Fig 1: Thiol levels increase on the leucocyte cell surface during immune activation. PBMCs of two donors were mixed at a 1 : 1 ratio and co-cultured for 96 h. (a) Cells were removed at the time points indicated for flow cytometry analysis and thiols visualized by Alexa-488-maleimide staining. (b) Quantitative analysis of flow cytometry data shown in (a). Values of four biological replicates relative to time point 0 h ± standard deviation. Example raw flow cytometry data gated on T cells (expressing a/ß TCR chain) showing the increase of thiol levels (Alexa-488-maleimide) correlates to T cell activation (increase in CD69 expression) (c) at time 0 and (d) after 96 h. (e) Quantitative analysis of flow cytometry data at time points taken throughout the MLR. Normalized mean values relative to time point 0 h for three replicates ± s.e. of the mean.
Fig 2: ARL3-Specific Release Localizes LCK to the Immune Synapse(A and F) T:B cell conjugates stained with anti ARL3, LCK, or ARL13B antibodies as indicated in the presence or absence of SEE.(B) LR of ARL3, ARL13B, and LCK of conjugates from (A) and (F): ARL3 shows no localization to the synapse in the presence (LR = 0.3) or absence of SEE (n = 38). LCK shows LR = 1.9 in the presence of SEE and 0.7 in its absence (n = 42; p = 0.0001). ARL13B shows LR = 2 in the presence of SEE (n = 32) and 0.6 in its absence (n = 16; p = 0.0001).(C) ARL3WTGFP and the constitutively active ARL3Q71LGFP were nucleofected into cells and T:B cell conjugates, in the presence of SEE, were stained with anti LCK antibodies.(D) LR of LCK of conjugates from (C). ARL3WTGFP expression had little effect on LR of LCK (n = 18) compared to cells not expressing ARL3WTGFP (LR = 2). ARL3Q71LGFP-expressing cells showed LR of LCK = 1 (n = 16, p = 0.0002). Differences in LR were analyzed using the Mann-Whitney U test (B and D).(E) CD69 expression was analyzed via flow cytometry for cells stimulated with anti CD28 and CD3 antibodies and overexpressing ARL2-GFP, ARL2Q70LGFP, ARL3Q71LGFP, ARL3-GFP, or GFP. ARL3Q71LGFP (p = 0.0001), ARL3WTGFP (p = 0.004), ARL2WTGFP (p = 0.02), and ARL2Q70LGFP (p = 0.03) p values refer to unpaired t test data.(G) Ciliated RPE cell stained with anti ARL13B antibody.Scale bars, 5 µm.
Fig 3: Thioredoxin inhibition affects labile disulfide bond reduction but not T cell activation during immune response. (a) MLRs were set up as described in figure 5 and in the presence of the thioredoxin inhibitor PX-12, and analysed by flow cytometry at the time points indicated. Thiol levels (Alexa-488-maleimide staining of T cells, expressing a/ß TCR chain) were followed. The data are from two biological replicates. (b) T cell activation state (CD69 expression) throughout the MLR for both the control and PX-12-treated samples.
Fig 4: Ruxolitinib does not alter apoptosis threshold or CD69 expression in the presence of ingenol-3,20-dibenzoate. Ruxolitinib (100 nM) did not significantly alter the percentage of resting CD4+ T cells expressing activated caspase 3 (a) or expression of CD69 (represented as the mean florescence intensity or MFI with the total percentage of cells in each culture condition expressing CD69 within each bar; b) in the presence of ingenol-3,20-dibenzoate (ingenol DB). Mean values and standard deviation are shown for four independent experiments using resting CD4+ T cells from aviremic ART-treated participants (n = 4)
Fig 5: RHBDL2 regulates human T cell activation(A) TaqMan assays for RHBDL2 mRNA levels in T cells transduced with virus encoding control or RHBDL2 shRNAs. Error bars represent RQ standard error.(B) T cell activation measured by quantification of surface CD69 expression by fluorescence-activated cell sorting (FACS). CD69 expression is compared between control and RHBDL2 shRNA transduced primary CD4 T cells after stimulation with CD3. Each trace represents three biological replicates. In the dashed box, the calculated EC50 of anti-CD3 for each shRNA. Error indicates SEM.(C) SOCE is monitored by cytosolic Fura-2 fluorescence and compared between control and RHBDL2 shRNA transduced T cells. T cells were stimulated with 2 µM thapsigargin in Ca2+-free buffer, followed by readmission of 2 mM external Ca2+.(D and E) Aggregate data from T cells treated as in (C) are plotted, analyzing the peak Ca2+ level in each condition (D) and rate of Ca2+ entry (E). Each bar in (D) and (E) represents between 34 and 45 cells. For two-tailed t tests, ***p < 0.001, compared with control shRNA transduced T cells. Error bars represent SEM.
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