Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2009 Sep 3.

Published in final edited form as: Nat Immunol. 2008 Mar 9;9(4):432–443. doi: 10.1038/ni1574

Impaired Ca²⁺ influx, cytokine production and proliferation in double-knockout T cells. (a) Store-operated Ca²⁺ influx in naive CD4⁺ T cells from littermate control mice (CTRL; Stim1^fl/flStim2^fl/fl) and double-knockout mice (DKO; Stim1^fl/flStim2^fl/fl CD4-Cre), stimulated with thapsigargin (top) or with anti-CD3 followed by crosslinking with streptavidin (SA; bottom) in nominally Ca²⁺-free Ringer’s solution, followed by perfusion with 2 mM Ca²⁺ in Ringer’s solution to induce Ca²⁺ influx (left). Middle, quantification of peak [Ca²⁺]_i in 2 mM Ca²⁺ in Ringer’s solution. Right, single-cell ‘false-color’ images showing [Ca²⁺]_i at the peak of the Ca²⁺ influx response. Original magnification, ×20. (b) Production of IL-2 and tumor necrosis factor (TNF) by naive CD4⁺ T cells from control (Stim1^+/+ CD4-Cre or Stim1^fl/flStim2^fl/fl) or double-knockout mice, stimulated for 6 h with PMA and ionomycin. (c) Expression of CD25 and CD69 on naive CD4⁺ T cells from control (Stim1^+/+CD4-Cre or Stim1^fl/flStim2^fl/fl) or double-knockout mice, left unstimulated (Unstim; control cells) or stimulated for 16 h with anti-CD3 and anti-CD28 (control (CTRL) or double-knockout (DKO) cells). (d) Proliferation of naive CD4⁺CD25^– T cells from control (Stim1^fl/flStim2^fl/fl) or double-knockout mice, stimulated for 72 h with anti-CD3 and anti-CD28 and assessed by CFSE labeling. Open histograms, stimulated cells; shaded histograms, unstimulated cells. Numbers above bracketed lines indicate number of cell divisions. (e) Cells undergoing zero to five cell divisions (from data in d). Stim1^+/+ or Stim2^+/+ CD4-Cre mice were used initially as controls, after which Stim1^+/+ CD4-Cre, Stim2^+/+ CD4-Cre and Stim1^fl/flStim2^fl/fl mice were used. Data are representative of three (a) or two (b—d) independent experiments (error bars (a), s.e.m.).

Impaired Ca²⁺ influx, cytokine production and proliferation in double-knockout T cells. (a) Store-operated Ca²⁺ influx in naive CD4⁺ T cells from littermate control mice (CTRL; Stim1^fl/flStim2^fl/fl) and double-knockout mice (DKO; Stim1^fl/flStim2^fl/fl CD4-Cre), stimulated with thapsigargin (top) or with anti-CD3 followed by crosslinking with streptavidin (SA; bottom) in nominally Ca²⁺-free Ringer’s solution, followed by perfusion with 2 mM Ca²⁺ in Ringer’s solution to induce Ca²⁺ influx (left). Middle, quantification of peak [Ca²⁺]_i in 2 mM Ca²⁺ in Ringer’s solution. Right, single-cell ‘false-color’ images showing [Ca²⁺]_i at the peak of the Ca²⁺ influx response. Original magnification, ×20. (b) Production of IL-2 and tumor necrosis factor (TNF) by naive CD4⁺ T cells from control (Stim1^+/+ CD4-Cre or Stim1^fl/flStim2^fl/fl) or double-knockout mice, stimulated for 6 h with PMA and ionomycin. (c) Expression of CD25 and CD69 on naive CD4⁺ T cells from control (Stim1^+/+CD4-Cre or Stim1^fl/flStim2^fl/fl) or double-knockout mice, left unstimulated (Unstim; control cells) or stimulated for 16 h with anti-CD3 and anti-CD28 (control (CTRL) or double-knockout (DKO) cells). (d) Proliferation of naive CD4⁺CD25^– T cells from control (Stim1^fl/flStim2^fl/fl) or double-knockout mice, stimulated for 72 h with anti-CD3 and anti-CD28 and assessed by CFSE labeling. Open histograms, stimulated cells; shaded histograms, unstimulated cells. Numbers above bracketed lines indicate number of cell divisions. (e) Cells undergoing zero to five cell divisions (from data in d). Stim1^+/+ or Stim2^+/+ CD4-Cre mice were used initially as controls, after which Stim1^+/+ CD4-Cre, Stim2^+/+ CD4-Cre and Stim1^fl/flStim2^fl/fl mice were used. Data are representative of three (a) or two (b—d) independent experiments (error bars (a), s.e.m.).