CD4⁺ T cells rescue the survival of CD8⁺ T cells upon co-culture in a trans-well.

<p>CD4⁺CD25<b>⁻</b> (1×10⁵) and CD8⁺CD25<b>⁻</b> (1×10⁵) T cells were isolated from wild-type B6 mice, and were cultured in the upper and lower chamber of a 24-well transwell plate respectively. Both populations of cells in upper and lower chambers were activated with 1∶1 (cells:beads) ratio of Dynabeads (Mouse T-activator CD3/CD28). In some wells, the two populations were cultured alone with Dynabeads in the lower chamber as indicated in the figure. Some culture wells were supplemented with 10 µg/mL anti-IL-2 mAb as shown in the figure. At 48 hrs post-culture, cells were harvested and stained with PI to analyze percent survival by flow cytometry. The bars (n = 3) show the frequency of PI negative cells within the CD4⁺<b>(white bars)</b> and CD8⁺<b>(grey bars)</b> T cells. The figure shows results from one representative experiment which was repeated three times with similar results. Bars indicate average value ± SD obtained from three culture wells (technical triplicates) in each experimental group (**denotes <i>p</i><0.005).</p

IL-21 is critical for maintaining survival of CD4 and CD8 T cells under IL-2 inhibition.

<p>CD4⁺CD25<b>⁻</b> and CD8⁺CD25<b>⁻</b> T cells were isolated from IL-21 KO mice, and were cultured together (2.5×10⁴ cells/well from each population) or individually (5×10⁴/well) as indicated in a 96-well plate. The cells were stimulated with anti-CD3/CD28 mAb’s in presence or absence of anti-IL-2 and recombinant murine IL-21 as indicated. The cells were harvested at 48 hrs post-culture and subsequently stained for CD4, CD8 and PI for measuring cell survival. (<b>A</b>) The bar graph shows frequency of PI negative cells within CD4⁺ T cell population when cultured alone (<b>black bars</b>) or with CD8⁺ T cells (<b>gray bars</b>). (<b>B</b>) Bars graph shows frequency of PI negative cells within CD8⁺ T cell population when cultured alone (<b>black bars</b>) or with CD4⁺ T cells (<b>gray bars</b>). The figure shows results from one representative experiment which was repeated three times with similar results. Bars indicate average value ± SD obtained from three culture wells (technical triplicates) in each experimental group (*denotes <i>p</i><0.05).</p

A combination of anti-TCRβ mAb and anti-LFA1 mAb inhibits an antigen-specific T cell response invivo.

<p>(<b>A</b>) A total of 1×10⁶ CFSE labeled splenocytes from a Rag1^−/−OT-II mouse were adoptively transferred into C57BL/6 recipients. The recipients were provided with 5 µg OVA_323–339 to stimulate the OT-II cells (except the No Stimulation group). Histograms show the CFSE⁺ population from 1×10⁶ total events collected from the spleens of recipients who received either (<b>i</b>) no treatment (No Treat), (<b>ii</b>) 5 µg IL2 and 25 µg anti-IL2 mAb complex treatment (IL2Cx), (<b>iii</b>) 250 µg CTLA4Ig, <b>(</b>i<b>v</b>) 20 µg anti-LFA1 mAb (Anti-LFA), (<b>v</b>) 20 µg anti-TCRβ mAb (Anti-TCR), (<b>vi</b>) no stimulation, or (<b>vii–x</b>) combinations of the above listed treatments as indicated. Each panel is a representative histogram from one of at least three recipients per treatment group. (<b>B</b>) Histograms from select groups in A were analyzed using FlowJo Software’s proliferation tool to calculate the division index, which is plotted in the bar graphs for each of the indicated treatment groups. Each bar represents the mean ± standard deviation obtained from three recipients (* indicates <i>p</i><0.05). (<b>C</b>) Bar graphs show the total CD4⁺ T cell numbers in spleens (left column), OT-II cell numbers in spleens (middle column), and Treg percentage within the CD4⁺ T cell population in lymph nodes (right column) from the adoptive transfer recipients described above. Each bar represents the mean ± standard deviation obtained from three mice (solid black bars labeled NT represent recipients receiving no treatment while patterned bars represent recipients who received the indicated treatments; * indicates <i>p</i><0.05).</p

The combination of anti-TCRβ mAb with anti-LFA1 mAb inhibits T cell responses in vitro.

<p>(<b>A)</b> (<b>i</b>) CFSE dilution analysis of labeled OT-II cells cultured with no stimulation (gray shaded histogram), with 5 µg/mL of the indicated mAbs alone (left column; overlay solid black line histograms), or with 5 µg/mL of OVA_323–339 alone or in the presence of 5 µg/mL of the indicated mAbs (right column; overlay solid black line histograms). (<b>ii</b>) Bar graphs show the percentage of dividing cells within the OT-II cell cultures. Each bar represents the mean ± standard deviation of three replicate wells that are representative of two independent experiments (* indicates <i>p</i><0.005 compared to OVA). (<b>B</b>) (<b>i</b>) CFSE dilution analysis of labeled OT-I cells cultured with no stimulation (gray shaded histogram), with 5 µg/mL of the indicated mAbs alone (left column; overlay solid black line histograms), or with 5 µg/mL of OVA_257–264 alone or in the presence of 5 µg/mL of the indicated mAbs (right column; overlay solid black line histograms). (<b>ii</b>) Bar graphs show the percentage of dividing cells within the OT-I cell cultures. Each bar represents the mean ± standard deviation of three replicate wells that are representative of two independent experiments (* indicates <i>p</i><0.005 compared to OVA).</p

Neutralizing IL-2 and IL-21 simultaneously reduces the survival of both CD4⁺ and CD8⁺ T cells.

<p>CD4⁺CD25<b>⁻</b> and CD8⁺CD25<b>⁻</b> T cells were isolated from wild-type B6 mice, and were cultured together (2.5×10⁴ cells/well from each population) or individually (5×10⁴ cells/well) as indicated in a 96-well plate. The cells were stimulated with anti-CD3/CD28 mAbs in the presence or absence of anti-IL-2 and IL-21R.Fc as indicated. The cells were harvested at 48 hrs post-culture and subsequently stained for CD4, CD8 and PI for measuring cell survival. (<b>A</b>) The bars graph shows frequency of PI negative cells within the CD4⁺ T cell population when cultured alone (<b>black bars</b>) or with CD8⁺ T cells (<b>gray bars</b>). (<b>B</b>) Bars graph shows frequency of PI negative cells within CD8⁺ T cell population when cultured alone (<b>black bars</b>) or with CD4⁺ T cells (<b>gray bars</b>). The figure shows results from one representative experiment which was repeated three times with similar results. Bars indicate average value ± SD obtained from three culture wells (technical triplicates) in each experimental group (*denotes <i>p</i><0.05).</p

Significant prolongation of allograft survival by the combination therapy of anti-TCRβ and anti-LFA1 mAbs.

<p>The plot shows the percent survival of BALB/c skin grafts transplanted onto C57BL/6 recipients either left untreated (solid line) or treated with anti-LFA1 mAb (dotted line), anti-TCRβ mAb (dashed line), or a combination of the two (dashed-dotted line). Numbers indicate the mean survival time of the six mice in each of the treatment groups plus or minus the standard deviation (* indicates <i>p</i><0.01 compared to all other groups).</p

Neutralizing IL-2 reduces the survival of CD8⁺ but not CD4⁺ T cells.

<p>CD4⁺CD25<b>⁻</b> and CD8⁺CD25<b>⁻</b> T cells were purified from wild-type B6 mice and stimulated with anti-CD3/CD28 mAbs in the presence or absence of neutralizing IL-2 mAb at two different concentrations as indicated. Some cells were left unstimulated (no anti-CD3/CD28) and were used as negative controls. A total of 5×10⁴ cells per well were cultured in flat-bottom 96-well plates and harvested for analysis at the indicated time points. (<b>A</b>) Harvested cells were stained with Propidium Iodide (PI) to measure cell survival by flow cytometry. Bar graphs show the percentage of PI negative cells in each group (n = 3). (<b>B</b>) Supernatants were harvested from the cultures at the indicated time points which were then analyzed for levels of IL-2 using ELISA. Bar graphs show the concentration of IL-2 in the culture supernatants. (<b>C</b>) Histograms show the expression of the anti-apoptotic protein Bcl-2 measured by flow cytometry in the cultured cells at the indicated time points. (<b>D</b>) Histograms represent the mitochondrial membrane potential measured by flow cytometry of harvested cells (with higher fluorescence depicting a hyper-polarized state of mitochondria) at the indicated time points. The figure shows results from one representative experiment which was repeated three times with similar results. Bars indicate average value ± standard deviation (SD) obtained from three culture wells (technical triplicates) in each experimental group (*denotes <i>p</i><0.05 and **denotes <i>p</i><0.005).</p

IL-21 rescues the survival of CD8⁺ T cells under IL-2 deprivation.

<p>CD8⁺CD25<b>⁻</b> T cells (5×10⁴) were purified from wild-type B6 mice and stimulated with anti-CD3/CD28 mAbs in the presence (<b>all grey bars</b>) or absence (<b>black bar</b>) of 10 µg/mL neutralizing IL-2 mAb. The cultures with anti-IL-2 were further supplemented with either 10 IU/mL (IL-2) or 10 ng/mL (all other cytokines), each of 61 different cytokines (as marked in the figure). The cells were harvested at 48 hrs and subsequently stained for PI for measuring cell survival. The bars graph shows frequency of PI negative cells under each culture condition as marked. The figure shows results from one representative experiment which was repeated three times with similar results. Bars indicate average value ± SD obtained from three culture wells (technical triplicates) in each experimental group.</p