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Type 3 innate lymphoid cells induce proliferation of CD94+ natural killer cells  Shuo Li, PhD, Hideaki Morita, MD, PhD, Beate Rückert, Sci Tec, Tadech.

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Presentation on theme: "Type 3 innate lymphoid cells induce proliferation of CD94+ natural killer cells  Shuo Li, PhD, Hideaki Morita, MD, PhD, Beate Rückert, Sci Tec, Tadech."— Presentation transcript:

1 Type 3 innate lymphoid cells induce proliferation of CD94+ natural killer cells 
Shuo Li, PhD, Hideaki Morita, MD, PhD, Beate Rückert, Sci Tec, Tadech Boonpiyathad, MD, Avidan Neumann, PhD, Cezmi A. Akdis, MD  Journal of Allergy and Clinical Immunology  Volume 140, Issue 4, Pages e7 (October 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Proliferation of ILC-PBMCCFSE coculture. ILC subsets were cultured with CFSE-labeled autologous PBMCs at a ratio of 1:20. The cultures were harvested on day 5 and analyzed by flow cytometry. A, FACS plots depicting proliferation of PBMCs in the presence of ILCs in general. Lymphocytes were gated on the basis of forward and side scatter, and gating out dead cells using a Fixable Viability Dye (eBioscience, Frankfurt, Germany). The region R1 (marked area) depicts the “dividing” (showing CFSE dilution) cells in the presence of ILCs. B, Of the total viable lymphocytes, the percentages (% in the y-axis) of “dividing” cells were shown for each coculture. FACS, Fluorescence-activated cell sorting; FSC-A, forward scatter-area; SSC-A, side scatter-area. Data derived from 6 blood samples in 4 independent experiments; ILC3 refers to NKp44− ILC3; Statistics by Mann-Whitney test, *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 ILC3 drives CD94+ cells expansion. A, Representative FACS plots depicting “dividing” cell subsets within PBMCs, gating on viable lymphocytes. B, Summary of data depicting the proliferation of various cell types in the presence of ILCs. Of each given coculture, the percentages (% in the y-axis) of various cell types within total viable lymphocytes were shown. C, Bar plot depicting the extent of CD94+ cell and CD3+ T-cell expansion in relevance to the parental cell populations, gating on viable lymphocytes: blue bars represent the percentage of proliferating CD94+ cells within total CD94+ cell; red bars the percentage of proliferating CD3+ cells within total CD3+ cell. FACS, Fluorescence-activated cell sorting. Data derived from 6 blood samples in 4 independent experiments; ILC3 refers to NKp44− ILC3; *P < .05, Wilcoxon matched-pairs signed rank test; Error bar = STDEV. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 ILC sorting strategy. Lymphocytes were gated on the basis of FSC and SSC, followed by the exclusion of dead cells using Fixable Viability Dye eFluor 780 (eBioscience). We focused on the lineage− CD127+ viable lymphocytes population, of which the CD161mid/+ cells were the main ILC-containing cell population. The 3 ILC subsets were then distinguished on the basis of their differential expression of c-kit, CRTH2, and NKp44: P1 is ILC1, P2 is ILC2, and P3.1 is NKp44− ILC3. EF780 stands for Fixable Viability Dye eFluor 780. FSC-A, Forward scatter-area; FSC-H, forward scatter-height; FSC-W, forward scatter-width; SSC-A, side scatter-area; SSC-H, side scatter-height; SSC-W, side scatter-width. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Additional characterization of proliferating cells in ILC-PBMC cocultures. FACS plots depicting proliferation profiles of CD4 and CD8 T cells gated on viable CD3+ lymphocytes (A), CD27 and CCR7 expression gated on viable lymphocytes (B), and the expression of CD16 in cocultured cells in relevance to CD94 expression, gated on viable lymphocytes (C). CD16+ cells are shown in red dots, and lymphocytes in blue dots. The missing panels indicate when there was not enough ILC to set up the culture. No ILC and coculture with ILC3 was used in all experiments. The data demonstrate that there is some CD4+ T-cell proliferation, but slight, if any, additional proliferation in CD8 T cells and CD27 and CCR7 positive cells over cells without ILC conditions. FACS, Fluorescence-activated cell sorting. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E2 Additional characterization of proliferating cells in ILC-PBMC cocultures. FACS plots depicting proliferation profiles of CD4 and CD8 T cells gated on viable CD3+ lymphocytes (A), CD27 and CCR7 expression gated on viable lymphocytes (B), and the expression of CD16 in cocultured cells in relevance to CD94 expression, gated on viable lymphocytes (C). CD16+ cells are shown in red dots, and lymphocytes in blue dots. The missing panels indicate when there was not enough ILC to set up the culture. No ILC and coculture with ILC3 was used in all experiments. The data demonstrate that there is some CD4+ T-cell proliferation, but slight, if any, additional proliferation in CD8 T cells and CD27 and CCR7 positive cells over cells without ILC conditions. FACS, Fluorescence-activated cell sorting. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E2 Additional characterization of proliferating cells in ILC-PBMC cocultures. FACS plots depicting proliferation profiles of CD4 and CD8 T cells gated on viable CD3+ lymphocytes (A), CD27 and CCR7 expression gated on viable lymphocytes (B), and the expression of CD16 in cocultured cells in relevance to CD94 expression, gated on viable lymphocytes (C). CD16+ cells are shown in red dots, and lymphocytes in blue dots. The missing panels indicate when there was not enough ILC to set up the culture. No ILC and coculture with ILC3 was used in all experiments. The data demonstrate that there is some CD4+ T-cell proliferation, but slight, if any, additional proliferation in CD8 T cells and CD27 and CCR7 positive cells over cells without ILC conditions. FACS, Fluorescence-activated cell sorting. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E3 Quantification of CD94 expression on ex vivo CD3+, CD4+, and CD16+ cells. Freshly isolated PBMCs were stained with Viability dye eFlour 780, followed by staining with fluorochrome-conjugated anti-CD3, anti-CD16, anti-CD45, and anti-CD94 mAbs. Cells were gated on live cells (Viability dye eFlour 780−) and then gated on CD45+CD94+ cells. CD45+CD3+ (T cells) and CD45+CD16+ (NK cells) were assessed for surface expression of CD94. The dot plot shows the representative gating strategy of CD94 expression on CD3+, CD4+, and CD16+ cell subsets. CD45+CD3+CD4+ (T helper cells) and CD45+CD3+CD16+ (NKT cells) expression on CD94 was gated on CD45+CD3+CD94+ cells and CD45+CD16+CD94+ cells, respectively. The numbers in the plot are averaged and represent the percentage of total CD45+CD94+ cells belonging to each cell subset. Representative data from N = 3 donors. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E4 Cytokine levels in supernatants. A-J, Cytokine concentrations were measured with a multiplex assay on day 5. Dots represent donor's cytokine measurement and the lines next to the dots indicate SEM (N = 3). Comparisons between no ILC, ILC1, ILC2, and ILC3 were performed using the Mann-Whitney test. *P < .05. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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