*Purpose: Preformed donor-specific anti-human leukocyte antigen antibodies (DSAs) have been associated with detrimental effects in transplant recipients. To eliminate preformed DSAs, we desensitized DSA-positive recipients by injecting anti-CD20 monoclonal antibodies (mAbs) and then performed plasmapheresis using a protocol for ABO-incompatible (ABO-I) transplant recipients. However, in contrast to ABO-I transplant recipients who did not affect anti-donor CD4⁺T-cell responses, hyper responses of anti-donor CD4⁺T-cell were observed in DSA-positive recipients after desensitization therapy. In this study, we investigated the effect of anti-CD20 mAb-mediated B-cell depletion on anti-donor T-cell responses and the inhibitory role of B-cells in a highly sensitized transplant model.

*Methods: Naïve Balb/c mice were transplanted with C57BL/6 skin grafts (twice; day 0, 14) to prepare a highly sensitized model. On day 42, sensitized Balb/c mice were injected with anti-CD20 mAb to eliminate B-cells (desensitization group). Seven days after the injection (day49), a mixed lymphocyte reaction assay using a carboxyfluorescein succinimidyl ester-labeling technique (CFSE-MLR) were performed to evaluate the effect of B-cell depletion on anti-donor T-cell responses. The control group was administered with isotype-matched control antibodies. Furthemore, to investigate the inhibitory effects of B-cells, CD19⁺whole B-cells, B1(CD5⁺CD19⁺) cells and non B1(CD5^–CD19⁺) cells were co-cultured in CFSE-MLR assay of desensitization group respectively, and the effect of various B-cell subsets on anti-donor T-cell responses were evaluated.

*Results: The average stimulation index (SI) values for the CD4⁺T-cell responses to donor stimulation were significantly higher in the desensitization group ​​than those in the control group. When co-cultured with various B-cells in CFSE-MLR assay of the desensitization group, the addition of whole CD19⁺B-cells and B1-cells significantly suppressed the anti-donor CD4⁺T-cell responses. However, the addition of non B1-cells did not suppress the anti-donor CD4⁺T-cell responses.

*Conclusions: These findings demonstrate that B-cell depletion with anti-CD20 mAb exacerbates anti-donor CD4⁺T-cell responses in a highly sensitized transplant model. It might be possible that the sensitized CD5⁺ CD19⁺B1-cells have an ability to inhibit anti-donor CD4⁺T-cell responses.

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