Gut Helicobacter presentation by multiple dendritic cell subsets enables context-specific regulatory T cell generation

  1. Emilie V Russler-Germain  Is a corresponding author
  2. Jaeu Yi
  3. Shannon Young
  4. Katherine Nutsch
  5. Harikesh S Wong
  6. Teresa L Ai
  7. Jiani N Chai
  8. Vivek Durai
  9. Daniel H Kaplan
  10. Ronald N Germain
  11. Kenneth M Murphy
  12. Chyi-Song Hsieh  Is a corresponding author
  1. Department of Internal Medicine, Division of Rheumatology, Washington University School of Medicine, United States
  2. Lymphocyte Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
  3. Department of Pathology, Division of Immunobiology, Washington University School of Medicine, United States
  4. Department of Dermatology, Department of Immunology, Pittsburgh Center for Pain Research, University of Pittsburgh, United States
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Migratory conventional dendritic cells (cDCs) present Helicobacter antigens to naïve T cells.

(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 …

Figure 1—figure supplement 1
Migratory conventional denditic cells (cDCs) present Helicobacter antigens to naïve T cells.

(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 …

Figure 2 with 1 supplement
Multiple subsets of migratory conventional dendritic cells (cDCs) present Helicobacter antigens to naïve T cells at homeostasis.

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 …

Figure 2—figure supplement 1
Multiple subsets of migratory conventional dendritic cells (cDCs) present Helicobacter antigens to naïve T cells at homeostasis.

(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).

Figure 2—figure supplement 1—source data 1

Migratory cDC1s are increased in the dMLN of wild-type mice relative to the proximal MLN (pMLN), Figure 2—figure supplement 1C raw data.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 1—source data 2

Comparable T cell activation by all subsets of migratory conventional dendritic cells (cDCs), Figure 2—figure supplement 1D raw data.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig2-figsupp1-data2-v2.xlsx
Figure 3 with 1 supplement
CD103+ CD11b cDC1s are not required for Helicobacter-specific peripheral regulatory T (pTreg) differentiation in vivo.

(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 …

Figure 3—source data 1

Deficiency of CD103+ CD11b– cDC1s in the distal mesenteric lymph node (dMLN) of Irf8Δ149en/Δ149en mice, Figure 3A raw data.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
CD103+ CD11b cDC1s are not required for Helicobacter-specific peripheral regulatory T (pTreg) differentiation in vivo.

(A) Frequency, but not number, of CD103cDC1s are reduced in the distal mesenteric lymph node (dMLN) of Batf3–/– mice. Representative fluorescence-activated cell sorting (FACS) plots and …

Figure 3—figure supplement 1—source data 1

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.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
CD103+ CD11b+ cDC2s are not necessary for in vivo Helicobacter-specific peripheral regulatory T (pTreg) differentiation.

(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 …

Figure 4—source data 1

Double positive CD103+ CD11b+ (DP) cDC2s are lost in the distal mesenteric lymph node (dMLN) of CD207-DTA mice, Figure 4A raw data.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
CD103+ CD11b+ cDC2s are not necessary for in vivo Helicobacter-specific peripheral regulatory T (pTreg) differentiation.

(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.

Figure 5 with 2 supplements
CD103 CD11b+ cDC2s are sufficient for in vivo Helicobacter-specific peripheral regulatory T (pTreg) differentiation.

(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 …

Figure 5—source data 1

CD103+ cDC1s and cDC2s are greatly reduced in the distal mesenteric lymph node (dMLN) of CD207-DTA::Irf8Δ149en/Δ149en mice, Figure 5A raw data.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
CD103– CD11bcDC2s are sufficient for in vivoHelicobacter-specific peripheral regulatory T (pTreg) differentiation.

(A) Variable frequencies of CD103conventional dendritic cells (cDCs) in the distal mesenteric lymph node (dMLN) of CD207-DTA::Batf3–/– mice. Fluorescence-activated cell sorting (FACS) plot and …

Figure 5—figure supplement 1—source data 1

Variable frequencies of CD103conventional dendritic cells (cDCs) in the distal mesenteric lymph node (dMLN) of CD207-DTA::Batf3–/–mice, Figure 5—figure supplement 1A raw data.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
CD103– CD11bcDC2s are sufficient for in vivo Helicobacter-specific peripheral regulatory T (pTreg) differentiation.

(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 …

Figure 6 with 2 supplements
CD11b+ SP cDC2s are tolerogenic by ‘nature’, not ‘nurture’.

(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 …

Figure 6—figure supplement 1
CD11bSP cDC2s are tolerogenic by ‘nature’, not ‘nurture’.

(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/…

Figure 6—figure supplement 1—source data 1

16S rRNA sequencing of whole colon lumen feces of conventional dendritic cell (cDC)-deficient mice, Figure 6—figure supplement 1B raw data.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
CD11bSP cDC2s are tolerogenic by ‘nature’, not ‘nurture’.

(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 …

Figure 7 with 1 supplement
Conventional dendritic cell (cDC)-mediated peripheral regulatory T (pTreg) cell differentiation in vivo is recessive to effector T cell development.

(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 …

Figure 7—source data 1

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.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig7-data1-v2.xlsx
Figure 7—source data 2

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.

https://cdn.elifesciences.org/articles/54792/elife-54792-fig7-data2-v2.xlsx
Figure 7—figure supplement 1
Conventional dendritic cell (cDC)-mediated peripheral regulatory T (pTreg) cell differentiation in vivo is recessive to effector T cell development.

(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 …

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyAnti-mouse CD3ε (clone# 145–2 C11) FITC, PE, and BV421Biolegend100305/07/35Dilution: 1:300
AntibodyAnti-mouse/human B220 (clone# RA3-6B2) APC/Cy7 and A700Biolegend103223/31Dilution: 1:750
AntibodyAnti-mouse CD19 (clone# 6D5) APC/Cy7Biolegend115529Dilution: 1:750
AntibodyAnti-mouse I-Ab (clone# AF6-120.1) APC and PerCP/Cy5.5Biolegend116417/15Dilution: 1:750
AntibodyAnti-mouse CD11c (clone# N418) PE/Cy7 and BV605Biolegend117317/33Dilution: 1:750
AntibodyAnti-mouse/human CD11b (clone# M1/70) BV711Biolegend101241Dilution: 1:750
AntibodyAnti-mouse CD103 (clone# 2E7) BV421Biolegend121421Dilution: 1:300
AntibodyAnti-mouse CD4 (clone# RM4-5) BV711, PE, and PBBiolegend100549/11/34Dilution: 1:750
AntibodyAnti-mouse CD25 (clone# PC61) APC, BV605, PerCP/Cy5.5, and PE-Cy7Biolegend102011/35/29/15Dilution: 1:750
AntibodyAnti-mouse/human CD44 (clone# IM7) APC/Cy7 and BV605Biolegend103027/47Dilution: 1:750
AntibodyAnti-mouse CD62L (clone# MEL-14) APC/Cy7 and BV605Biolegend104427/37Dilution: 1:750
AntibodyAnti-mouse FOXP3 (clone# FJK-16s) FITCThermo Fisher11-5773-82Dilution: 1:200
AntibodyAnti-mouse GATA3 (clone# 16E10A23) PEBiolegend653803Dilution: 1:30
AntibodyAnti-mouse RORγt (clone# B2D) APCThermo Fisher17-6981-80Dilution: 1:200
AntibodyAnti-mouse TBET (clone# 4B10) PE/Cy7Biolegend644823Dilution: 1:200
AntibodyAnti-mouse HELIOS (clone# 22F6) A647Biolegend137208Dilution: 1:200
AntibodyAnti-mouse CXCR3 (clone# CXCR3-173) BV421Biolegend126521Dilution: 1:300
AntibodyAnti-mouse Thy1.1 (clone# 30-H12) PE/Cy7Biolegend105325Dilution: 1:750
AntibodyAnti-mouse LAP (clone# TW7-16B4) BV421Biolegend141407Dilution: 1:100
AntibodyAnti-mouse CD51 (clone# RMV-7) PEBiolegend104105Dilution: 1:200
AntibodyAnti-mouse CD80 (clone# 16-10A1) APCBiolegend104713Dilution: 1:750
AntibodyAnti-mouse CD86 (clone# GL-1) BV605Biolegend105037Dilution: 1:300
AntibodyAnti-mouse CD40 (clone# 3/23) APCBiolegend124611Dilution: 1:750
AntibodyAnti-mouse CD273 (clone# TY25) APCBiolegend107210Dilution: 1:300
AntibodyAnti-mouse CD274 (clone# 10F.9G2) BV421Biolegend124315Dilution: 1:300
AntibodyAnti-mouse XCR1 (clone# ZET) APCBiolegend148205Dilution: 1:300
AntibodyAnti-mouse CD36 (clone# CRF D-2712) PEBecton Dickinson562702Dilution: 1:200
AntibodyAnti-mouse CD45.1 (clone# A20) PE, APC, and PE/Cy7Biolegend110707/14/29Dilution: 1:750
AntibodyAnti-mouse CD45.2 (clone# 104) PE, APC, PE/Cy7, and A700Biolegend109807/14/29/21Dilution: 1:750
AntibodyAnti-mouse VB6 (clone# RR4-7) PE and APCBiolegend140003/5Dilution: 1:750
AntibodyAnti-mouse Va2 (clone# B20.1) APC/Cy7 and PerCP/Cy5.5Biolegend127818/13Dilution: 1:750
AntibodyAnti-mouse VB5 (clone# MR9-4) PEBiolegend139503Dilution: 1:750
AntibodyAnti-mouse F4/80 (clone# BM8) PE/Cy7Biolegend123113Dilution: 1:750
AntibodyAnti-mouse SIRPα (clone# P84) A700Biolegend144021Dilution: 1:750
AntibodyAnti-mouse CD24 (clone# M1/69) PEBiolegend101807Dilution: 1:750
AntibodyAnti-mouse CD101 (clone# Moushi101) PE/Cy7Thermo Fisher50-112-3316Dilution: 1:300
Strain, strain background (Mus musculus)OT-II TCR transgenic miceThe Jackson Laboratory (JAX)#004194
Strain, strain background (Mus musculus)Ccr7GFP knockin/knockout miceThe Jackson Laboratory (JAX)#027913
Strain, strain background (Mus musculus)Itgax-Cre miceThe Jackson Laboratory (JAX)#008068
Strain, strain background (Mus musculus)Irf4fl/fl miceThe Jackson Laboratory (JAX)#009380
Strain, strain background (Mus musculus)CT2 TCR transgenic miceNutsch et al., 2016
Strain, strain background (Mus musculus)CT6 TCR transgenic miceNutsch et al., 2016
Strain, strain background (Mus musculus)Rag1–/– miceThe Jackson Laboratory (JAX)#002216
Strain, strain background (Mus musculus)Foxp3IRES-GFPmiceThe Jackson Laboratory (JAX)#006772
Strain, strain background (Mus musculus)Foxp3IRES-Thy1.1miceListon 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–/– miceHildner et al., 2008
Strain, strain background (Mus musculus)Zbtb46GFP miceSatpathy et al., 2012
Strain, strain background (Mus musculus)CD207-DTA mice; formerly huLangerin-DTAKaplan et al., 2005

Additional files

Download links