Cross-presentation of disialoganglioside GD3 to natural killer T cells

Abstract

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

Figure 1.

Lymphocyte responses induced against GD3 in immunized mice produce IL-4. Mice were immunized with 2 × 10⁶ SK-MEL-28 cells mixed with CFA on day 0, and boosted with 2 × 10⁶ SK-MEL-28 cells mixed with IFA on day 7. Control mice (Freund's adjuvant only) were injected with CFA on day 0 followed by IFA alone on day 7. (a) Splenocytes were collected on day 14 and stimulated with irradiated syngeneic APCs loaded with GD3, GM2, or without ganglioside (None) as indicated. IL-4 and IFN-γ production were detected by ELISPOT assay. (b) In other sets of mice, hepatic mononuclear cells and thymocytes were tested for IL-4 secretion. Data represent mean ± SEM of triplicate wells from four mice and similar results were observed in repeated experiments.

Figure 2.

GD3-loaded APCs induced GD3-reactive lymphocytes producing IL-4. Mice were injected with 10⁵ GD3-loaded APCs or unloaded APCs into the footpad, as indicated. Popliteal and inguinal LN (a) or splenocytes (b) were collected on day 7 and tested for reactivity against irradiated syngeneic APCs loaded with GD3 or unloaded APCs (as negative control) as indicated. IL-4 production was detected by ELISPOT assay. Each point represents the mean of triplicate wells from an individual mouse; horizontal lines indicate mean values of five mice.

Figure 3.

GD3-reactive cells are CD4⁺8⁻ NKT cells and CD4⁻8⁻ NKTs. Mice were immunized with 2 × 10⁶ SK-MEL-28 cells mixed with CFA on day 0, and boosted with 2 × 10⁶ SK-MEL-28 cells mixed with IFA on day 7. Splenocytes pooled from six to eight immunized mice were separated into subpopulations based on their surface expression of NK1.1, CD3, CD4, and CD8. Total cells (unseparated splenocytes), total NKT cells (CD3⁺ NK1.1⁺), total conventional T cells (CD3⁺ NK1.1⁻), CD4⁺ NKT cells (CD4⁺ CD8⁻ CD3⁺ NK1.1⁺), CD8⁺ NKT cells (CD4⁻ CD8⁺ CD3⁺ NK1.1⁺), DN NKT cells (CD4⁻ CD8⁻ CD3⁺ NK1.1⁺), and classic NK cells (CD3⁻ NK1.1⁺) populations were stimulated with irradiated syngeneic APCs in the presence (+ columns, closed squares) or absence (− columns, open squares) of GD3. IL-4 production by each cell population was detected by ELISPOT assay. Horizontal lines indicate mean values.

Figure 4.

GD3-reactive NKT cell response is blocked by anti-CD1d mAb. Irradiated APCs were preincubated with PBS alone (none) or with anti-CD1d (CD1d), anti–K^b/D^b (K^b/D^b), anti–I-A^b (I-A^b), or 10 μg/ml Ter119 (irrel.) mAb before being loaded with GD3. Splenocytes from mice immunized with SK-MEL-28 and Freund's adjuvant were used as effector cells and IL-4 production by splenocytes was detected by ELISPOT assay. Data represent mean ± SEM of triplicate wells from four mice and are indicative of replicate experiments.

Figure 5.

CD1d expression by APCs is required for presentation of GD3 to NKT cells. Splenocytes from mice immunized with SK-MEL-28 and Freund's adjuvant were used as effector cells and IL-4 production by splenocytes was detected by ELISPOT assay. APCs were isolated from the spleen of mCD1d knockout mice (CD1^−/−) or from wild-type mice (CD1^+/+) as indicated and loaded with GD3. Control wells received unloaded APCs. Each point represents the mean of triplicate wells from an individual mouse; horizontal lines indicate mean values.

Figure 6.

Detection of GD3-reactive NKT cells using CD1d-GD3 tetramers. Splenocytes from mice immunized with SK-MEL-28 cells and Freund's adjuvant (immunized) or mice injected with Freund's adjuvant only (control) were analyzed by flow cytometry for binding to FITC-conjugated anti-CD3 mAb, PE-conjugated anti-NK1.1 mAb and TC-conjugated GD3-loaded mCD1d tetramers or unloaded mCD1d tetramers. Gating was performed on CD3⁺ cells. (a) Double immunofluorescence staining of cell surface expression of NK1.1 (PE, abscissa) and tetramer (TC, ordinate) from representative immunized and control mice. Splenocytes from an immunized mouse were stained with unloaded tetramer (right). Double-positive cells are indicated by the framed window. (b) Percent tetramer⁺ cells among total CD3⁺ NK1.1⁺ splenocytes from immunized and control mice stained with GD3-loaded CD1d tetramers, and immunized mice stained with unloaded tetramer. Each point represents a single mouse. The difference in the mean values (horizontal lines) was highly significant (P < 0.001, Kruskal-Wallis test).

Figure 7.

Effects of αGalCer-reactive NKT cell depletion on GD3 reactivity. Splenocytes from immunized mice were either depleted of αGalCer-loaded CD1d tetramer-reactive cells and stained with TC-conjugated GD3-loaded CD1d tetramer (•), or mock-depleted of αGalCer-loaded CD1d tetramer-reactive cells and stained with TC-conjugated GD3-loaded CD1d tetramer (▪) or with TC-conjugated unloaded mouse CD1d tetramer (□). Gating was performed on CD3⁺ cells and each point represents a single mouse. P = 0.005, Kruskal-Wallis test. Horizontal lines indicate mean values.

Figure 8.

Cytokine production by GD3-reactive NKTs. Mice were immunized with 2 × 10⁶ SK-MEL-28 cells mixed with CFA on day 0 without further boost (a), or were immunized with 2 × 10⁶ SK-MEL-28 cells mixed with CFA on day 0 and boosted with 2 × 10⁶ SK-MEL-28 cells mixed with IFA on day 7 and day 14 (b). Splenocytes were collected at the time points indicated. Intracellular production of IL-4 (▪), IL-5 (▵), IL-10 (♦), IL-12 (○) and IFN-γ (▿) by NK1.1⁺ CD3⁺ cells was measured gating on CD3⁺ cells. Arrows indicate time of immunization. Data represent mean ± SEM from four mice, and similar results were observed in repeated experiments.