Th1 and Th2 cytokine production by sooty mangabey NKT clones.

<p>A) Intracellular cytokine staining of one representative NKT clone with antibodies against IFN-γ, TNF-α, IL-2, and IL-13 following 16 hours <i>in vitro</i> stimulation with PMA/Ca or the specific ligand α-GalCer presented on C1R.d cells (C1R.d/α-GC). Medium alone (R10) served as a negative control. Dot plots gated on NKT lymphocytes show intracellular expression of recent activation marker CD69 versus each cytokine with stimulation conditions denoted on the right of each panel. Numbers denote the % of NKT cells in each quadrant. B) Mean frequency of cytokine-positive NKT cells. Data on 9 NKT clones shown. Error bars denote SEM. ND: Not determined. C) Cytokine ELISA for IFN-γ, IL-4, IL-13, and IL-10 on culture supernatants collected at 24 hours post-stimulation. Mean and SEM of four clones shown. D) Cytokine profile of the NKT clones with regards to the concurrent production of IFN-γ, IL-2, and IL-13. Data on 9 NKT clones after 16 hours of stimulation shown. Pie charts (top panel) showing the proportion of one-, two-, and three-functional responses in cytokine-secreting 6B11⁺ cells. Bar charts (bottom panel) showing the mean proportion of responding cells for each of seven functional combinations. Boxes represent interquartile ranges. E) Representative dot plots (on the left) gated on NKT clones showing CD107a surface expression in CD1d/α-GalCer stimulated NKT lymphocytes in comparison to cells stimulated with C1R/α-GalCer or medium alone for 4 hours. Mean frequency of NKT clones degranulating after 4-hour stimulation (on the right). Data on surface upregulation of CD107a on four NKT cell clones shown. Error bars denote SEM.</p

Distribution of T lymphocyte subsets in circulating NKT cells and total T lymphocytes in sooty mangabeys.

<p>*Paired <i>t</i>-test.</p><p>^@Mean % and Standard Deviation in 13 SIV-negative sooty mangabeys.</p

Phenotypic comparison of NKT clones and <i>ex vivo</i> circulating NKT lymphocytes from one SIV-negative sooty mangabey.

<p>*Mean % of 27 clones.</p><p>ND- not determined.</p

Relationship between plasma SIV RNA and NKT lymphocytes.

<p>(A) Correlation between plasma SIV RNA and peripheral blood frequency of Vα24⁺CD1d TM⁺ NKT lymphocytes in 43 SIV-infected sooty mangabeys. (B) Percentage of DN NKT lymphocytes and (C) percentage of CD8⁺ NKT lymphocytes correlated with plasma SIV RNA in 15 SIV-infected sooty mangabeys. ‘r’ denotes correlation coefficient values determined by the Pearson correlation test.</p

T lymphocyte subset distribution of sooty mangabey NKT lymphocytes.

<p>A) Representative contour plots showing surface expression of CD4 and CD8 molecules on <i>ex vivo</i> circulating T and Vα24⁺CD1d TM⁺ NKT lymphocytes in two sooty mangabeys (SM #1 and SM #2). B) Contour plots showing surface expression pattern of CD4 and CD8 molecules on representative NKT clones. C) Distribution of CD4⁺ (CD4⁺8⁻), CD8⁺ (CD8⁺4⁻), DN (CD4⁻8⁻) and DP (CD4⁺8⁺) T lymphocyte subsets in <i>ex vivo</i> NKT lymphocytes in 13 SIV-negative sooty mangabeys (left panel) and in 57 NKT clones derived from sorted 6B11⁺ T lymphocytes of one SIV-negative sooty mangabey (right panel).</p

Identification of NKT lymphocytes in the peripheral blood of sooty mangabeys.

<p>A) Representative dot plots showing gating strategy for identification of NKT lymphocytes. On the CD3⁺ T lymphocyte population, the co-staining for Vα24 and CD1d tetramers loaded with PBS-57 (CD1d TM) and 6B11 are shown in the bottom panel. Unloaded tetramers (shown in the left of bottom panel) served as a control for non-specific staining. B) Representative dot plots of sorted NKT clones in one SIV-negative sooty mangabey stained with anti-Vα24 and CD1d TM or 6B11 antibody. C) Frequency of peripheral blood NKT lymphocytes in 50 SIV-negative sooty mangabeys. Horizontal bar denotes mean. The background staining with unloaded CD1d TM for one animal (which was above 0.002%) and the corresponding Vα24⁺CD1d TM⁺ frequency are shown in red. D) Positive correlation between the frequency of Vα24⁺CD1d TM⁺ T lymphocytes and Vα24⁺6B11⁺ T lymphocytes in the peripheral blood of 50 SIV-negative sooty mangabeys.</p

Phenotypic characterization of sooty mangabey NKT clones.

<p>A) Histogram plots gated on representative NKT clones showing surface expression of NK cell markers (CD16, CD56, NKG2D, and CD161), chemokine receptors (CCR5 and CXCR3), and memory markers (CD95, CD28, and CD45RA), and intracellular expression of cytolytic molecules (granzyme B and perforin). B) Mean % for each phenotypic marker on 5 to 71 NKT clones. Error bars denote standard error of mean (SEM). C) Memory phenotype of NKT lymphocytes in comparison to CD8⁺ and DN T lymphocytes in the peripheral blood of one SIV-negative sooty mangabey. Dot plots showing CD95 vs CD28 staining on each subset of T lymphocytes (top panel) and distribution of CCR7 and CD28 on the CD95⁺ memory lymphocytes (bottom panel).</p

Cytokine production by <i>ex vivo</i> peripheral blood NKT lymphocytes in sooty mangabeys.

<p>A) Cytokine ELISA for IFN-γ, IL-2, IL-10, IL-13, and IL-4 with culture supernatants collected 24 hours post-stimulation of PBMC (mean and SEM of 4 to 7 animals). B) Kinetics of cytokine secretion in PMA/Ca and C1R.d/α-GC stimulated PBMCs at 2 hour, 1 day, 2 days, and 7 days post-stimulation. C) IFN-γ production in C1R.d/α-GC stimulated lymphocytes in the presence of anti-human CD1d (clone 42.1) blocking antibody or isotype control Ab.</p

Sooty mangabey NKT lymphocytes respond to NKT ligands in a CD1d-restricted manner.

<p>A) IFN-γ ELISA on culture supernatants of NKT clones stimulated with C1R.d/α-GalCer (50 ng/ml) for 16 hours in the presence of anti-CD1d antibody (clone 42.1) at 0, 1, and 20 µg/ml. Data shows IFN- γ secretion from five clones. B) Representative histogram plots gated on NKT lymphocytes showing proliferation assessed by CFSE staining after 5 days of CD1d/α-GalCer stimulation in the presence or absence of anti-human CD1d mAb (clone 42.1). C) Dose-dependent reduction in proliferation of NKT cells with anti-CD1d. Mean and SEM of five NKT clones shown.</p

Activation and TCR downregulation in sooty mangabey NKT clones.

<p>Representative histogram plots on one NKT cell clone showing (A) surface expression of CD69 and (B) TCR detection by CD1d TM following 16 hours <i>in vitro</i> stimulation with the NKT-specific ligand α-GalCer presented on C1R cell lines expressing CD1d (C1R.d/α-GC), C1R cell lines pulsed with α-GalCer (C1R/α-GC), PMA/Ca or medium alone (R10).</p