Transient depletion of B cells in young mice results in activation of regulatory T cells that inhibit development of autoimmune disease in adults

Abstract

B-cell depletion therapy can be effective for treating B-cell lymphomas as well as many human and murine autoimmune diseases. B-cell-deficient mice are normally resistant to spontaneous autoimmune thyroiditis (SAT), but they develop SAT if regulatory T cells are transiently depleted during the first 3-6 weeks after birth. This was also a critical time when B-cell depletion effectively inhibited development of SAT in adult mice. The current study was undertaken to test the hypothesis that transient depletion of B cells using anti-CD20 would be sufficient to suppress SAT if B cells were depleted early in life and that inhibition of SAT would be due to the activity of Treg that functioned most effectively when B cells were absent or low. The results presented here support this hypothesis and indicate that development of autoimmune disease in adults is effectively inhibited when anti-CD20 is administered 1-3 weeks after birth. After 3 weeks, transient B-cell depletion is no longer effective, and B-cell depletion must be maintained to effectively suppress autoimmune disease. B-cell depletion in 1- to 3-week-old mice depletes all B-cell subsets, whereas B-cell depletion initiated in adults spares many marginal zone B cells. Following early B-cell depletion, splenic Treg increase in number, and depletion of Treg reverses the inhibitory effect of anti-CD20 on disease development. Early transient depletion of B cells could be useful for preventing autoimmune disease in individuals at high risk for developing autoimmune diseases as adults.

Fig. 1.

Early transient depletion of B cells is sufficient to inhibit development of SAT in NOD.H-2h4 mice. NOD.H-2h4 mice were given 100 μg anti-CD20 or isotype control i.p. and s.c. 9 and 16 days after birth. All mice were given NaI in their water at 8 weeks of age and thyroids were removed 2 months later. (a) Symbols represent SAT severity scores of individual mice 8 weeks after NaI water. The mean severity score for the group is indicated by the line. (b) Anti-MTg autoantibodies diluted 1/50 in serum from individual mice 8 weeks after NaI water. Results are expressed as mean OD₄₁₀ ± SEM. Significant differences between isotype control and anti-CD20-treated mice are indicated by the asterisk (P < 0.01).

Fig. 2.

Suppression of SAT after transient B-cell depletion is dependent on the age when B cells are depleted. NOD.H-2h4 mice were given 150 μg anti-CD20 beginning at the indicated ages and an additional 100 μg anti-CD20 3–7 days after the first injection. Mice were given NaI water at 8 weeks of age and thyroids were removed 8 weeks later to assess SAT severity. Results are pooled from two separate experiments and are representative of four different experiments. The 7 weeks group (‡) represents mice that were given anti-CD20 every 3 weeks beginning at 7 weeks of age to maintain B-cell depletion as previously described (24). (a) SAT severity scores of individual mice 8 weeks after NaI water; the mean severity score for each group is indicated by the line. (b) Anti-MTg autoantibodies expressed as mean OD₄₁₀ ± SEM of individual sera diluted 1/50. Groups that differ significantly from the isotype control are indicated by the asterisk (P < 0.05).

Fig. 3.

Effectiveness of B-cell depletion by anti-CD20 varies depending on the age when B cells are depleted. Flow cytometric analysis of B220+ B cells in the spleens of NOD.H-2h4 mice given isotype control or anti-CD20 10 and 16 days after birth (4 weeks groups) or at 8 weeks of age (10 weeks groups). Spleens were removed 14 days after injection of antibody and analyzed by flow cytometry. The results are representative of the groups shown in Table 2 and include six to eight individual mice assayed for each group.

Fig. 4.

Flow cytometric analysis of Foxp3+ Treg in anti-CD20-treated Foxp3 GFP NOD.H-2h4 mice. The top panels show representative results for Foxp3+ cells as a percentage of the total splenocytes. The percentage of CD4+ T cells for each group is the sum of the upper left and upper right quadrants. In the bottom panels, splenocytes are gated on CD4+ T cells and the percentage of Foxp3+ Treg expressed as a percentage of CD4+ T cells is indicated in the upper right quadrant. Results are representative of the groups shown in Table 3.

Fig. 5.

Suppression of SAT in mice given anti-CD20 is abrogated following transient depletion of Treg. Mice were given anti-CD20 or isotype control at 10 (100 μg) and 16 (150 μg) days of age. Some mice were given three weekly injections of anti-CD25 (PC 61) or rat Ig as a control beginning 1 week after the second injection of anti-CD20 or isotype control. All mice were given NaI water at 8 weeks of age, and thyroids were removed 8 weeks later. Results are pooled from two separate experiments and are representative of five similar experiments. (a) SAT severity scores of individual mice 8 weeks after NaI water; the mean severity score for each group is indicated by the line. (b) Anti-MTg autoantibody responses expressed as mean OD₄₁₀ ± SEM of individual sera diluted 1/50. Groups that differ significantly from the isotype control are indicated by the asterisk (P < 0.05).

Fig. 6.

T cells from isotype control-treated donors with SAT provide help to B cells from TCRα−/− mice, whereas T cells from anti-CD20-treated donors have minimal activity unless Treg are depleted. TCR-α−/− mice were irradiated (300 rad) and given 5 × 10⁶ splenic T cells (purified using nylon wool as described in Methods). T-cell donors were either naive NOD.H-2h4 mice or mice that were given the indicated treatment before receiving NaI water for 8 weeks (SAT donors). Recipients were given NaI in their water, and thyroids were removed 8 weeks later. Results are pooled from three separate experiments. Mice not given T cells did not develop SAT and their anti-MTg autoantibody responses were <0.100 OD units (data not shown). (a) SAT severity scores of individual mice 8 weeks after NaI water; the mean severity score for each group is indicated by the line. (b) Anti-MTg autoantibody responses expressed as mean OD₄₁₀ ± SEM of individual sera diluted 1/50. Groups differing significantly from the isotype control are indicated by the asterisk (P < 0.05).