iNKT Cells Suppress the CD8⁺ T Cell Response to a Murine Burkitt’s-Like B Cell Lymphoma

<div><p>The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8⁺ T cell response. Lymphoma cells transplanted into syngeneic wild type (WT) mice or Jalpha18^−/− mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8⁺ T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18^−/− mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8⁺ T cells. In contrast, lymphoma growth in CD1d1^−/− mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8⁺ T cell response to B cell lymphoma by opposing the effects of type II NKT cells.</p> </div

Jalpha18^−/− mice have enhanced CD8⁺ T cell responses to tumor-derived antigen.

<p>(A) The frequency of tumor reactive CD8⁺ T cells was determined by staining with K^b/SIINFEKL tetramers. Staining was performed in the LN, spleen, and IHL 8 days after transplant of TBL-OVA tumor cells. (B) The number of K^b/SIINFEKL reactive CD8⁺ T cells was determined by flow cytometry. (n = 8–10 pooled from two experiments). (C) WT, J<b>alpha</b>18^−/−, and CD1d1^−/− mice were transplanted with 10⁵ TBL-OVA cells i.v. The number of K^b/SIINFEKL-tetramer reactive CD8⁺ T cells in the spleen was determined by flow cytometry (One representative of three similar experiments is shown. n = 4–5/group. The J<b>alpha</b>18^−/− and WT groups in this experiment are also included in panel E, as the controls for these experiment were combined). (D and E) WT and J<b>alpha</b>18^−/− mice were given either 10⁵ TBL-OVA tumor cells or 10⁵ peptide pulsed BMDC intravenously. Spleen cells were analyzed 8 days later for the presence of K^b/SIINFEKL tetramer reactive CD8⁺ T cells. The (D) frequency and (E) number of tetramer  =  CD8⁺ T cells is shown (n = 6–7 from two experiments). (F–G) The number of (F) K^b/SIINFEKL reactive CD8⁺ T cells and (G) lymphoma cells was determined 8 days after administration of 10⁵ TBL-OVA cells, 10⁵ SIINFEKL-pulsed BMDC, or SIINFEKL-pulsed BMDC in combination with either TBL-OVA or TBL-GFP lymphoma cells. ***p<.001, **p<.01, nd  =  not detected.</p

In the absence of iNKT cells, CD8⁺ T cells are required for tumor clearance.

<p>(A and B) The frequency of tumor cells in the spleens of tumor transplanted Jalpha18^−/− mice treated with control (anti-human DR1), anti-NK1.1, anti-CD8, or both anti-NK1.1 and anti-CD8 antibodies was measured by flow cytometry. Plots in (A) are gated on CD49b^-NK1.1^-CD4^-CD8^- cells and the number on each plot indicates the frequency of tumor cells in that gate. (B) The frequency of tumor cells among splenocytes for the indicated antibody-treated Jalpha18^−/−mice is shown. (C) The number of tumor cells present in spleens of antibody treated mice was determined (n = 6–8 pooled from 2 independent experiments). *p<.05. **p<.01.</p

Tumor cells engraft in Jalpha18^−/− mice but are rejected.

<p>The frequency of tumor cells present among intrahepatic leukocytes was determined 6, 8, 10, 12, and 14 days after transplant in Jalpha18^−/− or WT mice. Data are (A) representative FACS plots and (B) combined data from 2 independent experiments (n = 2 for days 12 and 14, n = 3–7 for all other timepoints).</p

T cells promote tumor growth.

<p>(A and B) WT and CD3epsilon^−/− mice were analyzed 14 days after tumor transplant. Spleen and pooled LN (inguinal, axillary, brachial) cells were stained for flow cytometry. (A) Plots are gated on live cells and numbers indicate the frequency of GFP⁺CD19⁺ tumor cells present in each organ. (B) The number of tumor cells in the LN and spleen in WT and CD3epsilon^−/− mice was determined by flow cytometry (n = 11–19 pooled from at least 3 independent experiments). (C and D) The frequency (C) and number (D) of tumor cells present in the LN on day 14 was determined by flow cytometry for WT, TCRdelta^−/−, and TCRbeta^−/− mice (n = 13–14 pooled from 3 independent experiments). *p<.05, **p<.01, ***p<.001.</p

alphaGalCer enhances the suppressive effect of iNKT cells.

<p>WT mice were treated with either 1 microgram alpha-GalCer or vehicle control 1, 5, and 9 days after transplant of TBL-OVA tumor cells. Jalpha18^−/− mice were left untreated following tumor transplant. (A) The frequency of tumor-reactive CD8⁺ T cells was determined by staining with K^b/SIINFEKL tetramers on days 8 and 11 after transplant. (B) The number of SIINFEKL-reactive CD8⁺ T cells was determined on day 11 in the LN, spleen, and IHL. (C) The number of TBL-OVA lymphoma cells in the spleen was determined by flow cytometry 8, 11, and 14 days after transplant in Jalpha18^−/−, vehicle-treated WT, and alpha-GalCer-treated WT mice. Data are representative (A) FACS plots, (B) combined data from 3 independent experiments (n = 9–10), and (C) combined data from at least three independent experiments (n = 4–10/timepoint for both WT groups, n = 3–6 for Jα18^−/−). *p<.05, **p<.01, ***p<.001.</p

Reconstitution of iNKT cells promotes tumor growth in Jalpha18^−/− mice.

<p>(A) Liver mononuclear cells from WT (iNKT) and Jalpha18^−/− mice (Control) were adoptively transferred into Jalpha18^−/− recipients. 10⁵ lymphoma cells were transplanted one day later and tumor growth in the liver was analyzed on day 10 post-transplant. Representative flow plots are gated on CD11b^-Gr1^-CD4^-CD8^- cells, and the number on the plot represents the percentage of gated cells that were CD19⁺GFP⁺ tumor cells. (B-C) The frequency (B) of tumor cells among all IHL and the absolute number of tumor cells (C) from a total of 6–7 mice in three independent experiments are shown. *p<.05.</p

iNKT cells promote tumor growth.

<p>(A and B) The (A) frequency and (B) number of tumor cells was calculated for LN (and spleen in A) from WT, CD4^−/−, and CD8^−/− mice on day 14 after lymphoma transplant (n = 5–9 pooled from 2 independent experiments). (C) LN and spleen cells from tumor-transplanted WT and Jalpha18^−/− mice were analyzed by flow cytometry for the presence of tumor cells. The numbers on the plots indicate the percentage of all <b>TCRbeta^-</b>CD11b^- cells that were GFP⁺CD19⁺ tumor cells. (D) The numbers of tumor cells present in the LN and spleens of WT and Jα18^−/− mice were determined by flow cytometry (n = 9–11 pooled from three independent experiments). (E) Tumor-free survival of WT and Jalpha18^−/− mice was assessed (n = 8–10 from two experiments, p = .008). (F) WT and CD1d1^−/− mice were transplanted with 10⁵ TBL-GFP cells. Fourteen days later, the number of tumor cells present in the spleen and LN was determined. *p<.05, ***p<.001.</p