dock8 protein  (Proteintech)

Journal: Nature immunology

Article Title: Migration-induced cell shattering due to DOCK8-deficiency causes a type-2 biased helper T cell response

doi: 10.1038/s41590-020-0795-1

Figure Lengend Snippet: a, Peripheral blood mononuclear cells from either control or DOCK8-deficient humans restimulated with PMA/ionomycin; data are representative of 4 DOCK8-deficient and 13 healthy humans. b-d, Lung cells from wild type (WT) or Dock8 deficient (KO) mice 20 days post C. neoformans infection were restimulated ex vivo and production of IL-4, IL-5, IL-13, and TNFα assessed by flow cytometry. A representative flow cytometry plot (gated on CD4+ FoxP3− CD44hi T cells) from a WT and KO mouse where numbers indicate percent cells in each quadrant (b). Data are summarized from 4 independent experiments (c). Frequency of activated CD4+ T cells making any of the Th2 cytokines IL-4 or IL-5 or IL-13, determined as in (b,c) from 4 independent experiments (d). e, Representative histograms of T helper lineage transcription factor GATA-3 expression measured by flow cytometry in WT naïve (CD44lo, shaded) or WT versus KO activated (CD44hi) CD4+ T cells in the lung on day 20 post C. neoformans infection (left) and summary of relative fluorescent intensity of GATA-3 (RFI) from 3 independent experiments (right). f-h, Total C. neoformans colony forming units (CFU) in the lung (f), and number of total immune cells (g) or eosinophils (h) in the lung 20 days p.i. with C. neoformans compiled from 4-5 independent experiments. i, Representative hematoxylin- and eosin-stained lungs of C. neoformans-infected lung from WT and KO mice 20 days p.i. Scale bars are 1mm. Insets are magnified 14x. In all graphs error bars represent median ± min/max; each data point is from an individual mouse.

Article Snippet: Janssen et al described direct binding of DOCK8 to the WASP-interacting protein (WIP), suggesting that DOCK8 regulates F-actin assembly 21 .

Techniques: Control, Infection, Ex Vivo, Flow Cytometry, Expressing, Staining

Journal: Nature immunology

Article Title: Migration-induced cell shattering due to DOCK8-deficiency causes a type-2 biased helper T cell response

doi: 10.1038/s41590-020-0795-1

Figure Lengend Snippet: a, Percent of immune cells undergoing apoptosis (apop, AnnexinV+) or are dead (AnnexinV−, viability stain+) in the lung on day 8 or 20 post C. neoformans infection. Data from 2-3 independent experiments. b-d, Frequency of cleaved caspase-3+ cells analyzed by flow cytometry 8 or 20 days post-infection with C. neoformans in DCs, granulocytes, lymphocytes and non-DC MNPs (gated as in Supplementary Fig. 3b). Representative histograms from day 20 (b) and data summarized from 2 independent experiments for DCs and non-DC MNPs in WT and KO mice (c), and data from CX3CR1-Dock8−/− mice or controls treated as in Fig. 3f (d). e-h, WT and KO LifeAct-GFP+ BMDCs migrating in a 1.7mg/ml collagen matrix ex vivo. Still images taken at 0 and 3 hours from two representative movies, Supplementary Video 2 (e). Relative viability measured 45 min from start of migration assay as compared to cells kept in media for the same duration in 2 independent experiments (f). BMDC cell area, perimeter, major and minor axes quantified after 3 hours of migration, summarized from 5 movies and 3 mice per group; n=2106 WT, n=4479 KO cells (g). Example confocal images of individual WT and KO BMDCs (h). i-k, Dock8−/− mice were treated with a pan-caspase inhibitor (Q-VD-OPh) on day 1, 3, 5, and 7 post C. neoformans infection (i), and on day 8 post infection number of activated (CD44hi) CD4+ T cells (j) and frequency of activated CD4+ T cells making Th2 cytokines (IL-4, IL-5 or IL-13) or GM-CSF following ex vivo restimulation (k) was assessed in the lung. Data from 2 independent experiments. In all graphs error bars represent median ± min/max except in (f) where mean ± SD is shown; each data point is from an individual mouse except in (g) where each data point is from an individual cell.

Article Snippet: Janssen et al described direct binding of DOCK8 to the WASP-interacting protein (WIP), suggesting that DOCK8 regulates F-actin assembly 21 .

Techniques: Staining, Infection, Flow Cytometry, Ex Vivo, Migration

Journal: Nature immunology

Article Title: Migration-induced cell shattering due to DOCK8-deficiency causes a type-2 biased helper T cell response

doi: 10.1038/s41590-020-0795-1

Figure Lengend Snippet: a, Immune cell population counts found in the lung at day 20 p.i. with C. neoformans in WT (x-axis) compared to KO (y-axis) mice, gated as shown in Supplementary Fig. 3b. Dotted line is drawn where WT and KO cell numbers would be equal. Each point represents the mean ± S.D. of 15 (WT) and 17 (KO) mice determined in 3 independent experiments. b, Lung cells from WT, KO, or Dock8fl/fl mice crossed to Lck, CD11c, LysM or eosinophil-specific cre lines were harvested 20 days post C. neoformans infection, restimulated ex vivo and production of IL-4, IL-5, or IL-13 assessed by flow cytometry. c,d, Congenically labelled (CD45.1+) T cells from WT mice were transferred into DOCK8 KO (CD45.2+) recipients that were infected with C. neoformans 7 days later (c) and percent of Th2 cytokine- or GM-CSF- producing CD4+ T cells was assessed in 2 independent experiments for WT and KO mice, as well as WT T cells in KO recipients (d). e, CX3CR1 expression on lung cell populations 20 days post-infection with C. neoformans shown as a representative histogram from 3 independent experiments. f-h, WT, Dock8 KO, and CX3CR1-Dock8−/− mice were infected with C. neoformans, with a tamoxifen treatment schedule as shown (f). Number of activated CD4+ T cells (g) and frequency of type-2 cytokine and GMCSF producing lung CD4+ T cells (h) measured 8 days post-infection. Control group includes: WT mice (black circles), corn-oil treated Dock8fl/fl.CX3CR1-ERcre+ (white circles), and tamoxifen-treated Dock8fl/fl.CX3CR1-ERcre− (gold circles). Data are from 3 independent experiments. In all graphs except (a), error bars represent median ± min/max; each data point is from an individual mouse.

Article Snippet: Janssen et al described direct binding of DOCK8 to the WASP-interacting protein (WIP), suggesting that DOCK8 regulates F-actin assembly 21 .

Techniques: Infection, Ex Vivo, Flow Cytometry, Expressing, Control

Journal: Nature immunology

Article Title: Migration-induced cell shattering due to DOCK8-deficiency causes a type-2 biased helper T cell response

doi: 10.1038/s41590-020-0795-1

Figure Lengend Snippet: a,b, BMDC were kept in dissociated culture (non-migrating) or added to 1.7mg/ml collagen matrices. Caspase and MLKL activation was examined after 40 min by western blot. Pro-caspase 3 gel is shown at high and low exposure (hi exp, lo exp). Two independent replicates are shown per group (a). IL-1β released was quantified after 3 hours by ELISA in 2 independent experiments. Each data point represents a BMDC batch generated from an individual mouse (b). c-d, Cytokine production was measured in mice infected with C. neoformans (day 20) either in BAL fluid by cytokine array (c) or in whole lung by ELISA (d). Data in (c) is from 4 mice per group and shown as fold change relative to WT group mean. Data in (d) is from 2 independent experiments. e-g, WT, KO and Dock8/IL-1R double KO mice (KO x IL1R KO) were infected with C. neoformans and on day 20 post infection number of activated (CD44hi) CD4+ T cells (e), frequency of activated CD4+ T cells making Th2 cytokines (IL-4, IL-5 or IL-13) or GM-CSF following ex vivo restimulation (f), and eosinophil numbers (g) were evaluated in the lung. Data are from 2 independent experiments. h-k, Congenically labelled (CD45.2+) T cells from IL1R KO mice were transferred into CD45.1+ TCRβ KO or Dock8/TCRβ double KO recipients that were infected with C. neoformans (h) and on day 20 p.i. number of activated (CD44hi) CD4+ T cells (i), frequency of activated CD4+ T cells making Th2 cytokines (IL-4, IL-5 or IL-13) or GM-w ex vivo restimulation (j), and eosinophil numbers in the lung was assessed in both recipient groups compared to Dock8−/− mice (k). Data are from 3 independent experiments. l, Dock8−/− mice were treated with a pan-caspase inhibitor (Q-VD-OPh) with or without IL-1β on day 1, 3, 5, and 7 post C. neoformans infection, and on day 8 post infection the frequency of activated CD4+ T cells making Th2 cytokines (IL-4, IL-5 or IL-13) following ex vivo restimulation was assessed in the lung. Data from 2 independent experiments. For all graphs each data point is from an individual mouse. In (b,d) lines represent means; in all other graphs error bars represent median ± min/max.

Article Snippet: Janssen et al described direct binding of DOCK8 to the WASP-interacting protein (WIP), suggesting that DOCK8 regulates F-actin assembly 21 .

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Infection, Ex Vivo