Human lung-derived CD45RA^<b>+</b>CD27^<b>−</b> effector CD8^<b>+</b> T cells express low levels of CD28.

<p><b>A</b>) CD3<b>⁺</b>CD8<b>⁺</b> T cells were classified into naïve, effector and memory cells based on CD27 and CD45RA surface expression. These subsets were analyzed for the expression levels (MFI) of CD28 and NKG2D. <b>B</b>) Comparison of CD28 (left histogram) and NKG2D (right histogram) in effector (open bar), naïve (grey bar) and memory (black bar) CD8<b>⁺</b> T cells. Four independent samples were analyzed to obtain data presented in B and the P values were calculated using Student's t-test.</p

MICA is expressed in human tracheal epithelial cells.

<p>Paraffin sections of human <b>A</b>) trachea and <b>B</b>) lung tissue were processed and stained for MICA and the epithelial cell markers E-Cadherin and cytokeratin. <b>C</b>) Enlarged view of tracheal sections showing the intracellular localization of MICA (Arrow heads). <b>D</b>) CD4⁺ but not <b>E</b>) CD8 T cells express MICA. Lung-derived lymphocytes were stained for CD4<b>⁺</b> and CD8<b>⁺</b> T cells. CD4<b>⁺</b> and CD8<b>⁺</b> T cells were further stained and analyzed for MICA expression through confocal microscopy. Data shown are one representative image of seven independent sections analyzed for each lung tissue and trachea.</p

Analysis of CD28 expression on influenza-specific (M1 peptide, GFL9) CD8^<b>+</b> T cells in PBMC and lung.

<p><b>A</b>) Human PBMC and lung-derived lymphocytes were stained with anti-CD3, anti-CD8 and GFL9/HLA-A2 pentamer to detect antigen-specific CD8<b>⁺</b> T cells. Gated populations indicate percent GFL9 epitope-specific CD8<b>⁺</b> T cells. CD28 expression is higher in antigen-specific memory CD8⁺ T cells. <b>B)</b> GFL9/HLA-A2 pentamer-positive tracheal and <b>C)</b> lung tissue-resident CD8<b>⁺</b> T cells were analyzed for CD28 and NKG2D expression using confocal microscopy. Arrow heads mark some of the pentamer-positive CD8⁺ T cells. <b>D)</b> Lung tissue and <b>E)</b> PBMC were stained for CD27 and CD45RA in order to classify them into effector and memory cells. These cells were stained with anti-CD28 and GFL9/HLA-A2 pentamer. Histograms show the level of CD28 expression among the pentamer-positive effector or memory CD8⁺ subsets. Numbers in the histograms represent the MFI of CD28 expression.</p

Influence of CD28 and NKG2D-mediated co-stimulation in cytotoxicity.

<p>Negatively selected CD3<b>⁺</b>CD8<b>⁺</b> T cells were further sorted based on their CD28 expression levels into CD28^Lo and CD28^Hi CD8<b>⁺</b> T cells. These cells were activated with plate-bound antibodies directed against CD3, CD28 and NKG2D in different combinations. Cell surface expression of CD107a was analyzed through flow cytometry and used as a measure of cytotoxicity. Three independent samples were used to obtain data and P values were calculated using paired t-test.</p

Efficiency of CD28 and NKG2D co-stimulation in cytokine/chemokine production.

<p>Negatively selected CD8<b>⁺</b> T cells (N = 10) or sorted CD28^Lo and CD28^Hi CD8<b>⁺</b> T cells (N = 5) were activated with plate-bound antibodies in different combinations. Quantities of IFN-γ, TNF-α and IL-2 were estimated in the culture supernatants using multiplex assays. Cytokine generation from total CD8⁺ (<b>A–C</b>), effector CD28^Lo (<b>D–F</b>) and naïve/memory CD28^Hi (<b>G–I</b>) CD8⁺ T cells are shown. Bar diagram represents the mean ± standard deviation of cytokine production. P values were calculated using paired t-test.</p

Expression of NKG2D and CD28 on human CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells.

<p><b>A</b>) Analysis of NKG2D expression on CD3<b>⁺</b>CD4<b>⁺</b> and CD3<b>⁺</b>CD8<b>⁺</b> T cells by flow cytometry. Human PBMC were stained for CD3, CD4 or CD8 along with NKG2D or CD28. Background isotype control (open histograms) and NKG2D (grey histograms) stainings are shown. The mean fluorescence index (MFI) for NKG2D (N = 30) among the CD3<b>⁺</b>CD4<b>⁺</b> and CD3<b>⁺</b>CD8<b>⁺</b> T cells were compared and presented in the right panel. <b>B</b>) Similar analysis was performed for the expression of CD28 on CD3<b>⁺</b>CD4<b>⁺</b> and CD3<b>⁺</b>CD8<b>⁺</b> T cells. Open histograms represent negative controls. Since the expression of CD28 on CD8⁺ T cells was biphasic, a comparison of the percentage of CD28^HiCD4⁺ and CD28^HiCD8⁺ T cells was performed (N = 30). <b>C</b>) CD28 expression among CD3<b>⁺</b>CD8<b>⁺</b> T cells falls into two distinct subsets. Differences between the MFI of CD28<b>^Hi</b> and CD28<b>^Lo</b>CD8<b>⁺</b> T cells are shown. <b>D</b>) Co-expression of CD28 and NKG2D in CD3<b>⁺</b>CD8<b>⁺</b> T cells. <b>E</b>) Confocal microscopy analyses of the expression of CD28 and NKG2D on CD4<b>⁺</b> and CD8<b>⁺</b> T cells. PBMC were either stained for CD4 or CD8. They were also stained for CD28 and NKG2D. The stained cells were visualized by confocal microscopy and expression of each receptor was visualized individually and merged. Arrow heads indicate limited positivity of NKG2D in CD4⁺ T cells. P values for A–C were calculated using Student's t-test.</p