Delicate balance among three types of T cells in concurrent regulation of tumor immunity

Abstract

The nature of the regulatory cell types that dominate in any given tumor is not understood at present. Here, we addressed this question for regulatory T cells (Treg) and type II natural killer T (NKT) cells in syngeneic models of colorectal and renal cancer. In mice with both type I and II NKT cells, or in mice with neither type of NKT cell, Treg depletion was sufficient to protect against tumor outgrowth. Surprisingly, in mice lacking only type I NKT cells, Treg blockade was insufficient for protection. Thus, we hypothesized that type II NKT cells may be neutralized by type I NKT cells, leaving Tregs as the primary suppressor, whereas in mice lacking type I NKT cells, unopposed type II NKT cells could suppress tumor immunity even when Tregs were blocked. We confirmed this hypothesis in 3 ways by reconstituting type I NKT cells as well as selectively blocking or activating type II NKT cells with antibody or the agonist sulfatide, respectively. In this manner, we showed that blockade of both type II NKT cells and Tregs is necessary to abrogate suppression of tumor immunity, but a third cell, the type I NKT cell, determines the balance between these regulatory mechanisms. As patients with cancer often have deficient type I NKT cell function, managing this delicate balance among 3 T-cell subsets may be critical for the success of immunotherapy for human cancer.

Fig 1. Anti-CD25 treatment is sufficient to induce protection in WT and CD1d^−/− mice but not in Jα18^−/− mice

A, CT26 cells (5×10⁴) were injected s.c in the left flank of WT, CD1d^−/− and Jα18^−/− mice (5 mice/group). Five days before tumor challenge, 0.5 mg of anti-CD25 or Rat IgG was injected i.v. Tumor size was measured twice a week. The experiment was repeated 4 times. Anti-CD25 treatment significantly reduced tumor size in WT mice (p=0.0079 against Rat IgG-treated WT mice) and CD1d^−/− mice (p=0.0449 against Rat IgG-treated CD1d^−/− mice). Anti-CD25 was not effective in Jα18^−/− mice. B, Lymph node and spleen cells were prepared from WT, CD1d^−/− and Jα18^−/− mice (3 mice/group). Percentage of Tregs (CD3⁺CD4⁺CD25⁺FOXP3⁺) was evaluated by flow cytometry. The frequency of Tregs was evaluated in 3 mice per group. C, Varying numbers of lymph node CD4⁺CD25⁺T cells from WT or Jα18^−/− mice were cocultured with 5×10⁴ CD4⁺CD25⁻ T cells from each strain and 0.5 µg/ml of anti-CD3. Cells were cultured for 72 hr, with the presence of [³H]-thymidine for the last 16 hours. % of suppression was determined as 100 × [1- (CPM of Treg culture/ CPM of non-Treg culture)]. Data are presented as mean ± SD. These experiments were repeated 3 times with comparable results.

Fig 2. The balance between the 3 kinds of T cells in 3 strains of mice

A .Left panel, In WT mice type I and type II NKT cells counteract each other to cancel their effects, leaving Tregs to dominate the suppression. Middle panel, In CD1d^−/− mice, neither subset of NKT cells exists, and the dominant suppressors are again the Tregs. Right panel, In Jα18^−/− mice the effect of type II NKT cells is not counter-regulated by type I NKT cells, leaving both Tregs and the type II NKT cells able to suppress, so both need to be blocked concurrently to abrogate suppression. B. Livers from naïve BALB/c mice (WT) and Jα18^−/− mice were perfused and processed for lymphocyte enrichment. Total liver lymphocytes pooled from 3 mice were stained with anti-CD3 and sulfatide-loaded CD1d-dimer. Unloaded-CD1d-dimer-reactive cells and dead cells were excluded from the analysis. Left panel Representative density plot of sulfatide-loaded CD1d-dimer-reactive cells among total liver lymphocytes. Right panel, The frequency of sulfatide-loaded CD1d-dimer-reactive cells was evaluated in 6 pools of 3 livers each of WT mice and 8 pools of 3 livers each from Jα18^−/−. Data, pooled from 6 independent experiments, are presented as values from each pool (symbols) with bars showing mean ± SD. ** p = 0.002

Fig 3. A combination of Treg blockade and type II NKT cell blockade reduces tumor burden in Jα18^−/− mice

CT26 cells (5×10⁴) and anti-CD25 (0.5 mg) were injected into WT, CD1d^−/− and Jα18^−/− mice (5 mice/group) as described in Fig 1A. Some mice were also treated with 0.2 mg of anti-CD1d mAb or Rat IgG on days 1, 4, and 7. Tumor size was measured twice a week. Jα18^−/− mice that received the combined treatment developed significantly smaller tumors (p=0.0079 against Jα18^−/− mice that were treated with anti-CD25 alone or anti-CD1d alone). Mice that received the combined treatment (anti-CD25+anti-CD1d) and mice that received only anti-CD25 treatment developed similar tumor size in WT and CD1d^−/− groups. Data are presented as mean ± SD. The experiment was repeated 3 times with comparable results.

Fig 4. Adoptive transfer of type I NKT cells and Treg blockade reduce tumor burden in Jα18^−/− recipients

Livers from 40 naïve BALB/c mice were perfused and digested for lymphocyte enrichment. A, Total liver lymphocytes were stained with anti-CD3 and PBS57-CD1d-tetramer. A presented pseudo dot plot represents the entire lymphocyte population. B and C, PBS57-CD1d-tetramer positive cells were isolated by magnetic bead sorting. The sorted cells were stained with anti-CD3, anti-CD4, anti-CD25 and analyzed by flow cytometry. Presented pseudo dot plots represent the entire positive fraction (B and left panel of C) or CD4⁺CD25⁺ gated population (right panel of C). D and E, On day -1, 4×10⁵ PBS57-CD1d-tetramer positive cells were adoptively transferred into the tail vein of Jα18^−/− mice that were already treated on day -5 with 0.5 mg anti-CD25 i.v (5 mice/group). On day 0 mice were challenged s.c. with CT26 cells (5×10⁴). D, on day 5, liver lymphocytes and spleen cells of WT or Jα18^−/− mice which did or did not receive adoptively transferred NKT cells (3 mice/group) were stained with anti-CD3 and PBS57-CD1d-tetramer and analyzed by flow cytometry. Presented pseudo dot plots represent the entire lymphocyte population in each tissue. E, Tumor size was measured twice a week. Adoptive transfer of type I NKT cells into anti-CD25-treated Jα18^−/− mice significantly reduced tumor size (p=0.0035 against Jα18^−/− mice treated with Rat-IgG; p=0.0007 against Jα18^−/− mice treated with anti-CD25; p=0.0002 against Jα18^−/− mice adoptively transferred type I NKT cells). Data are presented as mean ± SD. The experiment was repeated 4 times with comparable results.

Fig 5. Sulfatide treatment after Treg blockade suppresses tumor immunity in WT mice

A. CT26 cells (5×10⁴) and anti-CD25 mAb (0.5 mg) were injected into WT mice as described in Fig 1A (5 mice/group). One hour after tumor challenge, mice were injected s.c with 30 µg sulfatide or control vehicle at a site adjacent to that of the tumor injection. Tumor size was measured twice a week. Sulfatide significantly increased tumor size in anti-CD25-treated mice (p=0.0079 vs vehicle+anti-CD25-treated mice). Data are mean ± SD. The experiment was repeated 3 times. B. Anti-CD25-treated mice were challenged with CT26 cells and injected with vehicle or 30 µg/mouse sulfatide. 72 hours after the injections, livers from the mice were perfused and processed to enrich lymphocytes. Total liver lymphocytes were stained with PercCP-Cy5.5- anti-CD3 and PE-PBS57-CD1d tetramer to enumerate type I NKT cells. The proportion of type I NKT cells in the mice with anti-CD25+sulfatide significantly lower than in the mice with anti-CD25 alone (mean 41% reduction, range 32–46%, p<0.01 by t-test with conservative variance estimation). Presented density plots are representative of four independent experiments.

Fig 6. Blockade of Treg and type II NKT cells decreases tumor burden in R331 tumor-challenged Jα18^−/− mice

A. R331 cells (5×10⁵) were injected s.c in the left flank of WT and Jα18^−/− mice (5 mice/group). Five days before tumor challenge, 0.5 mg of anti-CD25 or Rat IgG was injected i.v. Tumor size was measured every four days starting day 7. Anti-CD25 treatment significantly reduced tumor size in WT mice (p= 0.0075 vs Rat IgG-treated WT mice) but not in Jα18^−/− mice. B. R331 cells (5×10⁵) and anti-CD25 (0.5 mg) were injected into WT and Jα18^−/− mice (5 mice/group) as described above. Two groups of each strain of mice were also treated with 0.2 mg of anti-CD1d mAb or Rat IgG on days 1, 4, and 7. Tumor size was measured every 2–4 days starting on day 7. Jα18^−/− mice that received anti-CD25+anti-CD1d treatment had significantly slower tumor growth than Jα18^−/− mice that were treated with anti-CD25 alone or anti-CD1d alone. (p<0.05 by the repeated measures analysis of variance test). All group had five mice each except for the anti-CD25 treated group that had four mice. Data are presented as mean ± SD. The experiment was repeated 2 times with comparable results.