (FACS) plots of CD11b+ SP APCs showing low/intermediate F4/80 expression in Zbtb46GFP– cells. (B) Representative post-sort analysis of CD103+ CD11b– cDC1s (CD103+ SP), CD103+ CD11b+ cDC2s (DP), or CD11b+ SP migratory cDCs from the distal mesenteric lymph node (dMLN) used for experiments in Figure 2. (C) Migratory cDC1s are increased in the dMLN of wild-type mice relative to the proximal MLN. Migratory cDC subset compositions were analyzed by dividing the mesenteric LN chain into six sections, roughly corresponding to one lymph node per section, from the colon-draining dMLN (section 1, red) to the proximal LN (section 6). Data was normalized to the subset frequency in the dMLN (MLN1) (expt. = 2, n = 3). (D) There is comparable T cell activation by all subsets of migratory cDCs. T cell activation potential of migratory cDC subsets was quantified by coculturing cDCs with naïve OT-II T cells as in Figure 1A as well as indicated concentrations of OVA323-339 peptide (expt. = 5). Each dot represents an individual mouse (A) or the mean of indicated experiments (C and D). Mean ± SEM (A, C, and D) shown. p-values from Tukey’s multiple comparisons tests (A); two-way repeated measures ANOVA of cDC subset (C); mixed effects analysis of repeated measures of cDC subset with Tukey’s multiple comparisons tests (D); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The following source data are available for (A and B): Figure 2—figure supplement 1—source data 1 (C) and Figure 2—figure supplement 1—source data 2 (D).
Migratory cDC1s are increased in the dMLN of wild-type mice relative to the proximal MLN (pMLN), Figure 2—figure supplement 1C raw data.
Comparable T cell activation by all subsets of migratory conventional dendritic cells (cDCs), Figure 2—figure supplement 1D raw data.
(A) Deficiency of CD103+ CD11b– cDC1s in the distal mesenteric lymph node (dMLN) of Irf8Δ149en/Δ149en mice. Representative fluorescence-activated cell sorting (FACS) plot and quantification of …
Deficiency of CD103+ CD11b– cDC1s in the distal mesenteric lymph node (dMLN) of Irf8Δ149en/Δ149en mice, Figure 3A raw data.
(A) Frequency, but not number, of CD103+ cDC1s are reduced in the distal mesenteric lymph node (dMLN) of Batf3–/– mice. Representative fluorescence-activated cell sorting (FACS) plots and …
Frequency, but not number, of CD103+ cDC1s are reduced in the distal mesenteric lymph node (dMLN) of Batf3–/– mice, Figure 3—figure supplement 1A raw data.
(A) Double positive CD103+ CD11b+ (DP) cDC2s are lost in the distal mesenteric lymph node (dMLN) of CD207-DTA mice. Representative fluorescence-activated cell sorting (FACS) plot and quantification …
Double positive CD103+ CD11b+ (DP) cDC2s are lost in the distal mesenteric lymph node (dMLN) of CD207-DTA mice, Figure 4A raw data.
(A) Double positive CD103+ CD11b+ (DP) cDC2s are lost in the distal mesenteric lymph node (dMLN) of Irf4-ΔDC mice.
Representative fluorescence-activated cell sorting (FACS) plots and quantification of migratory conventional dendritic cell (cDC) subsets in the dMLN of littermate control (Irf4fl/fl or Irf4–/fl) and Irf4-ΔDC mice (expt. = 2). (B and C) CT2 and CT6 peripheral regulatory T (pTreg) cell differentiation is unaffected in Irf4-ΔDC mice. Congenically marked 5 × 104 naïve CT2 (B) or 105 naïve CT6 (C) were transferred into littermate Irf4fl/fl, Irf4–/fl, and Irf4-ΔDC mice and analyzed for Foxp3IRES-GFP or Thy1.1 expression and cell trace violet (CTV) dilution in the dMLN after 7 days (expt. = 3, except for CT6 CTV where expt. = 2). (D) Analysis of additional cDC markers shows that DP cDC2s are lost in CD207-DTA mice. Representative FACS plots and quantification of CD103+ CD11b+ and CD103+ CD101+ SIRPα+ cDC2s in the dMLN of littermate control and CD207-DTA mice (expt. = 1). (E) Deficiency of CD103+ CD11b+ cDC2s in the proximal MLN (pMLN) of CD207-DTA mice. Representative FACS plot and quantification of migratory cDC subsets in the pMLN of littermate control and CD207-DTA mice (expt. = 3). Each dot represents an individual mouse. Mean ± SEM shown. p-values from Student’s t-test (B and C) or Sidak’s multiple comparisons test (D and E); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
(A) CD103+ cDC1s and cDC2s are greatly reduced in the distal mesenteric lymph node (dMLN) of CD207-DTA::Irf8Δ149en/Δ149en mice. Representative fluorescence-activated cell sorting (FACS) plot and …
CD103+ cDC1s and cDC2s are greatly reduced in the distal mesenteric lymph node (dMLN) of CD207-DTA::Irf8Δ149en/Δ149en mice, Figure 5A raw data.
(A) Variable frequencies of CD103+ conventional dendritic cells (cDCs) in the distal mesenteric lymph node (dMLN) of CD207-DTA::Batf3–/– mice. Fluorescence-activated cell sorting (FACS) plot and …
Variable frequencies of CD103+ conventional dendritic cells (cDCs) in the distal mesenteric lymph node (dMLN) of CD207-DTA::Batf3–/–mice, Figure 5—figure supplement 1A raw data.
(A and B) Helicobacter-specific T cell activation and Treg cell differentiation are not dramatically altered in the cLP of CD103+ cDC1 and cDC2-deficient mice. Congenically marked 5 × 104 naïve CT2 …
(A) Retinal dehydrogenase (RALDH) activity as measured by Aldefluor is unchanged in CD11b+ SP cDC2s in CD207-DTA::Irf8Δ149en/Δ149en mice deficient in CD103+ cDC1s and cDC2s (expt. = 2). (B) LAP and …
(A and B) The colonic bacterial microbiome is not significantly different among littermate control (Irf8+/Δ149en), Irf8Δ149en/Δ149en, CD207-DTA (Irf8+/+ and Irf8+/Δ149en), and CD207-DTA:: Irf8Δ149en/…
16S rRNA sequencing of whole colon lumen feces of conventional dendritic cell (cDC)-deficient mice, Figure 6—figure supplement 1B raw data.
(A) Molecules involved in T cell activation are unchanged in CD11b+ SP cDC2s in CD207-DTA::Irf8Δ149en/Δ149en mice. Representative expression of I-Ab, CD80, CD86, CD40, CD273, and CD274 by flow …
(A) Migratory cDC induction of OT-II pTreg cells in vitro is dependent on T cell receptor (TCR) stimulation. Treg cell induction of OT-II cells by migratory cDC subsets with varying concentrations …
Migratory conventional dendritic cell (cDC) induction of OT-II peripheral regulatory T (pTreg) cells in vitro is dependent on T cell receptor (TCR) stimulation, Figure 7A raw data.
In vitro, CD103+ SP cDC1s have increased ability to induce OT-II peripheral regulatory T (pTreg) cells at higher levels of TCR activation, Figure 7B raw data.
(A) Migratory conventional dendritic cell (cDC) subsets induce low levels of Foxp3IRES-GFP or Thy1.1 in CT2 and CT6 in vitro. (B) The induction of Foxp3IRES-GFP or Thy1.1 in CT2 and CT6 cultured …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Anti-mouse CD3ε (clone# 145–2 C11) FITC, PE, and BV421 | Biolegend | 100305/07/35 | Dilution: 1:300 |
Antibody | Anti-mouse/human B220 (clone# RA3-6B2) APC/Cy7 and A700 | Biolegend | 103223/31 | Dilution: 1:750 |
Antibody | Anti-mouse CD19 (clone# 6D5) APC/Cy7 | Biolegend | 115529 | Dilution: 1:750 |
Antibody | Anti-mouse I-Ab (clone# AF6-120.1) APC and PerCP/Cy5.5 | Biolegend | 116417/15 | Dilution: 1:750 |
Antibody | Anti-mouse CD11c (clone# N418) PE/Cy7 and BV605 | Biolegend | 117317/33 | Dilution: 1:750 |
Antibody | Anti-mouse/human CD11b (clone# M1/70) BV711 | Biolegend | 101241 | Dilution: 1:750 |
Antibody | Anti-mouse CD103 (clone# 2E7) BV421 | Biolegend | 121421 | Dilution: 1:300 |
Antibody | Anti-mouse CD4 (clone# RM4-5) BV711, PE, and PB | Biolegend | 100549/11/34 | Dilution: 1:750 |
Antibody | Anti-mouse CD25 (clone# PC61) APC, BV605, PerCP/Cy5.5, and PE-Cy7 | Biolegend | 102011/35/29/15 | Dilution: 1:750 |
Antibody | Anti-mouse/human CD44 (clone# IM7) APC/Cy7 and BV605 | Biolegend | 103027/47 | Dilution: 1:750 |
Antibody | Anti-mouse CD62L (clone# MEL-14) APC/Cy7 and BV605 | Biolegend | 104427/37 | Dilution: 1:750 |
Antibody | Anti-mouse FOXP3 (clone# FJK-16s) FITC | Thermo Fisher | 11-5773-82 | Dilution: 1:200 |
Antibody | Anti-mouse GATA3 (clone# 16E10A23) PE | Biolegend | 653803 | Dilution: 1:30 |
Antibody | Anti-mouse RORγt (clone# B2D) APC | Thermo Fisher | 17-6981-80 | Dilution: 1:200 |
Antibody | Anti-mouse TBET (clone# 4B10) PE/Cy7 | Biolegend | 644823 | Dilution: 1:200 |
Antibody | Anti-mouse HELIOS (clone# 22F6) A647 | Biolegend | 137208 | Dilution: 1:200 |
Antibody | Anti-mouse CXCR3 (clone# CXCR3-173) BV421 | Biolegend | 126521 | Dilution: 1:300 |
Antibody | Anti-mouse Thy1.1 (clone# 30-H12) PE/Cy7 | Biolegend | 105325 | Dilution: 1:750 |
Antibody | Anti-mouse LAP (clone# TW7-16B4) BV421 | Biolegend | 141407 | Dilution: 1:100 |
Antibody | Anti-mouse CD51 (clone# RMV-7) PE | Biolegend | 104105 | Dilution: 1:200 |
Antibody | Anti-mouse CD80 (clone# 16-10A1) APC | Biolegend | 104713 | Dilution: 1:750 |
Antibody | Anti-mouse CD86 (clone# GL-1) BV605 | Biolegend | 105037 | Dilution: 1:300 |
Antibody | Anti-mouse CD40 (clone# 3/23) APC | Biolegend | 124611 | Dilution: 1:750 |
Antibody | Anti-mouse CD273 (clone# TY25) APC | Biolegend | 107210 | Dilution: 1:300 |
Antibody | Anti-mouse CD274 (clone# 10F.9G2) BV421 | Biolegend | 124315 | Dilution: 1:300 |
Antibody | Anti-mouse XCR1 (clone# ZET) APC | Biolegend | 148205 | Dilution: 1:300 |
Antibody | Anti-mouse CD36 (clone# CRF D-2712) PE | Becton Dickinson | 562702 | Dilution: 1:200 |
Antibody | Anti-mouse CD45.1 (clone# A20) PE, APC, and PE/Cy7 | Biolegend | 110707/14/29 | Dilution: 1:750 |
Antibody | Anti-mouse CD45.2 (clone# 104) PE, APC, PE/Cy7, and A700 | Biolegend | 109807/14/29/21 | Dilution: 1:750 |
Antibody | Anti-mouse VB6 (clone# RR4-7) PE and APC | Biolegend | 140003/5 | Dilution: 1:750 |
Antibody | Anti-mouse Va2 (clone# B20.1) APC/Cy7 and PerCP/Cy5.5 | Biolegend | 127818/13 | Dilution: 1:750 |
Antibody | Anti-mouse VB5 (clone# MR9-4) PE | Biolegend | 139503 | Dilution: 1:750 |
Antibody | Anti-mouse F4/80 (clone# BM8) PE/Cy7 | Biolegend | 123113 | Dilution: 1:750 |
Antibody | Anti-mouse SIRPα (clone# P84) A700 | Biolegend | 144021 | Dilution: 1:750 |
Antibody | Anti-mouse CD24 (clone# M1/69) PE | Biolegend | 101807 | Dilution: 1:750 |
Antibody | Anti-mouse CD101 (clone# Moushi101) PE/Cy7 | Thermo Fisher | 50-112-3316 | Dilution: 1:300 |
Strain, strain background (Mus musculus) | OT-II TCR transgenic mice | The Jackson Laboratory (JAX) | #004194 | |
Strain, strain background (Mus musculus) | Ccr7GFP knockin/knockout mice | The Jackson Laboratory (JAX) | #027913 | |
Strain, strain background (Mus musculus) | Itgax-Cre mice | The Jackson Laboratory (JAX) | #008068 | |
Strain, strain background (Mus musculus) | Irf4fl/fl mice | The Jackson Laboratory (JAX) | #009380 | |
Strain, strain background (Mus musculus) | CT2 TCR transgenic mice | Nutsch et al., 2016 | ||
Strain, strain background (Mus musculus) | CT6 TCR transgenic mice | Nutsch et al., 2016 | ||
Strain, strain background (Mus musculus) | Rag1–/– mice | The Jackson Laboratory (JAX) | #002216 | |
Strain, strain background (Mus musculus) | Foxp3IRES-GFPmice | The Jackson Laboratory (JAX) | #006772 | |
Strain, strain background (Mus musculus) | Foxp3IRES-Thy1.1mice | Liston et al., 2008 | ||
Strain, strain background (Mus musculus) | Irf8Δ149en/Δ149en mice; formerly Irf8 +32 5’–/– | Durai et al., 2019 | ||
Strain, strain background (Mus musculus) | Batf3–/– mice | Hildner et al., 2008 | ||
Strain, strain background (Mus musculus) | Zbtb46GFP mice | Satpathy et al., 2012 | ||
Strain, strain background (Mus musculus) | CD207-DTA mice; formerly huLangerin-DTA | Kaplan et al., 2005 |