CD161 DEFINES EFFECTOR T CELLS THAT EXPRESS LIGHT AND RESPOND TO TL1A-DR3 SIGNALING

Abstract

Expression of NK cell markers identifies pro-inflammatory T cell subsets in the liver and intestinal immune compartments. Specifically, CD161 is expressed on Th17 cells which play an important role in the regulation of mucosal inflammation. In this study, we characterized human peripheral blood CD161+ T cells as an effector population partially resembling a gut T cell phenotype. CD161+ CD4+ T cells express the gut-associated TNF family member, LIGHT, and respond to crosslinking of DR3, a receptor to another gut-associated cytokine, TL1A. Robust IFN-γ production in response to DR3 signaling correlated with enhanced expression of surface DR3 on CD161+ T cells and co-stimulation with IL12 and IL18. CD161+ T cell effector function was directly demonstrated by activation of responder monocytes in co-culture leading to CD40 upregulation and CD14 downregulation. CD161+ T cells reciprocally responded to activated monocytes, inducing expression of activation marker, CD69, and production of IL2 and IFN-γ, further demonstrating effective CD161+ T cell cross-talk with monocytes. Finally, CD161 defined a subset of T cells that co-express CD56, a second NK marker. Our findings implicate human CD161+ T cells in gut-associated signaling mechanisms, and suggest a monocyte mediated effector function in mucosal inflammation.

Fig. 1.

DC56 is persistently expressed only on T cells co-expressing CD161. Lymphocytes were isolated from human peripheral blood and surface stained with fluorescent conjugated anti-CD3, anti-CD161 and anti-CD56 antibodies, followed by flow cytometric analysis. A. Human CD3⁺ T cell subsets were purified from PBL preparations by flow cytometry sorting based on CD3, CD56 and CD161 staining characteristics. Gated viable, lymphocyte size, scatter plots are shown for representative samples, indicating staining profiles and sort gates. B. Sorted CD161⁺/CD56⁺, CD161^–/CD56⁺, CD161⁺/CD56^– and CD161^–/CD56^– were cultured separately and CD56 staining histograms are shown for the CD56⁺ (Shaded) and CD56^– (unshaded) staining in sorted populations prior to culture (top) or upon re-staining on day 3 (Bottom). Data are representative of at least five experiments

Fig. 2.

LIGHT is preferentially expressed on CD4⁺ T cells co-expressing CD161. PBL were isolated from a healthy donor blood and cultured for 18 hours in the presence of PMA and Ionomycin. Cells were surface stained with anti-CD3, anti-CD4/8, anti-CD161 and anti-LIGHT antibodies and analyzed by flowcytometry. Stimulated (shaded) and unstimulated (unshaded) histogram plots are shown for live CD3⁺ cells gated for CD161, and CD4/8 expression as indicated. Data are representative of at least five experiments

Fig. 3.

CD161 defines a T cell population responsive to TL1A-DR3 signal. Isolated PBL were stimulated with anti-DR3 antibodies for 18 hours followed by 6 hours in the presence of Golgi inhibitor, Brefeldin A. Cells were then surface stained with anti-CD3, anti-CD56 and anti-CD161 antibodies and intracellularly immunostained for IFNγ. Plots show percent of viable CD3⁺ gated T cells expressing INFγ following activation with anti-DR3 (top) or isotype control (bottom) antibodies in the presence of titrated exogenous IL18 and IL12. Spontaneous cytokine production by unstimulated cells was consistently less than 2%. Data are representative of at least five experiments

Fig. 4.

Surface DR3 protein expression is preferentially induced on CD161⁺ T cells at low IL12/IL18 concentrations. Isolated PBL were cultured for 24 hours in the presence of varying concentrations of exogenous IL12 and IL18, and then immunostained for surface CD3, CD8/4, CD161 and DR3 expression. Plots show percent of viable CD3⁺ gated CD161⁺ (♦) and CD161^– (o) T cells expressing DR3, which did not bind the DR3 isotype control. Data are representative of at least three experiments

Fig. 5.

Experimental setup for the analysis of effector–responder interaction in monocyte-T cell co-cultures. Monocytes were isolated from human leukocyte preparations by negative selection using magnetic beads and T cell subsets were purified by flow sorting (>99% pure by flow cytometric reanalysis). Effector cells were then activated as indicated, washed and co-cultured with responder cells at 1:1 ratio for 24 hours. Responder T cell or monocyte activation was determined as a function of cytokine and surface protein expression profiles evaluated by flow cytometric analysis gating on the responder cell subset

Fig. 6.

DR3 stimulated CD161⁺ T cells activate primary autologous monocytes. Flow sorted CD3⁺/CD161⁺, or CD3⁺/CD161^– T cells to be tested as effectors cells were activated with anti-DR3 antibodies (shaded) or isotype control (unshaded) in the presence of IL12 (0.2 ng/ml) and IL18 (10 ng/ml) for 24 hours, washed and co-cultured with monocytes as responder cells for an additional 24 hours. Co-cultured cells were analyzed by flow cytometry and histograms show gated monocytes stained for CD40 (top) or CD14 (bottom). Data are representative of at least three experiments

Fig. 7.

CD161⁺ T cells are preferentially activated by monocytes. PBL were co-cultured with activated monocytes for 24 hours in the presence (top) or absence (bottom) of IL12 and IL18. A. CD4⁺ and CD8⁺ T cell activation was analyzed as a function of surface staining for CD69 by flow cytometry, following co-culture with PMA activated monocytes. B. T cells were co-cultured with monocytes pre-stimulated by PMA, and T cell activation measured as a function intracellular staining for IFNγ and IL2 following 5 hours of culture in the presence of Golgi maturation inhibitor, Brefeldin A. Bar graphs represent percent positive cells in CD3⁺/CD161⁺ (closed), and CD3⁺/CD161^– (open) gates. Data are representative of a minimum of three experiments