NKT Cell Responses to B Cell Lymphoma

Abstract

Natural killer T (NKT) cells are a unique subset of CD1d-restricted T lymphocytes that express characteristics of both T cells and natural killer cells. NKT cells mediate tumor immune-surveillance; however, NKT cells are numerically reduced and functionally impaired in lymphoma patients. Many hematologic malignancies express CD1d molecules and co-stimulatory proteins needed to induce anti-tumor immunity by NKT cells, yet most tumors are poorly immunogenic. In this study, we sought to investigate NKT cell responses to B cell lymphoma. In the presence of exogenous antigen, both mouse and human NKT cell lines produce cytokines following stimulation by B cell lymphoma lines. NKT cell populations were examined ex vivo in mouse models of spontaneous B cell lymphoma, and it was found that during early stages, NKT cell responses were enhanced in lymphoma-bearing animals compared to disease-free animals. In contrast, in lymphoma-bearing animals with splenomegaly and lymphadenopathy, NKT cells were functionally impaired. In a mouse model of blastoid variant mantle cell lymphoma, treatment of tumor-bearing mice with a potent NKT cell agonist, α-galactosylceramide (α-GalCer), resulted in a significant decrease in disease pathology. Ex vivo studies demonstrated that NKT cells from α-GalCer treated mice produced IFN-γ following α-GalCer restimulation, unlike NKT cells from vehicle-control treated mice. These data demonstrate an important role for NKT cells in the immune response to an aggressive hematologic malignancy like mantle cell lymphoma.

Keywords: CD1d; NKT cells; mantle cell lymphoma; mouse models of lymphoma; α-galactosylceramide.

Figure 1

B cell lymphoma mediated activation of mouse NKT cell hybridomas. (A) Mouse B cell lymphoma lines (WEHI-231, CH31, and CH33) were cocultured with NKT cell hybridomas, DN32. D3 and N38-3C3. Recognition of CD1d was assessed by measuring IL-2 production in the supernatants by ELISA. L cells stably transfected with mCD1d1 (LCD1d) served as a positive control; (B) B cell lines were pulsed with the potent NKT cell agonist, α-GalCer (200 ng/mL for 2 h), washed extensively with PBS and co-cultured with NKT cell hybridomas; (C) Cell surface CD1d expression on mouse B cell lymphoma lines. Murine B cell lymphoma lines were analyzed for surface expression of CD1d by flow cytometry. Cells were stained with PE-labeled mouse CD1d specific clone 1B1 (red histograms), the gray filled histograms indicate the isotype control.

Figure 2

Healthy human NKT cells are stimulated by human B cell lymphomas, but circulating NKT cells are reduced in lymphoma patients. (A) Differential CD1d expression in human B cell lymphoma cell lines. Human B cell lymphoma cell lines were analyzed for surface expression of CD1d by flow cytometry. Cells were stained with PE-labeled human CD1d specific clone CD51.1 (red histograms). Isotype staining was performed to demonstrate specificity (black histograms). C1R-CD1d served as the positive control; (B) Primary human NKT cells were cocultured with human B cell lymphomas in the absence or presence of antigen- α-GalCer. Cytokine production following stimulation with C1R-CD1d served as the positive control; (C) Circulating NKT cells are reduced in MCL patients. PBMC were isolated from healthy donors (HD) and mantle cell lymphoma (MCL) patients and stained for flow cytometry; (D) Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors and cancer patients. Cells were stained for Vα24⁺Vβ11⁺ TCR and analyzed by FACS. Scatterplots demonstrate the variation in the percentages of NKT cells. MZL-marginal zone lymphoma; DLBCL-diffuse large B cell lymphoma; MCL- mantle cell lymphoma; FL-follicular lymphoma. % NKT cells of healthy donors vs. lymphoma patients; Statistical analysis was performed using one way ANOVA *** p < 0.001; (E) Primary NKT cells expanded from a healthy donor were co-cultured with B cells isolated from a healthy donor or a mantle cell lymphoma (MCL) patient in the presence or absence of α-GalCer. Culture supernatants were harvested and ELISA was used to measure IFN-γ production. Data are representative of three independent experiments.

Figure 3

NKT cell responses are impaired during lymphoma progression. (A) Splenic NKT cell profiles in WT, DTG and c-myc Tg mice; (B) Splenic NKT cell % in WT, DTG, and c-myc Tg mice at six to seven weeks and 10–15 weeks of age. The term “disease” means that the mice were sick, as indicated by splenomegaly; (C) Primary mouse splenocytes cultured in medium alone or α-GalCer. After 48 h, IFN-γ levels were measured in the supernatant by ELISA; (D) To assess T cell function, splenocytes were cultured with anti- CD3/CD28 microbeads or PMA/ionomycin for 48 h. IFN-γ production in the culture supernatant was measured by ELISA. Data shown from one experiment and are representative of seven similar experiments.

Figure 4

Activation of NKT cells reduces tumor burden in vivo. Eight-week old IL-14/c-myc double transgenic mice were treated with vehicle alone (DMSO) or α-GalCer (2 μg/mouse) in PBS i.v. and after six weeks, lymph nodes and spleens were harvested and examined for disease. (A) H&E staining of spleen sections shows improved splenic architecture and lower frequency of blastoid variant MCL cells as compared to vehicle treated controls; (B) spleens and (C) lymph nodes of α-GalCer treated mice show reduced splenomegaly and lymphadenopathy, respectively, as compared to vehicle-treated, littermate controls. These data are representative of three independent experiments of two to five mice per group; a total of 11 mice were treated with vehicle, and 10 were treated with α-GalCer.

Figure 5

Activation of NKT cells with a single dose of α-GalCer (i.v.) increases survival in a mouse model of MCL-BV. (A) DTG mice were treated with α-GalCer or vehicle alone as described above. Survival curves showing mice treated with vehicle alone (n = 11) or with α-GalCer (n = 10). Mice were euthanized upon detection of severe lymphadenopathy along with their non-terminal counterparts; (B) Percentage of TCRβ⁺, PBS57 loaded (α-GalCer)-CD1d tetramer⁺ NKT cells in vehicle treated or α-GalCer treated mice; (C,D) Splenocytes were cultured for 48 h ex vivo in medium alone, or with α-GalCer for restimulation. Both baseline and restimulated levels were higher for (C) IFN-γ but not (D) IL-4 in mice treated with α-GalCer.