Journal: The Journal of allergy and clinical immunology

Article Title: DOCK8-DEFICIENT CD4 + T CELLS ARE BIASED TO A TH2 EFFECTOR FATE AT THE EXPENSE OF TH1 AND TH17 CELLS

doi: 10.1016/j.jaci.2016.07.016

Figure Lengend Snippet: DOCK8 deficient patients

Article Snippet: Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Virus, Infection, Milk, Chocolate

Journal: The Journal of allergy and clinical immunology

Article Title: DOCK8-DEFICIENT CD4 + T CELLS ARE BIASED TO A TH2 EFFECTOR FATE AT THE EXPENSE OF TH1 AND TH17 CELLS

doi: 10.1016/j.jaci.2016.07.016

Figure Lengend Snippet: (A) The frequency of CD4+ T cells in normal donors and DOCK8-deficient patients. (B, C) Naïve (CD45RA+CCR7+), central memory (TCM; CD45RA−CCR7+) and effector memory (TEM; CD45RA−CCR7−) populations in normal donors (closed symbol; n = 25) and DOCK8-deficient patients (open symbol; n = 18) were enumerated based on expression of CD45RA and CCR7. (D-G) PBMCs were labelled with mAbs against CD4, CD45RA, CD25, CD127, CXCR5, CXCR3 and CCR6. (D) Treg cells were identified as CD25hiCD127lo. (E) Amongst the non-Treg population naïve and Tfh cells were identified as CXCR5−CD45RA+ and CXCR5+CD45RA−, respectively. (F) Th1, Th2 and Th17 populations were identified within the population of CXCR5−CD45RA− memory CD4+ T cells as CXCR3+ CCR6−, CCR6−CXCR3− and CCR6+CXCR3− cells, respectively. (G) Using this gating the frequency of Tregs, Tfh, Th1, Th2 and Th17 cells within the CD4+ T cell compartment was determined in normal individuals (closed symbol; n = 15 or 16) and in DOCK8-deficient patients (open symbol; n = 10 or 11). Each point represents an individual donor or patient. Statistics performed with Prism using Student t-test. (H-M) Naïve (CD45RA+CCR7+), central memory (TCM; CD45RA−CCR7+) and effector memory (TEM; CD45RA−CCR7−) populations in normal donors (closed symbol) and DOCK8-deficient patients (open symbol) were identified and assessed for expression of (H) CD27, (I) CD28, (J) CD127, (K) CD57, (L) CD95 and (M) PD1. Each point corresponds to the mean ± SEM % of cells expressing the indicated surface receptor, or MFI (mean fluorescence intensity) of expression (n = 4 – 12 normal donors or DOCK8-deficient individuals). Statistics performed with Prism using t-test.

Article Snippet: Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Fluorescence

Journal: The Journal of allergy and clinical immunology

Article Title: DOCK8-DEFICIENT CD4 + T CELLS ARE BIASED TO A TH2 EFFECTOR FATE AT THE EXPENSE OF TH1 AND TH17 CELLS

doi: 10.1016/j.jaci.2016.07.016

Figure Lengend Snippet: Naïve and memory CD4+ T cells were sorted from normal donors and DOCK8-deficient patients and cultured with TAE beads for 5 days. After this time, culture supernatants were examined for secretion of (A) IL-4 (B) IL-5, (C) IL-13, (D) IFNγ, (E) TNF, (F) IL-17A, (G) IL-17F, (H) IL-6, (I) IL-10, (J) IL-2, using a custom designed cytometric bead array (CBA; BD biosciences). Data represent the mean ± SEM of experiments using cells from 9 normal donors or DOCK8-deficient patients. Statistics performed with Prism using One-way ANOVA. (K-L) Naive (K) and memory (L) CD4+ T cells were isolated from normal donors (n = 4) and DOCK8-deficient patients (n = 4), labelled with CFSE and cultured with TAE beads for 5 days. After this time, the frequency of cells in each division was determined by dilution of CFSE. (M) Sorted naïve and memory CD4+ were immediately restimulated with PMA/ionomycin for 6 hours in the presence of Brefeldin A and IL-4 expression determined by intracellular staining and flow cytometry. (N, O) Naive and memory CD4+ T cells were labelled with CFSE, cultured with TAE beads for 5 days, and the proportion of cells expressing (L) IFNγ or (M) IL-4 was determined for each division interval by dilution of CFSE. Data represent the mean ± SEM of 2 – 4 normal donors and DOCK8-deficient patients.

Article Snippet: Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Isolation, Expressing, Staining, Flow Cytometry

Journal: The Journal of allergy and clinical immunology

Article Title: DOCK8-DEFICIENT CD4 + T CELLS ARE BIASED TO A TH2 EFFECTOR FATE AT THE EXPENSE OF TH1 AND TH17 CELLS

doi: 10.1016/j.jaci.2016.07.016

Figure Lengend Snippet: Naïve (CD45RA+CCR7+) and memory (CD45RA−CCR7+/−) CD4+ T cells were isolated from normal donors and DOCK8-deficient patients and cultured with TAE beads for 5 days. Cells were then re-stimulated with PMA/ionomycin for 6 hours in the presence of Brefeldin A for the last 4 hours. Intracellular expression of (A) IL-2, (B) TNFα, (C) IL-4, (D) IFNγ, (E) IL-17A, (F) IL-22, (G) IL-21 and (H) IL-10 was determined using saponin as the permeabilising agent followed by flow cytometric analysis. Data represent the mean ± SEM of 8 normal donors or 8 DOCK8-deficient patients. Statistics performed with Prism using One-way ANOVA.

Article Snippet: Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology.

Techniques: Isolation, Cell Culture, Expressing

Journal: The Journal of allergy and clinical immunology

Article Title: DOCK8-DEFICIENT CD4 + T CELLS ARE BIASED TO A TH2 EFFECTOR FATE AT THE EXPENSE OF TH1 AND TH17 CELLS

doi: 10.1016/j.jaci.2016.07.016

Figure Lengend Snippet: (A) Naïve and (B) memory CD4+ T cells were isolated from normal donors and DOCK8-deficient patients and activated under neutral conditions (Th0; TAE only), or Th1- (+ IL-12), Th2- (+ IL-4), or Th17- (+ IL-1β, IL-6, IL-21, IL-23, TGFβ, PG) polarising conditions. After 5 days, secretion of Th1 (IFNγ), Th2 (IL-5, IL-13) and Th17 (IL-17A, IL-17F) cytokines was determined by CBA. The data represent the mean ± SEM of experiments using cells from 5 – 9 normal donors and DOCK8-deficient patients. Expression of (C) TBET and (D) GATA3 was determined by flow cytometry; the data represent the fold change (mean ± sem) in expression of the indicated transcription factor relative to Th0 culture of the normal control. (E) expression of RORC was determined by QPCR. Data represent the mean and SEM of 2 – 3 normal donors and DOCK8-deficient patients. Statistics performed with Prism using two-way ANOVA

Article Snippet: Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology.

Techniques: Isolation, Expressing, Flow Cytometry, Control

Journal: The Journal of allergy and clinical immunology

Article Title: DOCK8-DEFICIENT CD4 + T CELLS ARE BIASED TO A TH2 EFFECTOR FATE AT THE EXPENSE OF TH1 AND TH17 CELLS

doi: 10.1016/j.jaci.2016.07.016

Figure Lengend Snippet: Plasma from normal donors and DOCK8-deficient patients was analysed for IgE specific for (A) a staple food mix (egg white, milk, codfish, wheat, peanut and soyabean) and (B) a house dust mite mix by ImmunoCAP. The data represent the mean ± SEM of 13 normal donors and 15 DOCK8-deficient patients. The dotted line refers to the upper limit of the negative reference interval (0.35 kUA/L).

Article Snippet: Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology.

Techniques: Clinical Proteomics