Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation

Abstract

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4⁺ and CD8⁺ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti-CD20 antibody-mediated B cell depletion on CD4⁺ and CD8⁺ memory T cell alloresponses after skin transplantation in wild-type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4⁺ T cells but enhanced that of CD8⁺ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT-II CD4⁺ anti-OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.

Keywords: T cell biology; basic (laboratory) research/science; immunobiology; rejection: T cell mediated (TCMR).

Figure 1.

Opposite effects of B cell depletion on CD4⁺ and CD8⁺ memory T cell responses after skin transplantation BALB/c mice were injected with an anti-CD20 murine IgG2a monoclonal antibody, 5D2 (250 μg given intraperitoneally) 5 days before placement of a B6 skin allograft and then at day 5, 15 and 30 post-transplantation. CD4⁺ and CD8⁺ T cells from the spleen of recipients were FACS-sorted 40 days post-transplantation (black bars). Control mice included naïve mice (white bars: no Ab treatment and no transplantation) and transplanted mice, which did not received anti-CD20 mAbs (grey bars). The frequencies of alloreactive memory CD4⁺ T cells (top panels) and CD8⁺ T cells (lower panels) producing IL-2 (left panels) or γIFN (right panels) inflammatory cytokines cultured for 48 hours without APCs (medium) or with irradiated donor or control syngeneic APCs (mixed lymphocyte reaction) were measured using a previously described 4-hour ELISPOT assay (23). The results are expressed as numbers of ⬜⬜⬜⬜⬜⬜⬜⬜ spots per 0.5 million T cells and represent 4–6 mice tested individually ± SD.

Figure 2.. B cell depletion impairs CD4⁺ memory T cell responses after skin transplantation in OT-II transgenic mice

Panel A. OT-II TCR transgenic mice whose CD4⁺ T cells recognize MHC class II A^b bound to OVA 323–335 peptide were injected with 5D2 anti-CD20 mAb 5 days prior to and then at day 5, 15 and 20 after placement of a skin graft from an Act mOVA donor mouse (MST: 16 ± 4 days). The generation of a memory T cell response against OVA-APCs was tested using a previously described 4-hour γIFN ELISPOT assay (23) (black bars). Naïve (white bars, non-transplanted) and transplanted mice (grey bars), which did not receive anti-CD20 mAbs, were used as controls. Panel B. OT-II mice were allosensitized through the placement of an OVA skin allograft (rejected within 16 days). Ninety five days later, these mice were injected or not with anti-CD20 antibodies and received 5 days later a second OVA skin allograft later. The reactivation of allospecific memory T cells was tested by ELISPOT as described for panel A. The results are expressed as numbers of ⬜⬜⬜⬜⬜⬜⬜⬜ spots per 0.5 million T cells and represent 6–8 mice tested individually ± SD.

Figure 3.. B cell depletion promotes donor hematopoietic chimerism in OT-II transgenic mice sensitized to ovalbumin

First, OT-II mice received a skin allograft from an Act mOVA donor mouse. Thirty days later these mice were either treated (solid circles) or not (open circles) with a single injection of anti-CD20 mAbs (5D2, 250 μg given intraperitoneally). Five days later, these mice were irradiated with a non-myoablative dose (3Gy), injected with anti-CD40L mAb (MR1, 1 mg given i.p) and injected intravenously with 10⁷ million bone marrow cells from an Act mOVA mouse. The presence of donor hematopoietic chimerism in the blood of mice was monitored at different time points by FACS using an antibody recognizing MHC class I K^b + OVA 254–267 SIINFEKL peptide (panel A-C). The frequencies of recipient TCR transgenic T cells were monitored using antibodies detecting the Vα2-Vβ5 variable regions of the TCR expressed by CD4⁺ T cells in OT-II mice (panel D). The frequencies of TCR transgenic T cells were also measured in control mice: conditioned OT-II mice that did not receive bone marrow cells (open squares), Act mOVA mice (open diamonds) and non-treated OT-II mice (open triangles). The results are representative of 4–8 mice tested individually ± SD. In each panel, p values are indicated as follows: * p < 0.05, ** p < 0.005 and *** P < 0.0005.