Article Figures & Data
Figures
- FIGURE 1.
ST2L is a type 2 marker, while IL-18R is a type 1 marker. Short-term polarized human cell lines were generated as described in Materials and Methods. Expression of ST2L and IL-18R on Th1 and Th2 lines (A), Tc1 and Tc2 lines (B), and NK1 and NK2 lines (C) is shown. Intracellular cytokine staining and three-color flow cytometric analysis of PMA- and ionomycin-activated alloantigen-specific cells were conducted as described in Materials and Methods. The expression of ST2L and IL-18R on NK cells is shown for unactivated cells. Similar results, but with slightly reduced signal, were also obtained with PMA-activated cells (data not shown). The numerical values in the dot plots denote percentage of cells within gated quadrants that were statistically set with negative control Abs (data not shown). All flow cytometric analyses were conducted with WinMDI version 2.8 or PC LYSYS (BD Biosciences). Results are representative of four experiments.
- FIGURE 2.
Cytokine secretion profile of ST2L+ and IL-18R+ PBMCs from healthy individuals. A, IL-18R+ lymphocytes produce only IFN-γ. Cytokine production was determined after 6-h activation with PMA/ionomycin in the presence of monensin. Three-color flow cytometric analysis of IFN-γ-positive cells was conducted on the IL-18R+-gated population (R2). B, The majority of ST2L+ lymphocytes produce only IL-4 or IL-5 and no IFN-γ, whereas a very small proportion coexpresses IFN-γ with IL-4 or IL-5. Three-color flow cytometric analysis of IFN-γ and IL-4 or IL-5 production by the ST2L+-gated population (R2) of PBMCs from an individual with hay fever after 6-h in vitro activation with PMA/ionomycin. The numerical values in the dot plots denote the percentage of cells within the respective gated quadrants that were statistically set with negative control Abs. The reagents used are the same as in Fig. 1. FACS data were analyzed from the 35,000 events acquired in the lymphocyte-gated population. Results are representative of three experiments.
- FIGURE 3.
Within the ST2L+ lymphocyte population in PBMCs, T (CD3+) lymphocytes do not produce IFN-γ. Three-color flow cytometric analysis using the same reagents as in Fig. 1, with the exception of PE-conjugated anti-hIFN-γ. R2 and R3 denote the respective ST2L+ CD3+- and ST2L− CD3+-gated populations that were analyzed for IL-4 and IFN-γ production. The PBMCs were PMA/ionomycin activated for 6 h (upper panel) and 1 h (lower panel) before staining for IL-4 and IFN-γ, respectively. A total of 35,000 events in the gated lymphocyte population is shown. Results are representative of three experiments.
- FIGURE 4.
CD56+ lymphocytes that express ST2L produce IL-4, IL-5, and IFN-γ, whereas the IL-18R+ cells produce only IFN-γ. Three-color flow cytometric analysis was performed on PBMCs that have been activated with PMA/ionomycin for 6 h (IL-4 and IL-5) and 1 h (IFN-γ), using the same reagents as in Fig. 1, with the exception of FITC-conjugated mAb CD56 (Serotec, Oxford, U.K.) and PE-conjugated anti-hIFN-γ. The cytokine-producing CD56+ lymphocytes were either ST2L+- or IL-18R+-gated cells in R2. A total of 35,000 events in the gated lymphocyte population is shown. Results are representative of three experiments.
- FIGURE 5.
ST2L and IL-18R are stable markers for memory T2 and T1 PBMCs. ST2L+IL-18R− and ST2L−IL-18R+ cells sorted from PBMCs and stimulated through two rounds of in vitro culture were PMA/ionomycin activated before two-color flow cytometric analysis. Results are representative of three experiments.
- FIGURE 6.
Predominance of type 2 lymphocytes in HIV-1-infected individuals. PBMCs were obtained from normal healthy controls (HIV−, n = 21) and HIV-1-infected individuals (HIV+, n = 22). The distribution of type 1 and 2 lymphocytes in the Th, Tc, and NK cell populations was determined by three-color flow cytometry using FITC-conjugated anti-CD3, with PE-conjugated anti-CD4, anti-CD8, or anti-CD56 and anti-hIL-18R, anti-hST2L, or control sera. hIL-18R and hST2L expression were detected with biotinylated goat anti-rabbit Ig or goat anti-mouse Ig and PerCP-streptavidin. PE- or FITC-conjugated isotype control Ig were used. The proportion of ST2L+ or IL-18R+ cells in the gated CD3+CD4+ (Th), CD3+CD8+ (Tc), or CD3−CD56+ (NK) populations was quantitated within gated quadrants that were statistically set with negative control Abs such that the PerCP-positive control cells were ≤0.3%. To ensure an unbiased estimation of the proportions of PerCP-stained IL-18R+ and ST2L+ cells within any one of the gated Th, Tc, or NK cell populations, similar levels of percentage of PerCP-positive control cells were set for both. Each ○ represents one HIV-positive individual, and each □ represents one HIV-negative individual. To show the T1:T2 relationship, the percentages of ST2L+ and IL-18R+ cells within each of the Th, Tc, and NK cell populations of an individual are connected by lines. Dashed lines represent the mean. The significance of the difference in distribution of type 1 vs type 2 lymphocytes was determined using the paired sample t test. A total of 25,000 events in the gated lymphocyte population is shown.
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