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1.
Figure 5

Figure 5. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Cytotoxic T-lymphocyte antigen (CTLA)-4–CD80/CD86 interactions modulate intracellular Ca2+ concentration ([Ca2+]i) in CD4+ T cells activated with antiCD3. [Ca2+]i was measured after different times of activation with soluble anti-CD3 (sol aCD3) or plate-bound anti-CD3 (pb aCD3). Mean fluorescence intensities (MFIs) normalized to the baseline level in unactivated cells, which was taken as unity, are shown in (a). [Ca2+]i was measured at 12 and 24 hr in the presence or absence of CTLA-4–CD80/CD86 blockade. The MFIs were normalized to that of cells activated with anti-CD3, which was taken as unity (b and c). Data shown are mean ± standard error for three independent experiments. **P < 0·05.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
2.
Figure 9

Figure 9. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Model depicting the distinct effects of cytotoxic T-lymphocyte antigen (CTLA)-4–CD80/CD86 interactions under low and high signal strengths of T-cell activation. Activation of T cells with plate-bound anti-CD3 (pb aCD3), compared with soluble anti-CD3 (sol aCD3), demonstrates higher intracellular Ca2+ concentration ([Ca2+]i), reactive oxygen species (ROS) level and interleukin (IL)-2 production. The reduction in [Ca2+]i was greater in T cells activated with sol aCD3, compared with pb aCD3, and blockade of CTLA-4–CD80/CD86 interactions. Under activation with both pb and sol aCD3 and CTLA-4 blockade, the reductions in ROS amounts were similar. Despite the role of CTLA-4–CD80/CD86 interactions in enhancing [Ca2+]i and ROS levels, the functional outcomes with respect to proliferation are distinct.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
3.
Figure 6

Figure 6. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Increasing intracellular Ca2+ concentration ([Ca2+]i) changes the strength of signal (SOS) and the profile of cytotoxic T-lymphocyte antigen (CTLA)-4 interactions. Increasing concentrations of Thapsigargin (TG) were added to soluble anti-CD3 (sol aCD3) or plate-bound anti-CD3 (pb aCD3) in the absence and presence of anti-CD28, anti-CTLA-4 or mouse CTLA-4. Proliferation was measured by [3H]thymidine incorporation (a) to cells activated with sol aCD3 and (b) to cells activated with pb aCD3. Also, at the end of 48 hr, cells were stained with propidium iodide and the percentage of cells in the S/G2M phase was determined by flow cytometry. The percentages were normalized to that of cells activated with just sol aCD3 or pb aCD3 and fold differences are shown in (c) and (d). The left-hand panel shows data for sol aCD3 and the right-hand panel data for pb aCD3 activation. *P < 0·1; ***P < 0·05. c.p.m., counts per minute.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
4.
Figure 7

Figure 7. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Intracellular reactive oxygen species (ROS) increases with signal strength and cytotoxic T-lymphocyte antigen (CTLA)-4–CD80/CD86 interactions. Amounts of ROS were measured by 2′, 7′-dichlorofluorescein diacetate (DCFDA) fluorescence conversion at different times in cells activated with soluble anti-CD3 (sol aCD3) and plate-bound anti-CD3 (pb aCD3) ± anti-CD28. Mean fluorescence intensities (MFIs) under different conditions were normalized to the MFI of unactivated cells, which was taken as unity (a). A similar experiment was performed with blockade of CTLA-4–CD80/CD86 interactions and ROS was measured after 12 and 24 hr of activation. MFIs were normalized to that of cells activated with anti-CD3, which was taken as unity (b and c). Data shown are mean ± standard error for three independent experiments. P< 0·0005; **P< 0·05.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
5.
Figure 2

Figure 2. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Cell surface expression of CD3, costimulatory receptors and ligands is modulated upon T-cell activation. CD4+ T cells were activated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 0·1 or 0·5 μm Ionomycin (I). Cell surface expression of CD3, CD28, CD80, CD86 and cytotoxic T-lymphocyte antigen (CTLA)-4 was studied 12 and 36 hr post activation by flow cytometry using appropriate antibodies. The broken lines are for expression on unactivated cells, thin black lines are for expression on cells activated with PMA + 0·1 μm I, and thick black lines are for cells activated with PMA + 0·5 μm I. The mean fluorescence intensity (MFI) on unactivated cells was taken as unity and compared with the MFI of cells after activation. The MFI value at the top is for unactivated T cells, the middle value (italicized) is for T cells activated with PMA + 0·1 μm I and the bottom value (bold) is for PMA + 0·5 μm I activated cells.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
6.
Figure 8

Figure 8. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Opposing roles of reactive oxygen species (ROS) in cells activated with soluble anti-CD3 (sol aCD3) and those activated with plate-bound anti-CD3 (pb aCD3). Sol and pb aCD3-activated cells were cultured with the indicated concentrations of catalase. Interleukin (IL)-2 was measured after 36 hr (a and b). At the end of 48 hr, cells were stained with propidium iodide and cell cycle analysis was performed. The fold differences in the percentages of cells in the S/G2M (c and d) and hypodiploid (e and f) phases under different activation conditions were calculated with respect to that of cells activated with sol aCD3 or pb aCD3, which was taken as unity. The left-hand panel shows data for sol aCD3 and the right-hand data for pb aCD3. Data shown are the mean ± standard error for three independent experiments. *P< 0·1; **P < 0·05.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
7.
Figure 4

Figure 4. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Cytotoxic T-lymphocyte antigen (CTLA)-4-CD80/CD86 interactions modulate interleukin (IL)-2 production and CD25 expression. CD4+ T cells were activated with phorbol 12-myristate 13-acetate (PMA) (10 ng/ml) plus 0·1 or 0·5 μm Ionomycin (I) (a) or 0·001 or 0·01 μm Thapsigargin (TG) (b) in the presence or absence of anti-CD28, anti-CTLA-4 or mouse CTLA-4. Amounts of IL-2 were measured by enzyme-linked immunosorbent assay (ELISA) 24 hr post activation. Cell surface expression of CD25 expression was studied after 36 hr by flow cytometry (c). The numbers alongside the histograms in (c) are the mean fluorescence intensities (MFIs). The grey histograms are for controls and the black histograms show CD25 expression under the indicated activation conditions. The fold differences in MFIs under different activation conditions at 36 hr were normalized to the MFI of cells activated with PMA + 0·1 μm I (d). Similar fold differences in MFI were calculated for cells activated with PMA + 0·001 or 0·01 μm TG (e). Data shown are mean ± standard error for three independent experiments. †P < 0·0005; *P < 0·1; **P < 0·05; ***P < 0·01.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
8.
Figure 3

Figure 3. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

Blockade of cytotoxic T-lymphocyte antigen (CTLA)-4–CD80/CD86 interactions modulates activation of CD4+ T cells stimulated with phorbol 12-myristate 13-acetate (PMA) + Thapsigargin (TG). (a) CD4+ T cells were activated with 10 ng/ml PMA plus different concentrations of TG in the presence or absence of anti-CD28, anti-CTLA-4 or mouse CTLA-4. Cells were cultured for 36 hr and then pulsed with [3H]thymidine for an additional 12 hr. At the end of the pulse period, cells were harvested and incorporated [3H]thymidine levels were measured. (b, c) Cells activated with 10 ng/ml PMA plus 0·001 or 0·01 μm TG with or without the indicated antibodies were permeabilized for cell cycle staining after 48 hr of culture. The percentage of cells in the S/G2M (b) and hypodiploid (c) phases was determined by flow cytometry. Fold differences in percentages of S/G2M and hypodiploid cells under different activation conditions were normalized to that of cells activated with PMA + TG (0·001 μm) alone. Data shown are the mean ± standard error for three independent experiments. *P <0·1; **P< 0·05; ***P< 0·0005.c.p.m., counts per minute.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.
9.
Figure 1

Figure 1. From: Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4+ T lymphocytes.

The intracellular Ca2+ concentration ([Ca2+]i) is modulated by cytotoxic T-lymphocyte antigen (CTLA)-4–CD80/CD86 interactions in CD4+ T cells activated with phorbol 12-myristate 13-acetate (PMA) + Ionomycin (I). (a) CD4+ T cells were activated with 10 ng/ml PMA + 0·1 μm I in the presence or absence of anti-CD28, anti-CTLA-4 or mouse CTLA-4. At different times after activation, [Ca2+]i was measured using fluro-3 acetoxymethyl ester (Fluo-3AM). The mean fluorescence intensities (MFIs) obtained for different conditions were normalized to the MFI of cells activated with just PMA + I, which was taken as unity. Data shown are the mean ± standard error (SE) for three independent experiments. (b–e) Next, CD4+ T cells were activated with I (0·1 μm) plus the indicated concentrations of PMA (b) or 10 ng/ml PMA plus the indicated concentrations of I (c) in the presence or absence of anti-CD28, anti-CTLA-4 or mCTLA-4, and [3H]thymidine incorporation was measured. Cells activated with 10 ng/ml PMA plus 0·1 or 0·5 μm I in the presence of the indicated reagents were permeabilized and stained with propidium iodide after 48 hr of culture. The percentage of cells in the S/G2M and hypodiploid phases was determined by flow cytometry. Fold differences in percentages of S/G2M (d) and hypodiploid cells (e) under different activation conditions were normalized to that of cells activated with PMA + I (0·1 μm) alone, which was taken as unity. Data shown are the mean ± SE for three independent experiments. *P < 0·1; **P < 0·05. c.p.m., counts per minute.

Asma Ahmed, et al. Immunology. 2009 March;126(3):363-377.

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