Journal: The Journal of Experimental Medicine

Article Title: A positive feedback loop reinforces the allergic immune response in human peanut allergy

doi: 10.1084/jem.20201793

Figure Lengend Snippet: A positive feedback loop between the adaptive and innate immune systems in food allergy. Allergen exposure induces antigen-specific CD4 + T cells to secrete IL-4 and IL-13 in food-allergic patients. These Th2 cytokines drive the differentiation of monocytes into CD209 + MDDCs and the up-regulation CD23 on myeloid CD11c + DCs. This in turn facilitates the uptake and HLA presentation of allergen, reinforcing the adaptive immune response to allergen.

Article Snippet: In some cases, PBMCs were cultured with cow’s milk protein (100 µg/ml), recombinant human IL-4 (500 U/ml; R&D Systems), recombinant human IL-13 (50 U/ml; R&D Systems), recombinant human IL-5 (500 U/ml; R&D Systems), recombinant human GM-CSF (1,000 U/ml; R&D Systems), or human IL-4 combined with human GM-CSF for 3 d.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: A positive feedback loop reinforces the allergic immune response in human peanut allergy

doi: 10.1084/jem.20201793

Figure Lengend Snippet: Peanut allergen augments CD23 expression on myeloid CD11c + DCs. (A) Representative flow cytometry plots gated on lineage (CD3, CD19, CD56) negative (Lin − ) HLA-DR + cells show the effect of peanut protein on CD14 − CD16 − CD23 + CD11c + DCs for one NA and one PA nontwin participant. (B) Box plots overlaid with dot plots show the percentage of CD23 + CD11c + DCs per total PBMCs from five NA and nine PA nontwin participants with or without peanut stimulation. (C) Representative mass cytometry plots gated on lineage (CD3, CD19, CD56, CD123) negative (Lin − ) HLA-DR + cells show the effect of peanut protein on CD14 − CD16 − CD23 + CD11c + DCs for one NA and one PA nontwin participant. (D) Percentage CD23 + CD11c + DCs per total PBMCs incubated with or without peanut protein are shown in box plots overlaid with dot plots for five NA and five PA nontwin participants. (E) Top: Representative flow cytometry plots showing the effect of peanut protein on the expression of CD23 and CD209 by Lin − HLA-DR + CD14 − CD16 − CD11c + DCs from one PA nontwin participant. The frequencies of the gated populations are shown. Bottom left: Distribution of CD209 and CD23 expression among CD11c + DCs that expressed one or both markers. Bottom right: Among CD11c + DCs that expressed CD209 and/or CD23, pie charts showing the average proportion of cells expressing one or the other marker. PBMCs from PA individuals were incubated with peanut protein ( n = 9). (F and G) Left: Representative flow cytometry plots gated on Lin − HLA-DR + CD14 − CD16 − CD11c + DCs show the expression of CD23 (F) or CD209 (G) on CFSE − or CFSE + cells for one PA nontwin participant. The cells from the same subject stimulated with IL-4 for 3 d are evaluated as the positive control for the expression of CD23 or CD209 on CFSE + or CFSE − cells. Right: Percentage of CD23 + (F) or CD209 + (G) DCs segregated by CFSE staining for four PA nontwin participants. Box plots in B, D, F, and G represent IQR and median; whiskers extend to the farthest data point within a maximum of 1.5× IQR. Paired sample sets were analyzed using the Wilcoxon signed rank test (two sided). Each pair of points connected by a line represents one subject. Unpaired sample sets were analyzed using the Wilcoxon rank sum test (two sided). *, P < 0.05; **, P < 0.01.

Article Snippet: In some cases, PBMCs were cultured with cow’s milk protein (100 µg/ml), recombinant human IL-4 (500 U/ml; R&D Systems), recombinant human IL-13 (50 U/ml; R&D Systems), recombinant human IL-5 (500 U/ml; R&D Systems), recombinant human GM-CSF (1,000 U/ml; R&D Systems), or human IL-4 combined with human GM-CSF for 3 d.

Techniques: Expressing, Flow Cytometry, Mass Cytometry, Incubation, Marker, Positive Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: A positive feedback loop reinforces the allergic immune response in human peanut allergy

doi: 10.1084/jem.20201793

Figure Lengend Snippet: Th2 cytokines promote monocyte differentiation into CD209 + DCs, and blocking GM-CSF partially reverses the decrease in monocyte frequency induced by peanut protein in PA participants. (A) The expression levels of secreted Th2 cytokines from the PBMCs cultured with or without peanut protein from six pairs of discordant twin siblings for peanut allergy. Each dot represents a single participant, colored according to different treatment. Each pair of points connected by a line represents one subject. (B) Representative flow cytometry plots gated on lineage (CD3, CD19, CD56) negative cells show the change of CFSE + monocytes and CFSE + CD209 + CD11C + DCs by IL-4, IL-13, IL-5, GM-CSF, or IL4 + GM-CSF from buffy coats. (C–G) Percentages of CFSE + monocytes in Lin − CFSE + cells (left) and of CFSE + CD209 + CD11c + DCs in Lin − CFSE + cells (middle) or in CFSE + CD11c + DCs (right) from the PBMCs treated with IL-4 (C), IL-13 (D), IL4 + GM-CSF (E), GM-CSF (F), or IL-5 (G) from three buffy coats. (H) Percentages of monocytes in PBMCs are shown in box plots overlaid with dot plots from five PA participants treated with peanut protein + anti-GM-CSF antibody and peanut protein + isotype control for anti-GM-CSF antibody (mouse IgG1). Box plots in A and C–H represent the interquartile range (IQR) and median; whiskers extend to the farthest data point within a maximum of 1.5× IQR. Each pair of points connected by a line represents one subject. Paired sample sets were analyzed using the Wilcoxon signed rank test (two sided). Unpaired sample sets were analyzed using the Wilcoxon rank sum test (two sided). *, P < 0.05; **, P < 0.01.

Article Snippet: In some cases, PBMCs were cultured with cow’s milk protein (100 µg/ml), recombinant human IL-4 (500 U/ml; R&D Systems), recombinant human IL-13 (50 U/ml; R&D Systems), recombinant human IL-5 (500 U/ml; R&D Systems), recombinant human GM-CSF (1,000 U/ml; R&D Systems), or human IL-4 combined with human GM-CSF for 3 d.

Techniques: Blocking Assay, Expressing, Cell Culture, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: A positive feedback loop reinforces the allergic immune response in human peanut allergy

doi: 10.1084/jem.20201793

Figure Lengend Snippet: Signaling through IL4RA is responsible for the expression of CD209 and CD23 by DCs after peanut allergen stimulation. (A) Representative flow cytometry plots gated on Lin − cells show the effect of peanut protein, anti-IL4RA blocking antibody, and IL-4 on CFSE-labeled monocytes and CD209 + CD11c + DCs for one PA nontwin participant. (B and C) The percentage of CFSE + CD209 + CD11c + DCs per Lin − CFSE + cells (B, left) or Lin − CFSE + CD11c + DCs (B, right), as well as the percentage of CFSE + monocytes per Lin − CFSE + cells (C), are shown for seven PA nontwin participants. (D) Representative mass cytometry plots gated on Lin − HLA-DR + CD14 − CD16 − cells show the effects of peanut protein, IL-4, GM-CSF, and blocking antibodies to IL-4 or GM-CSF on CD23 + CD11c + DCs for one PA nontwin participant. (E–G) Percentages of CD23 + CD11c + DCs per total PBMCs from five PA nontwin participants treated with different combinations of peanut protein, IL-4, GM-CSF, and blocking antibodies against IL-4RA or GM-CSF. Box plots in B, C, and E–G represent IQR and median; whiskers extend to the farthest data point within a maximum of 1.5× IQR. Each pair of points connected by a line represents one subject. Paired sample sets were analyzed using the Wilcoxon signed rank test (two sided). Unpaired sample sets were analyzed using the Wilcoxon rank sum test (two sided). For the Wilcoxon signed rank comparisons, the lowest possible P value attainable for our analysis with five PA individuals is 0.0625. *, P < 0.05.

Article Snippet: In some cases, PBMCs were cultured with cow’s milk protein (100 µg/ml), recombinant human IL-4 (500 U/ml; R&D Systems), recombinant human IL-13 (50 U/ml; R&D Systems), recombinant human IL-5 (500 U/ml; R&D Systems), recombinant human GM-CSF (1,000 U/ml; R&D Systems), or human IL-4 combined with human GM-CSF for 3 d.

Techniques: Expressing, Flow Cytometry, Blocking Assay, Labeling, Mass Cytometry

Journal: The Journal of Experimental Medicine

Article Title: A positive feedback loop reinforces the allergic immune response in human peanut allergy

doi: 10.1084/jem.20201793

Figure Lengend Snippet: T cells are the major source of IL-4 and IL-13 responsible for monocyte and DC differentiation following peanut protein stimulation. (A and B) Representative mass cytometry plots showing CD4 + activated T cells as measured by CD25 and CD154 expression, identified by backgating from IL4 + cells (A) and IL-13 + cells (B) for one NA and one PA participant. (C and D) Percentage of IL-4 + (C) and IL-13 + (D) cells per total PBMCs incubated with or without peanut protein for NA versus PA participants. (E and F) Percentage of different cell types (T cells, B cells, natural killer cells, CD11c + DCs, and monocytes) in IL-4 + cells (left) or IL-13 + cells (right; E). Percentage of CD4 + and CD8 + T cells in IL-4 + CD3 + T cells (left) or IL-13 + CD3 + T cells (right; F). Each dot represents a single subject color-coded according to allergic status and peanut protein stimulation status. NK, natural killer. (G–I) Left: Representative mass cytometry plots show the effect of peanut protein on CD4 + T cells for the expression of CD25 and IL-4 (G), IL-13 (H), or CD154 (I) for one NA and one PA participant. Right: Percentage of CD25 + IL-4 + (G), CD25 + IL-13 + (H), and CD25 + CD154 + (I) CD4 + T cells for PBMCs from NA and PA participants incubated with or without peanut protein. (J and K) Mass cytometry backgating based on IL-4 and IL-13: Percentage of IL-4 + CD25 + CD154 + CD4 + T cells (J) and IL-13 + CD25 + CD154 + CD4 + T cells (K) per total PBMCs incubated with or without peanut protein for NA versus PA participants. Box plots in C, D, and G–K represent IQR and median; whiskers extend to the farthest data point within a maximum of 1.5× IQR. Blue circles represent twin participants ( n = 6 for each group); open circles represent nontwin participants (NA, n = 20; PA, n = 16). Each pair of points connected by a line represents one subject. Paired sample sets were analyzed using the Wilcoxon signed rank test (two sided). Unpaired sample sets were analyzed using the Wilcoxon rank sum test (two sided). ***, P < 0.001.

Article Snippet: In some cases, PBMCs were cultured with cow’s milk protein (100 µg/ml), recombinant human IL-4 (500 U/ml; R&D Systems), recombinant human IL-13 (50 U/ml; R&D Systems), recombinant human IL-5 (500 U/ml; R&D Systems), recombinant human GM-CSF (1,000 U/ml; R&D Systems), or human IL-4 combined with human GM-CSF for 3 d.

Techniques: Mass Cytometry, Expressing, Incubation

Journal: The Journal of Experimental Medicine

Article Title: A positive feedback loop reinforces the allergic immune response in human peanut allergy

doi: 10.1084/jem.20201793

Figure Lengend Snippet: CD209 promotes Th2 cytokine expression by peanut-specific T cells and correlates negatively with peanut OIT. (A–C) Blocking CD209 expression in CD11c + DCs reduces the production of Th2 cytokines (IL-4 and IL-13) from peanut-activated CD4 + T cells by peanut protein in PA participants. Representative flow cytometry plots gated on lineage (CD3, CD19, CD56) negative (Lin − ) HLA-DR + CD14 − CD16 − cells show the effect of anti-CD209 blocking antibody on CD209 + CD11c + DCs for one PA nontwin participant (A). Representative flow cytometry plots gated on CD4 + T cells show the effect of anti-CD209 blocking antibody on the expression of CD25, IL-4, and IL-13 for one PA nontwin participant (B). Percentage of CD4 + T cells expressing CD25 and either IL-4 (left) or IL-13 (right) for six PA nontwin participants (C). (D–G) The induction of CD209 + CD11c + DCs and peanut-specific CD4 + T cells after 3-d incubation with peanut protein is reduced by OIT. Representative flow cytometry plots gated on lineage (CD3, CD19, CD56) negative (Lin − ) HLA-DR + cells showing the effect of OIT on CD209 + CD11c + DCs for one PA participant (D). Percentage of CD209 + CD11c + DCs per total PBMCs (top) and per CD11c + DCs (bottom) are shown for six PA participants before and during OIT (E). Representative flow cytometry plots gated on CD4 + T cells show the effect of OIT on expression of the activation markers CD25 and CD154 for one PA participant (F). Percentage of CD25 + CD154 + cells per total CD4 + cells for six PA subjects before and during OIT (G). Box plots in 9, C, E, and G represent IQR and median; whiskers extend to the farthest data point within a maximum of 1.5× IQR. Each pair of points connected by a line represents one subject. Paired sample sets were analyzed using the Wilcoxon signed rank test (two sided). Unpaired sample sets were analyzed using the Wilcoxon rank sum test (two sided). *, P < 0.05.

Article Snippet: In some cases, PBMCs were cultured with cow’s milk protein (100 µg/ml), recombinant human IL-4 (500 U/ml; R&D Systems), recombinant human IL-13 (50 U/ml; R&D Systems), recombinant human IL-5 (500 U/ml; R&D Systems), recombinant human GM-CSF (1,000 U/ml; R&D Systems), or human IL-4 combined with human GM-CSF for 3 d.

Techniques: Expressing, Blocking Assay, Flow Cytometry, Incubation, Activation Assay