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Journal Articles

Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation

In Special Collection:
Immunology: New Insights and Resources , Cytokines Collection 2020
Yajing Gao, Krystin Deason, Aakanksha Jain, Ricardo A. Irizarry-Caro, Igor Dozmorov, Laura A. Coughlin, Isabella Rauch, Bret M. Evers, Andrew Y. Koh, Edward K. Wakeland, Chandrashekhar Pasare
Journal: Journal of Experimental Medicine
J Exp Med (2020) 217 (4): e20190476.
https://doi.org/10.1084/jem.20190476
Published: 22 January 2020
View article titled, Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation
Open the PDF for Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation in another window
Includes: Supplementary data
Images
An in vitro priming approach to generate functional pathogen-specific Th cells.(A) Top: Purity of isolated naive CD4 T cells (CD4+MHCII−CD62L+CD44−) from two independent experiments. Bottom: Purity of CD11c+ DCs before or after isolation. (B) Overlay histograms of CFSE dilution curves from naive CD4 T cells primed with Cr-stimulated DCs (pathogen primed) or LPS stimulated DC + TCR ligation (LPS + anti-CD3). (C) CFSE−CD90+ percentage in Cr-primed CD4 T cells where 10 µg/ml blocking antibodies to mouse IA/IE (MHCII) or CD80/CD86 were added to the coculture. n = 2–3. (D) IFNγ+ percentage of Cr-primed CFSE−CD90+ T cells in the presence or absence of anti–IL-12p40 antibodies during differentiation. n = 3. (E) Proliferation of T cells in the priming system with the indicated pathogens, shown by CFSE dilution. Un, unstimulated. (F) Proliferation of pathogen- or commensal-specific T cells primed by DCs stimulated with heat-killed commensal bacteria Bf, La (MOI = 3), or 10 µg/ml Lm/Cr lysate. n = 2–3. (G and H) IFNγ (G) and IL-17A (H) quantities in the culture supernatant of CD4 T cells previously primed with Lm (left) or Cr (right) that were restimulated for 48 h with unstimulated (Un) or Lm/Cr-fed, irradiated B cells. Lm and Cr concentrations used for restimulation were titrated at 3, 10, and 30 µg/ml. Culture supernatants from anti-CD3 (30 ng/ml)-stimulated T cells were also assessed for IFNγ production as a positive control. n = 2 technical replicates. Data are representative of three independent experiments. (I) Histogram showing CD25+ percentage or mean fluorescence intensity (MFI) of CD69, ICOS, and CD44 (percentage and MFI shown on upper left corner) on the CD90+ T cells from the same experiments in G and H denoting up-regulation of indicated activation markers in response to Lm/Cr rechallenge. Lysate concentration = 10 µg/ml. (J) Quantified fold expansion of transferred (in vitro–primed with Lm or Cr) T cells (CD45.2+) in the peripheral lymph nodes (pLNs) of mice from the same experiments shown in Fig. 1 G. n = 7 mice per group. (K) Quantified MFI of surface ICOS on recipient endogenous CD45.1+ T cells from the same experiments shown in Fig. 1 G. n = 7 mice per group. Data are pooled from or representative of two to four independent experiments. Statistics represent mean ± SEM, and P values were determined by repeated-measures two-way ANOVA with Tukey correction (C), one-way ANOVA with Tukey correction (F), or paired Student’s t test (D and J). *, P < 0.05; **, P < 0.01.

An in vitro priming approach to generate functional pathogen-specific Th ce...

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure S1. An in vitro priming approach to generate functional pathogen-specific Th cells. (A) Top: Purity of isolated naive CD4 T cells (CD4+MHCII−CD62L+CD44−) from two independent experiments. Bottom: Purity of CD11c+ DCs before or after More about this image found in An in vitro priming approach to generate functional pathogen-specific Th ce...
Images
Validation of an in vitro–priming approach to generate functional pathogen-specific Th cells.(A) Schematic overview of the priming system and workflow. (B) Top row: Representative CFSE dilution graph and cytokine (IFNγ and IL-17A) staining from CFSE+ and CFSE− populations. Bottom row: Histogram of CD25, CD44, and ICOS from CFSE+ and CFSE− populations. Cells were cocultured for 10–12 d before analysis. (C) Representative intracellular cytokine staining of IFNγ- and IL-17A–producing Th cells following priming by CD11c+ DCs stimulated with Lm, Cr, or Sa lysates. (D) Secreted protein levels of IFNγ, IL-17A, and IL-13 in the supernatants of cocultures as in C, measured by ELISA. n = 5. Un, unstimulated. (E) Scatter plot of mRNA expression values (log10[reads per kilobase of transcript per million mapped reads/RPKM]) obtained from RNA-seq of Lm- or Cr-specific CFSE− Th cells. Data were averaged from two independent samples. Blue data points indicate differentially expressed genes (fold change ≥2). (F) Percentages of IFNγ- and IL-17A–producing cells in pathogen- or commensal-specific Th cells. Naive WT CD4 T cells were primed by DCs stimulated with heat-killed commensal bacteria Bf, La (multiplicity of infection [MOI] = 3) or 10 µg/ml Lm/Cr lysate. Intracellular cytokine was examined at day 10. n = 2–4. (G) Experimental design for testing in vivo specificity (top) and the mismatch scheme (bottom). (H) Expansion of transferred (in vitro primed with Lm or Cr) CD4 T cells in the spleen at day 5 after infection of Lm (shown as fold change comparing CD45.2+ percentage of infected mouse to paired PBS control). n = 7 mice per group. (I) Mean fluorescence intensity (MFI) of surface ICOS on donor CD45.2+ T cells from the same experiment as G and H. n = 7 mice per group. Data are representative or combined from two to five independent experiments. All plots are pregated on live cells. Error bars represent mean ± SEM, and P values were determined by paired Student’s t test (D, H, and I) or two-way ANOVA with Tukey correction (F). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Validation of an in vitro–priming approach to generate functional pathogen-...

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure 1. Validation of an in vitro–priming approach to generate functional pathogen-specific Th cells. (A) Schematic overview of the priming system and workflow. (B) Top row: Representative CFSE dilution graph and cytokine (IFNγ and IL-17A) More about this image found in Validation of an in vitro–priming approach to generate functional pathogen-...
Images
Comparative transcriptional analysis reveals caspase-1 as a T cell–intrinsic regulator of Th17 differentiation.(A) tdT+ percentage of unstimulated (Un), Lm-primed, Cr-primed, or Th17-polarized 17A-fm CD4 T cells. Un, n = 4; Lm and Cr, n = 5; Th17, n = 2. (B) Heatmap of Th17-associated cytokine and TF expression in CFSE+, CFSE−tdT−, and CFSE−tdT+ populations from Cr-primed T cells. (C) Representative flow plots showing IFNγ-YFP+ percentage of CFSE−tdT+ population, from 17-γ double reporter T cells under ppTh17 (Cr-primed) or cdTh17 conditions at early (day 5) and late (day 10) stages of differentiation. (D) Representative flow plots showing IFNγ-YFP+ percentage of CD4+CD90+tdT+ population, from mLN of 17-γ double reporter mice at 10 dpi of Cr. 17-fm mice that do not carry the YFP allele were used as negative signal control. (E) Confirmation of representative differentially expressed genes shown in Fig. 2 by qRT-PCR, n = 2. (F) Expression levels of Casp1 and Casp11 in murine T cell populations, extracted from the Immgen database. n = 2–3; heatmap is shown as mean expression value. SPN, Spleen; sLN, skin-draining lymph node; PP, Peyer’s patches. (G) WT or Casp1Δ10 naive T cells were primed for 10 d with WT DCs stimulated with heat-killed Bf (MOI = 3). Flow plot shows the percentage of IL-17A– and IFNγ-producing cells in live CFSE−CD90+ population. n = 2–3. (H) Overlay of CFSE dilution histograms (left) 10–12 d after Cr-priming and quantified CD4 T cell proliferation (CFSE−%; right). n = 5. Δ10, Casp1Δ10. (I) tdT+ percentage in CFSE−CD90+ CD4 T cell population primed with Cr-stimulated DCs in vitro for the indicated time. (J) Western blot analysis of procaspase-1 (p45) and cleaved caspase-1 (p20) of Cr-primed CD4 T cells. Cells are sorted into CFSEhigh (hi), CFSEintermediate (int), and CFSEnegative (neg) populations and compared with WT macrophages (MΦ) undergoing inflammasome activation (4 h LPS + 30 min ATP [L+A]). (K) WT naive CD4 T cells were primed with Cr-stimulated WT DCs. At day 7, indicated inhibitors were added to the culture. Casp1 expression in FACS-sorted CFSE−CD90+ T cells was determined at day 10. Concentration of the inhibitors used were p38i (SB203580), 5 µM; JNKi (CAS 129–56-6), 2 µM; MEKi (U0126), 2 µM; JAKi (Ruxolitinib), 500 nM; IRAK1/4i (CAS 509093–47-4), 5 µM; IKKi (CAS 873225–46-8), 500 nM. n = 2–3. (L) IL-17A+ percentage of CFSE−CD90+ WT or Casp1Δ10 CD4 T cells primed with Cr-stimulated WT DCs in vitro. DMSO or Z-YVAD-fmk (20 µM) was present in the culture medium throughout the priming period. n = 3. (M) IL-17A+ percentage of CFSE−CD90+ WT or Casp1Δ10 CD4 T cells primed with Cr-stimulated WT DCs in vitro. Cultures were supplemented with 10 ng/ml IL-1α or IL-1β. n = 4. (N) Bicistronic hCD2 expression on the surface of Th17 cells transduced with indicated vector. Data are pooled from or representative of at least two independent experiments. Statistics represent mean ± SEM, and P values were determined by paired Student’s t test (A and H) or paired two-way ANOVA (L and M). ns, not significant; *, P < 0.05.

Comparative transcriptional analysis reveals caspase-1 as a T cell–intrinsi...

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure S2. Comparative transcriptional analysis reveals caspase-1 as a T cell–intrinsic regulator of Th17 differentiation. (A) tdT+ percentage of unstimulated (Un), Lm-primed, Cr-primed, or Th17-polarized 17A-fm CD4 T cells. Un, n = 4; Lm and More about this image found in Comparative transcriptional analysis reveals caspase-1 as a T cell–intrinsi...
Images
Comparative transcriptional analysis reveals major divergence in programming between ppTh17 and cdTh17 cells. (A) Experimental design for transcriptional profiling of CFSE+ (naive), ppTh17 (Cr-specific, day 12), cdTh17 (day 5), or ex vivo tdT+ cells (10 dpi/peak of infection with 5 × 108 CFU of Cr). (B) Euclidean distance between global gene expression in naive CFSE+, ppTh17, cdTh17, or ex vivo tdT+ cells. CFSE+ and ex vivo tdT+, n = 2; ppTh17 and cdTh17, n = 3. (C) Number of genes shared or uniquely expressed by indicated three Th17 populations (compared with CFSE+ naive T cells) and their functional annotation enrichment analyzed by DAVID. FDR, false discovery rate. (D) Heatmap and hierarchical analysis of key T cell TFs, cytokines, and cytokine receptor expression from transcriptional profiling described in A. (E) Heatmap and hierarchical analysis of gene expression for gene cluster involved in in vivo T cell motility, migration, chemokine and chemokine receptor signaling, T cell positioning, and antigen sampling. (F) Heatmap and hierarchical analysis of gene expression for genes involved in metabolic processes. (G) Heatmaps of genes representing cell cycle/DNA-replication pathways and amino acid metabolism pathways. (H) GSEA analysis of ppTh17 and cdTh17 cells compared with Molecular Signature dataset of effector versus memory T cells. (I) Flow cytometry analysis of CD127 (IL-7Rα), CD215 (IL-15Rα), and c-Myc in ppTh17 and cdTh17 cells. Data are representative or combined from two to three independent experiments. In heatmaps, each row/column represents one independent sample. Hierarchical clustering was determined by Euclidean distance and pairwise average-linkage (B and D–G). Heatmap represents global expression z-score.

Comparative transcriptional analysis reveals major divergence in programmin...

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure 2. Comparative transcriptional analysis reveals major divergence in programming between ppTh17 and cdTh17 cells . (A) Experimental design for transcriptional profiling of CFSE+ (naive), ppTh17 (Cr-specific, day 12), cdTh17 (day 5), or ex More about this image found in Comparative transcriptional analysis reveals major divergence in programmin...
Images
Caspase-1 promotes the differentiation of Th17 lineage independent of its enzymatic activity or inflammasome activation.(A) Differentially expressed transcripts between ppTh17 and cdTh17 cells. Each dot represents the average of three independent experiments. Blue dots indicate differentially regulated genes (fold change >1.5, false discovery rate <0.05). Black dots indicate differentially regulated transcripts described in Fig. 2. Red dots indicate Casp1 and Casp11 transcripts. (B) mRNA expression of Casp1 relative to 18s rRNA in sorted naive (CFSE+), ppTh17, cdTh17, or ex vivo tdT+ cells from mLNs of Cr-infected 17A-fm mice (10 dpi), quantified by independent qRT-PCR experiments. n = 2. (C) Naive CD4 T cells from WT or Casp1Δ10 (Δ10) mice were primed in vitro by Cr lysate–stimulated WT splenic CD11c+ DCs, and IL-17A– and IFNγ-producing cells were measured by intracellular cytokine staining and flow cytometry analysis of CD90+CFSE− live CD4 T cells (left); IL-17A+ percentages were quantified (right). n = 7. (D) IL-17A in the supernatant from experiments in C, measured by ELISA. n = 3. Δ10, Casp1Δ10. (E) IFNγ+ and IL-13+ percentage of CD90+CFSE− live cells, quantified from experiments in C. n = 7. Δ10, Casp1Δ10. (F) Relative expression (normalized to 18s rRNA and relative to day 3) of Casp1 mRNA at indicated time points after Cr-priming. n = 4. (G) Western blot analysis of procaspase-1 (p45) and cleaved caspase-1 (p20) from naive CD4 T cells, sorted CD90+CFSE− Cr-primed Th cells, or WT bone marrow–derived macrophages (MΦ) that were unstimulated or under conventional inflammasome activation (4 h LPS + 30 min ATP [L+A]). (H) Naive WT, Casp1Δ10 (Δ10), Pycard−/−, Il1b−/− CD4 T cells were primed with Cr-stimulated WT DCs. IL-17A+ percentage of CFSE−CD90+ live cells was measured by intracellular cytokine staining and quantified. n = 5. (I) Casp1Δ10 CD4 T cells were differentiated to Th17 lineage and retrovirally reconstituted with MSCV-IRES-hCD2 alone (Vector), full-length Casp1 (FL), Casp1 deficient of CARD (Casp1ΔCARD), or enzymatically (Enz) inactive form of Casp1 (EnzDead, C284A) and quantified for IL-17A+ percentage (gated on live, hCD2+ population). n = 4. (J) Log2(fold change) of major Th cell cytokine and TF expression comparing WT and Casp1Δ10 Cr-specific Th cells. WT or Casp1Δ10 naive CD4 T cells were primed with Cr-stimulated WT DCs for 10 d. CFSE−CD90+ population was FACS-sorted and subjected to mRNA-seq and analysis. n = 2. (K) Heatmap and hierarchical clustering of differentially expressed (>1.5 fold) genes comparing WT and Casp1Δ10 (Δ10) Cr-specific Th cells. Genes of interest were labeled by the heatmap. Each column represents one independent replicate. (L) Activation z-score of pathways enriched in WT or Casp1Δ10 Cr-specific Th cells. Pathway analysis was performed using Ingenuity Pathway Analysis. Data are representative of or combined from two to seven independent experiments. Statistics represent mean ± SEM, and P values were determined by paired Student’s t test (C–E and H) or one-way ANOVA with Tukey correction (I). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Caspase-1 promotes the differentiation of Th17 lineage independent of its e...

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure 3. Caspase-1 promotes the differentiation of Th17 lineage independent of its enzymatic activity or inflammasome activation. (A) Differentially expressed transcripts between ppTh17 and cdTh17 cells. Each dot represents the average of More about this image found in Caspase-1 promotes the differentiation of Th17 lineage independent of its e...
Images
T cell–intrinsic caspase-1 is required for host protection and Th17-mediated autoinflammatory disease.(A) Naive CD4 T cells from WT or Casp1Δ10 (Δ10) mice were isolated and polarized to cdTh17 cells. Representative flow cytometry plot (left) and quantifications of IL-17A+ percentage (right) are shown. n = 4. (B) Representative histogram overlay of CFSE dilution from experiments in A. (C) CFSE− percentage of transferred CD4 T cells, examined at 10 dpi. n = 8–9 mice per group. Δ10, Casp1Δ10. (D) Percentage of cytokine-positive cells in CD90+CD4− population (ILCs) from the same experiments in Fig. 4. n = 5 mice per group. (E) Colonic crypt length measured from histology images in Fig. 5 D. n = 3–5 mice per group. NT, nontransferred (i.p. PBS). Δ10, Casp1Δ10. (F and G) WT and Casp1Δ10 CD45RBhi CD4 cells were transferred to Rag1−/− mice. 3 wk after transfer, LPL cells were isolated and stained with FAM-FLICA to detect activation of caspase-1. FLICA+PI+ indicates inflammasome-activated cells. WT and Casp1Δ10 bone marrow–derived DCs (BMDCs) that were treated with inflammasome-activating ligands (4 h LPS [100 ng/ml] and 30-min 5 mM ATP) were used as positive and negative controls for FLICA staining. Representative flow plot is shown in F and quantified in G; n = 2–4/group. (H and I) IL-17A+IFNγ− percentage (H) and IFNγ+IL-17A− percentage (I) of CD4+CD90+CD44+ T cells in the mLN or colonic LP (Colon-LPL) of Rag1−/− mice 4 wk after transfer of WT or Casp1Δ10 naive CD4 T cells. n = 8–13 mice per group. (J) Percentages and numbers of CD4+CD90+ T cells from Colon-LPL of Rag1−/− mice 4 wk after transfer of WT or Casp1Δ10 naive CD4 T cells. n = 6–7 mice per group. Data are representative of or pooled from two to four independent experiments. Statistics represent mean ± SEM, and P values were determined by paired Student’s t test (A), one-way ANOVA with Tukey correction (E), or unpaired Student’s t test (G–I). ns, not significant; *, P < 0.05; ***, P < 0.001.

T cell–intrinsic caspase-1 is required for host protection and Th17-mediate...

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure S3. T cell–intrinsic caspase-1 is required for host protection and Th17-mediated autoinflammatory disease. (A) Naive CD4 T cells from WT or Casp1Δ10 (Δ10) mice were isolated and polarized to cdTh17 cells. Representative flow cytometry More about this image found in T cell–intrinsic caspase-1 is required for host protection and Th17-mediate...
Images
T cell–intrinsic caspase-1 is required for host protection against Cr.(A) Schematic of the experiment. (B) Representative intracellular cytokine staining of IFNγ, IL-22, and IL-17A in CD4+CD90+CD44+ T cells in the mLNs at 10 dpi of Cr. (C) Quantification of IL-17A+IL-22−, IL-17A+IL-22+, IL-17A−IL-22+ percentages (left) and IFNγ+IL-17A+%, IFNγ+IL-17A− percentages (right). n = 7–8 mice per group. (D) Cr burden (quantified as CFU) in the cecum at 10 dpi (left). Also shown is the Cr burden when normalized to cecum content weight (right). NT, nontransferred (i.v. PBS). n = 4–9 mice per group. (E) Representative image of stool appearance (left) and quantified stool water content (right, as percentage of water in each pellet). n = 4–7 mice per group. Data are representative of or combined from two independent experiments. Each data point represents one biological replicate. Statistics represent mean ± SEM, and P values were determined by unpaired Student’s t test (C) or one-way ANOVA (D and E). ns, not significant; *, P < 0.05; **, P < 0.01.

T cell–intrinsic caspase-1 is required for host protection against Cr

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure 4. T cell–intrinsic caspase-1 is required for host protection against Cr. (A) Schematic of the experiment. (B) Representative intracellular cytokine staining of IFNγ, IL-22, and IL-17A in CD4+CD90+CD44+ T cells in the mLNs at 10 dpi of More about this image found in T cell–intrinsic caspase-1 is required for host protection against Cr
Images
T cell–intrinsic caspase-1 is required for Th17-mediated colitis.(A) Weight change of Rag1−/− mice that received WT, Casp1Δ10, or Il1b−/− naive CD4 T cells (CD45RBhi) at indicated time points (n = 6–10 mice for each group). NT, nontransferred (i.p. PBS). (B) Percentage of initial body weight 4 wk after transfer. n = 12–19 mice per group. (C) Representative image of colons (left) and measured colon length (right) of Rag1−/− mice at 4–5 wk after transfer. n = 4–8 mice per group. (D) Representative H&E staining of colon sections from NT, WT, or Casp1Δ10 (Δ10) naive T cell–transferred Rag1−/− mice (left) and histology score (right). Images are displayed at 20× magnification. Ruler bar represents 100 µm. n = 12–14 mice per group. (E) Representative flow plots showing the percentages of IL-17A+IFNγ+ of CD4+CD90+CD44+ T cells in the mLNs or colonic LP (Colon-LPL) of Rag1−/− mice 4 wk after transfer of WT or Casp1Δ10 (Δ10) naive CD4 T cells. n = 8–13 mice per group. (F) The number of CD4+CD90+IL-17A+ T cells in the spleens of the Rag1−/− mice that received WT or Casp1Δ10 (Δ10) naive CD4 T cells. n = 6 per group. (G) Serum IL-17A levels at the indicated time points from the mice in Fig. 5 F. n = 5 mice per group. Data are representative of or combined from two to four independent experiments. Each data point represents one biological replicate. Statistics represent mean ± SEM and P values were determined by two-way repeated ANOVA with Bonferroni correction (A), one-way ANOVA (B and C), Mann-Whitney U test (D), unpaired Student’s t test (E and F) or multiple t tests with Holm-Sidak correction (G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

T cell–intrinsic caspase-1 is required for Th17-mediated colitis. (A) Wei...

in Transcriptional profiling identifies caspase-1 as a T cell–intrinsic regulator of Th17 differentiation > Journal of Experimental Medicine
Published: 22 January 2020
Figure 5. T cell–intrinsic caspase-1 is required for Th17-mediated colitis. (A) Weight change of Rag1−/− mice that received WT, Casp1Δ10, or Il1b−/− naive CD4 T cells (CD45RBhi) at indicated time points (n = 6–10 mice for each group). NT, More about this image found in T cell–intrinsic caspase-1 is required for Th17-mediated colitis. (A) Wei...
Journal Articles

Human IgA binds a diverse array of commensal bacteria

In Special Collection:
Immunology: New Insights and Resources , Tissue Immune Responses: Immune Cells and Mechanisms
Delphine Sterlin, Jehane Fadlallah, Olivia Adams, Claire Fieschi, Christophe Parizot, Karim Dorgham, Asok Rajkumar, Gaëlle Autaa, Hela El-Kafsi, Jean-Luc Charuel, Catherine Juste, Friederike Jönsson, Thomas Candela, Hedda Wardemann, Alexandra Aubry, Carmen Capito, Hélène Brisson, Christophe Tresallet, Richard D. Cummings, Martin Larsen, Hans Yssel, Stephan von Gunten, Guy Gorochov
Journal: Journal of Experimental Medicine
J Exp Med (2019) 217 (3): e20181635.
https://doi.org/10.1084/jem.20181635
Published: 31 December 2019
View article titled, Human IgA binds a diverse array of commensal bacteria
Open the PDF for Human IgA binds a diverse array of commensal bacteria in another window
Includes: Supplementary data
Images
Carbohydrate-binding profile of polyclonal IgA1 and IgA2 antibodies.(A) Glycan reactivity of serum polyclonal IgA1 and IgA2 (n = 5 healthy donors pooled in one experiment). Each peak represents an individual glycan recognized by IgA1 (blue line) or IgA2 (red line). (B) Heatmap diagram depicting glycans recognized by IgA1 or IgA2. Each row represents an individual glycan. (C) Preferential recognition of distinct terminal carbohydrate moieties by serum polyclonal IgA1, IgA2, or both. Terminal carbohydrate moieties equally recognized by both IgA1 and IgA2 are depicted in white. Terminal moieties preferentially recognized by IgA1 or IgA2 are showed in gray or black, respectively. (D) Isotype-dependent recognition of Galα-terminal structures of bacterial origin and other bacterial antigens excluding Galα-terminal antigens (without Galα) by serum polyclonal IgA, as compared with entire glycan microarray dataset. Terminal carbohydrate moieties equally recognized by both IgA1 and IgA2 are depicted in white. Terminal moieties preferentially recognized by IgA1 or IgA2 are showed in gray or black, respectively. (E) Differential isotype binding to bacterial attachment sites.

Carbohydrate-binding profile of polyclonal IgA1 and IgA2 antibodies. (A) ...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure 1. Carbohydrate-binding profile of polyclonal IgA1 and IgA2 antibodies. (A) Glycan reactivity of serum polyclonal IgA1 and IgA2 (n = 5 healthy donors pooled in one experiment). Each peak represents an individual glycan recognized by IgA1 More about this image found in Carbohydrate-binding profile of polyclonal IgA1 and IgA2 antibodies. (A) ...
Images
Gut bacteria segregate into IgAbright and IgAlow fractions in healthy humans. Related to Figs. 1 and 2. (A) The secretory component binds a modest range of carbohydrates. Glycan reactivity with the secretory component was assessed using glycan microarray technology (660 structures). Representative median RFUs are shown. Glycans specifically recognized by secretory component are colored in red. (B) Anti-IgA1 and anti-IgA2 antibodies do not cross-react. Flow cytometry analysis of IgA1 and IgA2 expression on peripheral B cells from a healthy donor. (C) IgA-coated bacteria split into IgAbright and IgAlow fractions depending on IgA1 and IgA2 coating. Representative flow cytometry analysis of IgA1 and IgA2 coating in IgAbright-coated bacteria (red lines), IgAlow-coated bacteria (blue lines), and IgA-unbound bacteria (gray lines). (D) Representative flow cytometric analysis of colon and ileum microbiota (left and central panels, respectively) with anti-IgA FITC or isotype-matched control antibody, as indicated. Numbers indicate percentage of positive cells. Data are cumulative from three independent experiments. Boxes extend from the 25th to the 75th percentiles. Error bars represent minimum and maximum values. P values were defined using the Mann-Whitney test. ***, P < 0.001. SSC-A, side scatter area. Quantification of IgA-coated bacteria in stool (n = 20) and ileum (n = 5).

Gut bacteria segregate into IgAbright and IgAlow frac...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure S1. Gut bacteria segregate into IgAbright and IgAlow fractions in healthy humans. Related to Figs. 1 and 2 . (A) The secretory component binds a modest range of carbohydrates. Glycan reactivity with the secretory component was More about this image found in Gut bacteria segregate into IgAbright and IgAlow frac...
Images
IgA1 targets IgA2-coated bacteria.(A) Representative flow cytometry analysis of endogenous IgA1 and IgA2 fecal or ileal microbiota coating. (B) Endogenous IgA1 and IgA2 microbiota coating levels in 20 fecal and 5 ileal healthy samples. Data are cumulative from three independent experiments. Error bars represent minimum and maximum values. P values were calculated using the Mann-Whitney test (colon vs. ileum) or Wilcoxon test (*, P < 0.05; ***, P < 0.001). (C) Endogenous IgM microbiota coating levels in 20 fecal and 5 ileal healthy samples (two independent experiments). Dark bars indicate medians. Red dotted line represent significant the positive cutoff. P values were calculated using the Mann-Whitney test (***, P < 0.001). (D) Left: Representative flow cytometric analysis of endogenous IgM-coated fecal or ileal microbiota. Right: Representative flow cytometric analysis of endogenous IgA1 and IgA2 binding in IgM-coated fecal or ileal microbiota (two independent experiments). SSC-A, side scatter area. (E) Endogenous IgM microbiota coating levels in 20 fecal and 5 ileal healthy samples (two independent experiments). Error bars represent minimum and maximum values. P values were calculated using the Mann-Whitney test (**, P < 0.01; *, P < 0.05). (F) Model Venn diagram showing overlap among endogenous IgA1, IgA2, and IgM binding in IgA-coated microbiota. (G) Endogenous IgA1 and IgA2 microbiota–coating levels in eight fecal samples from formula-fed neonates (two independent experiments). Error bars represent minimum and maximum values. P values were calculated using the Wilcoxon test (*, P < 0.05; **, P < 0.01). (H) Flow cytometric analysis of IgA1 and IgA2 expression on colonic lamina propria B cells from a 3-mo-old infant (one experiment).

IgA1 targets IgA2-coated bacteria. (A) Representative flow cytometry anal...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure 2. IgA1 targets IgA2-coated bacteria. (A) Representative flow cytometry analysis of endogenous IgA1 and IgA2 fecal or ileal microbiota coating. (B) Endogenous IgA1 and IgA2 microbiota coating levels in 20 fecal and 5 ileal healthy More about this image found in IgA1 targets IgA2-coated bacteria. (A) Representative flow cytometry anal...
Images
IgA1+IgA2+- and IgA2+-sorted fractions show distinct compositions within the same donor and between donors. Related to Fig. 4. (A) Sorting strategy of IgA1- and IgA2-coated bacteria (representative of five independent experiments). SSC-A, side scatter area. (B) Relative composition of phyla in fecal samples (input). Each column corresponds to one healthy donor (HD) sample out of five analysed (HD1 to HD5, as indicated). (C) Genera diversity of input, IgA1+, and IgA2+ fractions calculated using the Shannon index. Boxes extend from the 25th to the 75th percentiles. Error bars represent minimum and maximum values. (D) Relative composition of genera in fecal samples (input). Each column corresponds to one sample. The 25 most abundant genera are shown. (E) Relative abundance of families in input and sorted fractions from five healthy donors. The top 16 most abundant families are shown.

IgA1+IgA2+- and IgA2+-sorted fractions sho...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure S2. IgA1+IgA2+- and IgA2+-sorted fractions show distinct compositions within the same donor and between donors . Related to Fig. 4 . (A) Sorting strategy of IgA1- and IgA2-coated bacteria (representative of five independent More about this image found in IgA1+IgA2+- and IgA2+-sorted fractions sho...
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IgA1 coordinates with IgA2 to coat distinct commensal bacteria.(A) Median relative abundance of the four most frequent families in sorted fractions from five healthy donors. Each dot represents one donor. Boxes extend from the 25th to the 75th percentiles. Error bars represent minimum and maximum values. (B) Median relative abundance of genera from IgA1+IgA2+ and IgA2+ fractions from five healthy donors. Each dot represents one donor. (C) Relative abundance of indicated top 19 most abundant genera in sorted fractions from one healthy donor. (D) Specificity of IgA subclass targeting for all individuals analyzed (n = 5). Number of samples in which a given genera had a positive indicated IgA subclass EI, divided by the total number of samples. The formula is detailed in Materials and methods. (E)Flavobacterium enrichment in IgA2+ as compared with IgA1+IgA2+ fractions. Each dot represents one donor. P values were defined using the Wilcoxon test (*, P < 0.05). (F) Binding of purified breast milk IgA to indicated bacterial strains. Monoclonal IgA isotype control (gray-filled histogram) was included as negative flow cytometry control. The same experiment was repeated twice. ns, not significant.

IgA1 coordinates with IgA2 to coat distinct commensal bacteria. (A) Media...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure 3. IgA1 coordinates with IgA2 to coat distinct commensal bacteria. (A) Median relative abundance of the four most frequent families in sorted fractions from five healthy donors. Each dot represents one donor. Boxes extend from the 25th More about this image found in IgA1 coordinates with IgA2 to coat distinct commensal bacteria. (A) Media...
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IgA1- and IgA2-coated bacteria promote cytokine production by macrophages.(A) One out of three representative flow cytometric analyses of human gut microbiota purified from an IgA-deficient donor incubated with breast milk IgA and subsequently with anti-IgA FITC (three independent experiments). Numbers indicate percentage of positive cells. (B) Cytokine levels measured using Simoa technology in supernatants of macrophages incubated for 24 h with heat-killed S. haemolyticus opsonized with IgA1 (blue) or IgA2 (red) or without IgA (gray). The Mann-Whitney test was used to calculate P values; ns, not significant (n = 3 healthy donors, two independent measurements). Error bars indicate maximum values.

IgA1- and IgA2-coated bacteria promote cytokine production by macrophages. ...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure S3. IgA1- and IgA2-coated bacteria promote cytokine production by macrophages. (A) One out of three representative flow cytometric analyses of human gut microbiota purified from an IgA-deficient donor incubated with breast milk IgA and More about this image found in IgA1- and IgA2-coated bacteria promote cytokine production by macrophages. ...
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Human monoclonal IgA bind a broad but nevertheless private pattern of commensals. Related to Fig. 4. (A) Flow cytometric sorting of intestinal IgA+ memory B cells (defined as CD19+, cell surface IgA+ IgD−). Sort gate among CD19+ B cells is shown. Doublets and dead cells were excluded before CD19 gating, CD19+ cells were gated among CD45+ cells (not shown). Representative images from three independent sorts are shown. (B) Transduced B cells exhibited a stable germinal center–like phenotype and maintained IgA expression. Transduced B cells were surface-labeled with anti-CD38, anti-CD95, or anti-IgA (orange lines) or appropriate isotype antibody controls (gray dotted lines). Representative images of eight monoclonal B cell lines, evaluated in three independent experiments, are shown. (C) Monoclonal B cell lines produced dimeric IgA. Representative immunoblotting showing high molecular weight dimeric mAb in nonreducing conditions for mAb 8. Representative image of eight mAbs and two independent experiments are shown. (D) Representative flow cytometric plot of microbiota B stained with anti-IgG Alexa Fluor 647 and anti-IgA FITC. The same experiment was repeated twice. (E) Heatmap diagram of EI of the 50 most frequent genera from microbiota A. Hierarchical clustering grouped mAb+ fractions and genera. (F) Heatmap diagram of EI of the 50 most frequent genera from microbiota B. Hierarchical clustering grouped mAb+ fractions and genera. (G) Flow cytometric analysis of mAb or negative control (mAb− supernatant [left] or irrelevant IgG [right, anti-TNFα IgG1]) staining of pure bacterial strains (two independent experiments).

Human monoclonal IgA bind a broad but nevertheless private pattern of comme...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure S4. Human monoclonal IgA bind a broad but nevertheless private pattern of commensals. Related to Fig. 4 . (A) Flow cytometric sorting of intestinal IgA+ memory B cells (defined as CD19+, cell surface IgA+ IgD−). Sort gate among CD19+ B More about this image found in Human monoclonal IgA bind a broad but nevertheless private pattern of comme...
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Human monoclonal IgA target highly diverse commensal bacteria.(A) Representative flow cytometry plot of microbiota reactivity for mAb#2 and human IgA2 anti-TNP. mAbs 1–8 are gut monoclonal IgA2 expressed as dimeric IgA. SSC-A, side scatter area. (B) mAb coating of IgA-free microbiota. mAbs are classified in increasing fluorescence intensity order (median fluorescence intensity [MFI], range 2,080–10,724). The same experiment was repeated twice. (C) Somatic mutations of mAbs are not correlated to IgA staining intensity. Somatic mutations in the V-region of IGH gene were analyzed. Nonparametric Spearman correlation was calculated. (D) Representative flow cytometry plot of microbiota reactivity for mAb 10 and human IgG1 anti-TNFα. mAbs 9–16 are gut monoclonal IgA2 expressed as IgG1 (Benckert et al., 2011). (E) mAb coating of IgA and IgG-free microbiota. MAbs are classified in increasing fluorescence intensity order (MFI, range 3,585–17,683). The same experiment was repeated twice. (F) Somatic mutations of mAbs are not correlated to mAb staining intensity. Somatic mutations in V-region of IGH gene were analyzed. Nonparametric Spearman correlation was calculated. (G) mAb+ and mAb− fractions of IgA-free gut microbiota were sorted by flow cytometry, and their composition was analyzed by 16S rRNA sequencing. (H) Relative abundance of phyla in whole microbiota (input) and mAb+ fractions. Microbiota A is IgA-free, while microbiota B is IgA- and IgG-free. 16S rRNA sequencing data were from two independent experiments. (I) Relative abundance of genera in whole microbiota A (input) and mAb+ fractions. (J) Relative abundance of genera in whole microbiota B (input) and mAb+ fractions. (K) Scatter dot plot of median relative abundance of genera from mAb+ and mAb− fractions. ns, not significant.

Human monoclonal IgA target highly diverse commensal bacteria. (A) Repres...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure 4. Human monoclonal IgA target highly diverse commensal bacteria. (A) Representative flow cytometry plot of microbiota reactivity for mAb#2 and human IgA2 anti-TNP. mAbs 1–8 are gut monoclonal IgA2 expressed as dimeric IgA. SSC-A, side More about this image found in Human monoclonal IgA target highly diverse commensal bacteria. (A) Repres...
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Microbial surface glycans are common targets of gut human IgA.(A) Glycan reactivity for five mAbs (5 µg/ml) was assessed using glycan microarray technology (660 structures). Representative median RFUs of mAb1 are shown. Glycan ID numbers of top-bound glycans are indicated. (B) The median RFUs for top-bound glycans (above background) are shown for all five mAbs tested. (C) The ABR of glycans showing positive reactivity (above background, as in I) was calculated using an isotype control and visualized in an ordered (by dendrogram algorithm) reactivity matrix (heatmap). Each column represents a glycan. Color key is shown. (D) Representative flow cytometric dot plot of microbiota-reactivity for mAb 6 after whole-microbiota enzymatic deglycosylation (left). Decrease of mAb binding to microbiota after enzymatic deglycosylation (right). Columns and error bars represent median and maximum values, respectively. The same experiment was repeated twice. SSC-A, side scatter area. (E) Representative dot blots of purified PG and lipoteichoic acid (TA) from S. aureus and S. haemolyticus probed with irrelevant IgG1 (anti-TNFα IgG1), mAb 9, and mAb 10 (gray, red, and green circles, respectively). mAbs 9 and 10 were generated with IgG1 Fc domain. The same experiment was repeated twice.

Microbial surface glycans are common targets of gut human IgA. (A) Glycan...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure 5. Microbial surface glycans are common targets of gut human IgA. (A) Glycan reactivity for five mAbs (5 µg/ml) was assessed using glycan microarray technology (660 structures). Representative median RFUs of mAb1 are shown. Glycan ID More about this image found in Microbial surface glycans are common targets of gut human IgA. (A) Glycan...
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Self-reactivity and glycan reactivity of antigen-selected secretory IgA.(A) Median frequency of nonsilent (black) and silent (gray) somatic mutations in CDRs and VH FWRs in mAb IGH genes (n = 16 mAbs, four independent experiments). (B) Self-reactivity was tested by IFA with HEp-2000 cells: (1) negative control; (2) positive control: autoimmune human serum containing anti-DNA; (3) purified IgA from fecal water (20 µg/ml); (4) negative staining representative of 15 nonreactive mAbs; and (5) mAb 4. Representative images of three independent experiments are shown.

Self-reactivity and glycan reactivity of antigen-selected secretory IgA . ...

in Human IgA binds a diverse array of commensal bacteria > Journal of Experimental Medicine
Published: 31 December 2019
Figure S5. Self-reactivity and glycan reactivity of antigen-selected secretory IgA . (A) Median frequency of nonsilent (black) and silent (gray) somatic mutations in CDRs and VH FWRs in mAb IGH genes (n = 16 mAbs, four independent More about this image found in Self-reactivity and glycan reactivity of antigen-selected secretory IgA . ...

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