Chronic cat allergen exposure induces a TH2 cell–dependent IgG4 response related to low sensitization Amedee Renand, PhD, Luis D. Archila, MSc, John.

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Presentation transcript:

Chronic cat allergen exposure induces a TH2 cell–dependent IgG4 response related to low sensitization Amedee Renand, PhD, Luis D. Archila, MSc, John McGinty, BSc, Erik Wambre, PhD, David Robinson, MD, Belinda J. Hales, PhD, Wayne R. Thomas, PhD, William W. Kwok, PhD Journal of Allergy and Clinical Immunology Volume 136, Issue 6, Pages 1627-1635.e13 (December 2015) DOI: 10.1016/j.jaci.2015.07.031 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Fel d 1– and Fel d 4–specific memory TH2 cells are predominant during cat allergy. A, CD154 expression after stimulation with cat allergen peptides or dimethyl sulfoxide (none). Numbers indicate the frequency of memory CD45RA−CD154+ T cells per 106 CD4+ T cells. B and C, Frequency of Fel d 1, Fel d 4, Fel d 7, and Fel d 8 (Fig 1, B) and combined Fel d 1 and Fel d 4 (Fig 1, C) memory CD154+ T cells after cat allergen peptide stimulation. D, CRTH2, CD27, CCR4, CXCR3, and CD154 expression on gated memory Fel d 1– or Fel d 4–specific T cells from 2 different allergic adults. The percentage of each marker on CD154+ T cells is as indicated. E, Frequency comparison of combined Fel d 1– and Fel d 4–specific memory CD154+CRTH2+ (top) or CD27− (bottom) T cells between allergic and nonallergic subjects. Data were compared by using the Mann-Whitney test. *P ≤ .05 and ***P ≤ .001. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Cat allergen exposure induces specific IgG4 and allergen-specific CD4+ TH2 cell responses in allergic adults. A, Combined Fel d 1– and Fel d 4–specific IgG4 levels in the serum of nonallergic and allergic adults. B, Specific IgG4/specific IgE ratio (log-transformed) in subjects with cat allergy with and without cat ownership. C, Combined frequency of Fel d 1– and Fel d 4–specific memory CD45RA−, CRTH2+, or CD27− CD154+ T cells. D, CD154 and cytokine expression on gated CD4+ T cells after Fel d 1 peptide stimulation. E, Comparison of cytokine expression of CD154+ T cells in allergic adults. F, Intracellular cytokine staining of DRB5∗0101 (DRB5)-restricted Fel d 1 chain 117-36–specific CD4+ T cells in a DRB5 cat-owning allergic adult by using tetramer staining. Percentages of IL-4+, IL-5+, IL-17+, IFN-γ+, and IL-10+ Fel d 1–specific CD4+ T cells are indicated. G, Cytokine expression of tetramer-positive Fel d 1–specific CD4+ T cells in allergic adults. Data were compared by using the Student unpaired t test (Fig 2, B), the Kruskal-Wallis test with the Dunn multiple comparisons test (Fig 2, A and C), or the Mann-Whitney test (Fig 2, E and G). *P ≤ .05, **P ≤ .01, and ***P ≤ .001. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Correlation between the TH2 cell response and specific IgE or specific IgG4 level depends on allergen exposure. A and B, Correlation between the combined frequency of Fel d 1– and Fel d 4–specific memory CD154+CRTH2+CD45RA−CD4+ T cells and the total cat dander–specific IgE level (Fig 3, A) or the combined Fel d 1– and Fel d 4–specific IgG4 level (Fig 3, B) in allergic adults with or without a cat. TRF, Time-resolved fluorometry. C and D, Comparison of the combined frequency of Fel d 1– and Fel d 4–specific memory CD154+CRTH2+ or CD27− cells and specific IgE levels between asthmatic and nonasthmatic allergic adults without (Fig 3, C) or with (Fig 3, D) a cat. Spearman R and P values were determined to analyze the correlation. Data were compared by using the Mann-Whitney test. **P ≤ .01. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Fel d 1 sTH2 cells support IgG4 production by allergen-experienced B cells. A-C, Total IgG4 levels in the culture supernatants of B cells from DRB5 and DR3 nonallergic (3 subjects, 10 clones; Fig 4, C) and allergic subjects without cat ownership (3 subjects, 14 clones; Fig 4, A) or with cat ownership (3 subjects, 16 clones; Fig 4, B) in the presence or absence of Fel d 1 chain 117-36– or Fel d 1 chain 217-36–specific CD4+ T clones from DRB5 or DR3 allergic subjects without a cat with or without the Fel d 1 chain 117-36 or Fel d 1 chain 217-36 peptide. D-F, CD38 expression on B cells is shown in the bottom panels. Each clone was tested on duplicate or triplicate wells, and a minimum of 4 experiments were done per group. Data were compared by using the Mann-Whitney test. ***P ≤ .001. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 TH2 cell–dependent IgG4 production in response to Fel d 1 recombinant protein. Total IgG4 levels in culture supernatants of B cells isolated from DRB5 and DR3 allergic (3 subjects, 11 clones; A) or nonallergic (3 subjects, 12 clones; B and C) subjects with or without CD4+ TH2 clones and Fel d 1 recombinant protein (40 μg/mL) and with autologous PBMCs depleted of B cells (APC; Fig 5, C). Each clone was tested in duplicate or triplicate wells, and a minimum of 4 experiments were done per group. Data were compared by using the Mann-Whitney test. ***P ≤ .05. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Gating strategies and methods for detection of allergen-specific CD4+ T cells. CD4+ T cells were gated after removing doublets and CD14+, CD19+, and dead cells (viability positive). PE-CD154+ or PE–peptide-MHC class II tetramer-positive cells were detected after anti-PE bead enrichment. FSC, Forward scatter; SSC, side scatter. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Characterization of the Fel d 1–specific T-cell response by using tetramer staining. A, DRB5/Fel d 1 chain 117-36 tetramer–positive CD4+ T cells in DRB5 nonallergic and allergic subjects. Frequencies of Fel d 1–specific CD4+ T cells per million CD4+ T cells are as indicated. B, Correlation between the frequency of Fel d 1–specific memory CD154+CD45RA−CD4+ T cells and the frequency of DRB5/Fel d 1 chain 117-36 and DRB3/Fel d 1 chain 217-36 tetramer–positive CD4+ T cells in DRB5 and DR0301 allergic adults. C, Frequency of Fel d 1–specific CD4+ T cells in DRB5 and DR0301 nonallergic and allergic adults. D, DRB5/Fel d 1 chain 117-36 tetramer–positive CD4+ T cells in a DRB5 cat-owning allergic subject. Frequencies of Fel d 1–specific CD4+ T cells per million CD4+ T cells and percentages of CRTH2, CD27, CCR4, and CXCR3 on Fel d 1–specific CD45RA−CD4+ T cells are as indicated. Spearman R and P values were determined to analyze the correlation. Data were compared by using the Mann-Whitney test. **P ≤ .01. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Frequency of cat allergen–specific naive CD45RA+CD4+ T cells in allergic and nonallergic subjects. Frequency of total naive CD45RA+CD154+CD4+ T cells after cat allergen peptide stimulation of PBMCs in 14 nonallergic and 24 allergic subjects. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Frequencies of Fel d 1 and Fel d 4 sTH2 cells do not correlate during cat allergy. A, Frequencies of Fel d 1– and Fel d 4–specific memory CD154+ T cells in each allergic subject. B, CD154 expression on gated CD4+ T cells after 3 hours of PBMC stimulation with or without cat allergen peptides. Results shown are after anti-PE bead enrichment in an allergic subject with a strong response against Fel d 4. Numbers indicate frequencies of CD154+CD4+ T cells per 106 CD4+ T cells. C, Absence of correlation between frequencies of Fel d 1– and Fel d 4–specific memory CD154+CD45RA−CD4+ T cells in allergic subjects. Spearman R and P values were determined to analyze the correlation. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 TH2 phenotype of Fel d 1–, Fel d 4–, and Fel d 7–specific memory T cells during cat allergy. A, Correlation between the frequency of memory CD154+CD45RA−CD4+ T cells and the percentage of CRTH2+, CD27−, and CCR4+ cells in CD154+CD45RA− memory CD4+ T cells after Fel d 1 or Fel d 4 peptide stimulation of allergic PBMCs. B, Correlation between the frequency of memory CD154+CD45RA−CD4+ T cells and the percentage of CRTH2+ and CD27− cells in CD154+CD45RA− memory CD4+ T cells after Fel d 7 peptide stimulation of allergic PBMCs. C and D, Frequency comparison of Fel d 7–specific memory CD154+CRTH2+ (Fig E5, C) or CD27− (Fig E5, D) CD4+ T cells between 24 allergic and 14 nonallergic subjects. Spearman R and P values were determined to analyze the correlation. Data were compared by using the Mann-Whitney test. ns, Not significant. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Fel d 1– and Fel d 4–specific IgG4 and Fel d 1–specific T-cell responses in relation to allergic status and cat ownership. A, Fel d 1–specific (left) and Fel d 4–specific (right) IgG4 levels from the serum of nonallergic subjects with or without a cat and allergic adults with or without a cat. TRF, Time-resolved fluorometry. B, Frequency of Fel d 1–specific memory CD154+CD45RA−, CD4+ T cells in nonallergic subjects without or with a cat, non–cat-owning allergic adults, and cat-owning allergic adults. Numbers indicate the frequency of CD154+CD4+ T cells per 106 CD4+ T cells. Data were compared by using the Kruskal-Wallis test with the Dunn multiple comparisons test. *P ≤ .05 and ***P ≤ .001. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 Analysis of TFH cell markers (CXCR5 and IL-21) on cat allergen–specific CD4+ T cells. A, CXCR5 and CD154 expression on gated memory CD45RA−CD4+ T cells from a cat-owning (left) or non–cat-owning (right) allergic adult. The percentage of CXCR5+ cells on CD154+CD45RA−CD4+ T cells is as indicated. B, Fel d 1 chain 117-36–specific CD4+ T cells in a DRB5 cat-owning allergic subject. Percentage of CXCR5 on Fel d 1–specific CD45RA−CD4+ T cells is as indicated. C, Intracellular IL-21 staining of Fel d 1 chain 117-36–specific CD4+ T cells in a DRB5 cat-owning allergic adult. Percentages of IL-21+ Fel d 1–specific CD4+ T cells are indicated. Data were compared by using the Mann-Whitney test. ns, Nonsignificant. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E8 Selection of Fel d 1 chain 117-36–specific CD4+ T clones after single-cell sorting and expansion. A, Fel d 1 chain 117-36–specific CD4+ T clones were selected based on their capacity to bind DRB5/Fel d 1 tetramer compared by using DRB5/empty tetramer. Dot plots represent examples of positive staining of 4 different Fel d 1 chain 117-36–specific CD4+ T clones by using the DRB5/empty (top) and DRB5/Fel d 1 (bottom) tetramer. B, Specificity of clones (n = 3) was also confirmed by using a proliferation assay (carboxyfluorescein succinimidyl ester [CFSE] dilution) with irrelevant peptide (Fel d 1 chain 11-20 peptide), Fel d 1 chain 117-36 peptide, Fel d 4 recombinant protein (rFel d 4), or Fel d 1 recombinant protein (rFel d 1) after 4 days of culture. Similar data were obtained with Fel d 1 chain 217-36 CD4+ T-cell clones derived from DR3 allergic subjects. **P ≤ .01. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E9 Gating strategy for CD38 expression on B cells during coculture with TH2 clones and peptide. CD38 expression on gated CD19+ B cells during culture with or without TH2 clones and peptides. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E10 TH2 profile of Fel d 1 chain 117-36–specific CD4+ T-cells clones from allergic adults with or without a cat. Fel d 1 chain 117-36–specific CD45RA−CD4+ T cells from DRB5 allergic subjects with or without a cat were ex vivo sorted and expanded in vitro (single-cell sorting). Dot plots represent examples of intracellular cytokine staining of 6 different Fel d 1 chain 117-36–specific CD4+ T-cell clones. Percentages of IL-4, IL-5, IL-10, IFN-γ, IL-17, and CD4 Fel d 1–specific T-cell clones are indicated. Histograms show CRTH2 expression (black) compared with isotype antibody (gray). Graphs summarize the CRTH2 mean fluorescence intensity (MFI) and cytokine profiles of TH2 clones from allergic adults with (10 clones) or without (12 clones) cat ownership. Data were compared by using the Mann-Whitney test. **P ≤ .01. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E11 Fel d 1 sTH2 clones from allergic adults with or without cat ownership support IgG4 production by B cells from allergic adults. Total IgG4 levels in the culture supernatant (left) and CD38 expression (right) on B cells from DRB5 or DR3 allergic subjects in the presence or absence of Fel d 1 chain 117-36– or Fel d 1 chain 217-36–specific CD4+ T-cell clones from DRB5 or DR3 allergic subjects without or with a cat with or without the Fel d 1 peptide (3 subjects, 7 clones). Each clone was tested on duplicate or triplicate wells. Data were compared by using the Mann-Whitney test. ***P ≤ .001. ns, Not significant. Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E12 Antigen specificity of Fel d 1 sTH2 cells in induction of IgG4. Total IgG4 levels were analyzed after 14 days in isolated B-cell culture from DRB5 allergic (A) or nonallergic subjects (without a cat; B) with or without autologous PBMCs (APC) depleted of B cells with or without DRB5 Fel d 1 chain 117-36–specific CD4+ TH2 clones, Fel d 1 (40 μg/mL), or Fel d 4 (40 μg/mL). Journal of Allergy and Clinical Immunology 2015 136, 1627-1635.e13DOI: (10.1016/j.jaci.2015.07.031) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions