The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion - PubMed (original) (raw)

The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion

Michael T Wilson et al. Proc Natl Acad Sci U S A. 2003.

Abstract

CD1d-restricted natural killer T (NKT) cells are a subset of regulatory T cells that react with glycolipid antigens. Although preclinical studies have effectively targeted NKT cells for immunotherapy, little is known regarding the early in vivo response of these cells to antigenic stimulation. We have analyzed the early response of NKT cells to glycolipid antigens and bacterial infection by using specific reagents for tracking these cells. Our results demonstrate dramatic in vivo expansion and surface phenotype alterations after NKT cell activation with alpha-galactosylceramide. In addition, we show significant NK1.1 down-modulation on NKT cells in the setting of oral Salmonella infection. Our results indicate that in vivo activation of NKT cells leads to a dynamic response characterized by surface receptor down-modulation and expansion. These findings alter current understanding of NKT cell biology and should aid in the rational design of NKT cell-based immunotherapies.

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Figures

Fig. 2.

Reappearance of NKT cells on in vitro culture. (A) Spleen cells from uninjected mice (0) and from mice injected 8 h earlier with α-GalCer (5 μg, i.p.) were cultured in vitro in the presence or absence of α-GalCer (50 ng/ml). Three days later, proliferation was measured by [3H]thymidine uptake, and IL-4 and IFN-γ levels were measured by ELISA. (B) Splenocytes were harvested at the indicated time points after α-GalCer administration (5 μg, i.p.), labeled with CFSE, and cultured in vitro without further stimulation for 72 h. NKT cells were analyzed by flow cytometry before and after culture. Numbers indicate the percentage of TCRβ+ CD1d/α-GalCer tetramer+ cells among B220– cells. CFSE plots on tetramer+ cells were generated for cells cultured for 72 h.

Fig. 3.

NKT cells quickly down-regulate their surface TCRs on activation. (A and B) Spleen cells from mice injected i.p. with 5 μgof α-GalCer were killed at the indicated time points and stained with anti-TCRβ antibodies and CD1d/α-GalCer tetramers (A). The percentage of TCR expression for cells gated in A is expressed relative to tetramer binding on cells from uninjected mice (B). (C) Kinetic analysis of TCR surface down-regulation on NKT cells activated in vitro with 100 ng/ml of α-GalCer or plate-bound anti-CD3 antibodies. (D) Analysis of TCR surface down-regulation on NKT cells activated in vitro for 6 h with titrated doses of α-GalCer. TCR surface levels are expressed as the percentage of TCR surface expression on unstimulated NKT cells. (E) Spleen and liver mononuclear cells were harvested from uninjected mice and 6 h after i.p. injection of 5 μgof α-GalCer, and cells were stained with anti-CD3-PerCP-Cy5.5 and anti-NK1.1-allophycocyanin antibodies. An excess of unlabeled anti-TCRβ antibodies were added to the staining mixture to saturate surface TCRs. Cells were then fixed and permeabilized, stained with anti-TCRβ–phycoerythrin antibodies, and analyzed by flow cytometry. Flow cytometry plots represent cells electronically gated for intermediate levels of NK1.1 expression (Lower), which is typical of NKT cells (e.g., see Fig. 1 A and C). TCRβint CD1d/α-GalCer tetramer+ cell populations were identified in parallel stainings (Upper). (F) Recapitulation of the dynamics of NKT cell activation in vitro. Spleen cells were labeled with CFSE, cultured without (–) or with (+) 100 ng/ml α-GalCer for 24 h, then washed and cultured for an additional 72 h. Cells were stained at the 0-, 24-, and 96-h time points with anti-TCRβ and anti-B220 antibodies and CD1d/α-GalCer tetramers and analyzed by flow cytometry as in Fig. 1_B_. CFSE plots are shown for TCRβint CD1d/α-GalCer tetramer+ cells from the 24- (thin lines) and 96- (heavy lines) h time points.

Fig. 1.

NKT cell population dynamics in the spleen after in vivo activation with α-GalCer. (A–D) Splenic NKT cell populations were analyzed by flow cytometry at the indicated times after i.p. injection of 5 μg of α-GalCer. Numbers in A–C indicate the average percentage of TCRβint NK1.1+ (A), TCRβint CD1d/α-GalCer tetramer+ (B), and NK1.1+ CD1d/α-GalCer tetramer+ (C) cells among B220– lymphocytes of two mice at each time point. The average absolute numbers of TCRβint CD1d/α-GalCer tetramer+ cells in the spleen of two α-GalCer-injected mice at each time point were calculated (D). (E) DNA was harvested from spleen tissue at different time points after in vivo activation and Vα14-Jα18 DNA copy number was measured by quantitative real-time PCR. (Inset) Real-time PCR data averaged for three independent experiments at the 0- and 8-h time points. (F) Total RNA was harvested from spleen tissue at different time points after injection of α-GalCer and analyzed for IFN-γ, IL-4, and IL-10 mRNA expression levels by quantitative real-time RT-PCR.

Fig. 4.

Precursors for NKT cell expansion. (A) Thymectomized and shamthymectomized mice were injected 1 mo postsurgery with α-GalCer (5 μg, i.p.), killed at the indicated time points, stained, and analyzed by flow cytometry as in Fig. 1_B_.(B) NK1.1+ and CD4+NK1.1– cells were isolated from spleens of Vα14 transgenic mice by magnetic beads as described in Materials and Methods. These two populations were placed in parallel cultures with spleen antigen-presenting cells from TCRα–/– mice in the presence of 100 ng/ml of α-GalCer. Cells were analyzed for down-modulation of surface TCRs as in Fig. 3_A_, and data were plotted as in Fig. 3_B_. (C) Enriched NK1.1+ and CD4+NK1.1– cells were labeled with CFSE and cultured as in B. After 24 h, α-GalCer-containing medium was replaced with fresh medium lacking α-GalCer for the remainder of the culture period. CFSE dilution among gated CD1d/α-GalCer tetramer+ NKT cells was assessed in parallel cultures of isolated NK1.1– and NK1.1+ cells at the indicated time points. Dot plots within the histograms show levels of NK1.1 expression (y axis) vs. CFSE dilution (x axis) in cultures of the NK1.1+ fraction of cells.

Fig. 5.

NKT cell dynamics during oral Salmonella infection. Control mice (uninfected) and mice infected with S. typhimurium were killed at 0, 2, or 5 days postinfection, and spleen cells were stained and analyzed by flow cytometry as in Fig. 1 A–C. Representative data at 5 days postinfection are shown in A–C. Numbers indicate the percentage of TCRβint NK1.1+ (A) and TCRβint CD1d/α-GalCer tetramer+ (B) cells among B220– lymphocytes for one uninfected and the average of three infected mice. NK1.1 levels on TCRβint tetramer+ cells were also evaluated (C). The data from several separate experiments are summarized in D.

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The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion - PubMed (original) (raw)