Depletion of CD4+ CD25+ regulatory T cells inhibits local tumour growth in a mouse model of B cell lymphoma

Abstract

Regulatory T cells (T(regs)) may inhibit immunity against cancer. Induction and expansion of T(regs) in the immunosuppressive microenvironment created by a growing tumour appear to be one of the mechanisms by which it can evade host defence. We studied the impact of CD25+ T(regs) in a B cell lymphoma model in which Rag2-/- mice received adoptive transfer of wild-type spleen cells with or without CD25+ cells, and concurrently subcutaneous inoculation of the B cell lymphoma cell line A20. We also examined the effect of engaging the glucocorticoid-induced tumour necrosis factor receptor (GITR) - an approach reported previously to abrogate the suppressive effects of T(regs). Mice that received spleen cells depleted of CD25+ T(regs) showed significantly slower tumour growth and increased survival compared with mice that received unsorted spleen cells. The T(reg)-depleted group also had significantly more CD8+ T cells infiltrating the tumours and higher levels of serum immunoglobulin G subclasses. The anti-GITR treatment had no significant effect on tumour growth, survival or immunoglobulin production. In the CD25-depleted group four of 10 mice developed clinical signs of autoimmunity, in contrast to none in the non-depleted group. Forkhead box P3+ T cells were found in tumour-draining lymph nodes in mice in the CD25-depleted group, suggesting an in vivo induction or expansion of rare transferred donor T(regs). Thus, our study showed that removal of CD25+ T(regs) enhanced anti-tumour immunity against local growth of a B cell lymphoma and that induction or expansion of T(regs) could be one mechanism by which the growing tumour evades immune surveillance.

Fig. 1

Flow-cytometric analysis of CD25 and FoxP3 expression in unsorted wild-type Balb/c spleen cells before (a) and after CD25-depletion (b). Plots are gated on CD3⁺ CD4⁺ cells and show a near complete removal of CD25⁺ FoxP3⁺ cells by the magnetic CD25 depletion procedure. Results are representative of at least five independent experiments. FoxP3, forkhead box P3; PE, phycoerythrin; APC, allophycocyanin.

Fig. 3

Survival in the same three groups of mice described in Fig. 2a. Kaplan–Meier plot shows significantly increased survival in mice that received CD25-depleted cells compared with mice that received unsorted wild-type spleen cells and controls (P = 0·036, log-rank test).

Fig. 2

(a) Growth of B cell lymphoma tumours in control Rag2^–/– mice that did not receive adoptive spleen-cell transfer (n = 5), and the two groups that received either unsorted wild-type spleen cells (n = 10) or CD25-depleted cells (n = 10) (*P < 0·05, **P < 0·01, Mann–Whitney test, two-tailed). Statistical comparisons are between the groups that received unsorted or CD25-depleted cells. Error bars indicate standard error of the mean. (b) Tumour growth shown as tumour size (diameter measured in mm) at various time-points in individual mice of the group that received CD25-depleted spleen cells. Each line represents one animal. Horizontal lines indicate no measurable tumour.

Fig. 4

Serum levels of IgG1, IgG2a and total IgG in Rag2^–/– mice that received unsorted wild-type spleen cells or CD25-depleted cells and showed a survival > 48 days or < 48 days. Serum was collected on the day the mice were killed and measurements were performed by enzyme-linked immunosorbent assay. Significance levels indicated for Mann–Whitney two-tailed test. Ig, immunoglobulin.

Fig. 5

Number of tumour-infiltrating CD8⁺ T cells was increased significantly in Rag2^–/– mice that had received CD25-depleted cells compared with unsorted wild-type spleen cells. Data show the number of CD8⁺ cells mm² of tumour tissue (P = 0·038, Mann–Whitney two-tailed test).

Fig. 6

Immunohistochemistry of tumour-draining lymph node in Rag2^–/– mice that had received CD25-depleted wild-type spleen cells. Paired immunofluorescence staining shows the presence of CD3⁺ (red) cells concurrently expressing nuclear FoxP3 (green); ×400 magnification.