Alloantibodies prevent the induction of transplantation tolerance by enhancing alloreactive T cell priming

Abstract

Circulating alloantibodies in transplant recipients are often associated with increased Ab-mediated as well as cellular rejection. We tested the hypothesis that alloantibodies facilitate cellular rejection by functioning as opsonins to enhance T cell activation using a BALB/c to C57BL/6 heart or skin transplant model. Long-term heart and skin survival induced with anti-CD154 alone or in combination with donor-specific transfusion (DST), respectively, was abrogated by the presence of anti-K(d) mAbs, and alloreactive T cell activation as well as acute rejection was observed. The prevention of graft acceptance in the skin model was dependent on anti-K(d) binding to and converting DST from tolerigenic to immunogenic. Adoptive transfer of CFSE-labeled TCR-transgenic T cells into B6 recipients treated with anti-CD154/DST revealed the ability of anti-K(d) to enhance the proliferation of anti-K(d)-specific T cells via the indirect pathway as well as of non-K(d)-reactive, recipient MHC-restricted CD4(+) and CD8(+) T cells. Thus, alloantibodies with restricted specificity are able to facilitate the indirect presentation as well as the cross-presentation of a larger repertoire of "linked" donor-derived Ags. These observations highlight the ability of alloantibodies to function not only in classical humoral rejection but also as opsonins that facilitate the CD40-CD154-independent activation of alloreactive T cells.

Figure 1.

Anti-K^d mAbs elicits CD154-independent cardiac and skin allograft rejection. a. Rejection of BALB/c heart grafts by B6 recipients treated with anti-CD154 and anti-K^d mAbs (N = 4) ( closed square) compared to B6 recipients treated with anti-CD154 alone (N = 15) (closed triangle) or untreated recipients (N = 4) (open circle). b. Rejection of BALB/c skin grafts by B6 recipients treated with anti-K^d mAbs plus anti-CD154/DST (N = 20) (closed triangle) in comparison to long-term graft survival of BALB/c grafts by B6 recipients treated with anti-CD154/DST (N = 11) (open diamond) and rejecting untreated B6 recipients of BALB/c grafts (N = 9) (closed square). Acceptance of BALB/c grafts by B6 recipients treated with anti-CD154/DST and denatured anti-K^d mAbs (N = 5) (closed circle), or mouse IgG isotype control (N = 4) (asterisk), and C3H grafts by B/6 recipients treated with anti-K^d mAbs and anti-CD154/DST (N = 4) (half closed downward triangle). c. The transfer of serum from pre-sensitized mice (200 μl/mouse; day −2, 0 and 2 post-transplantation; Pre-Sen), but not from naïve mice, prevented BALB/c skin graft acceptance in B6 mice treated with anti-CD154/DST (N=4/group).

Figure 2.

Role of T cells in anti-K^d mAb-dependent graft rejection a. Frequencies of alloreactive IFN-γ producing cells in untreated B6 recipients (N = 3) (black), or in recipients of cardiac transplants treated with anti-CD154/DST (N = 4) (white), or anti-CD154/DST with anti-K^d (N = 4) (grey). Splenocytes were stimulated in vitro with irradiated B6 (Syn), BALB/c (Allo), C3H (Third-Party) stimulators, and controls were anti-CD3 or media. Data are presented as mean ± standard error. b. The frequency of alloreactive T cells secreting IFN-γ were measured in untreated B6 recipients (N = 3) (black), anti-CD154/DST treated B6 recipients (N = 3) (white), and anti-CD154/DST with anti-K^d mAbs (N = 4) (grey). c. Acceptance of BALB/c grafts by TCRβδ^−/− recipients treated with anti-CD154/DST and anti-K^d mAbs (N = 6) (open square) compared to rejection by similarly treated B6 recipients (N = 20; p = 0.0003) (closed triangle). Comparably treated B6.CD8^−/− recipients exhibited delayed rejection (N = 4; p = 0.0027) (closed diamond).

Figure 3.

Allospecific TCR75 CD4⁺ T cells proliferate in the presence of anti-K^d mAbs. CFSE-labeled TCR75 TCR-Tg T cells (0.25 x 10⁶) were injected i.v. at the time of skin transplantation. TCR75 T cells were harvested from the draining lymph nodes five days later and proliferation was measured by flow cytometry. Representative histogram of TCR75 T cell proliferation from untransplanted B6 recipients (naïve B6) ( 1^st Panel), B6 recipients of BALB/c grafts without treatment (No R_x) (2^nd Panel), treated with anti-CD154/DST (αCD154/DST ) (3^rd Panel), or with anti-CD154/DST and anti-K^d mAbs (αCD154/DST + αK^d) (4^th Panel). The percentage of dividing TCR75Tg T cells from individual mice in each group are summarized (N = 3/ group). Significantly higher percentages of proliferating TCR75 Tg T cells were observed in B6 recipients treated with anti-K^d mAbs and anti-CD154/DST compared to recipients treated with anti-CD154/DST (p = 0.0067).

Figure 4.

Anti-K^d mAbs primes T cells specific for alloantigens not recognized by anti-K^d mAbs and enhances presentation of exogenously derived alloantigens. All animals were injected with 5uM CFSE labeled 0.8 x 10⁶- 10⁷ OT-II and OT-I TCR Tg T cells at the time of skin transplantation. Actin-OVA BALB/c mice were used as donors for all skin transplants. Flow cytometric analysis of OT-II (top row) and OT-I (bottom row) TCR Tg T cells was measured in the draining lymph nodes five days post-skin transplantation of untransplanted B6 recipients (1^st Column), B6 recipients of BALB/c-OVA grafts without treatment (2^nd Column), B6 recipients of BALB/c-Ova grafts treated with anti-CD154/DST (3^rd Column), and B6 recipients of BALB/c-OVA grafts treated with anti-CD154/DST and anti-K^d mAbs (4^th Column). Significantly higher percentages of proliferating cells were observed in B6 recipients treated with anti-K^d mAbs and anti-CD154/DST compared to recipients treated with anti-CD154/DST (for OT-1 p = 0.0001, for OT-II; p = 0.0015).

Figure 5.

CTLA4Ig overrides the effects of Anti-K^d mAbs and facilitates graft acceptance in anti-CD154/DST-treated recipients. Acceptance of BALB/c skin grafts by B6 recipients treated with anti-CD154/DST + CTLA4Ig (N =4) (closed square) anti-CD154/DST + CTLA4Ig with anti-K^d mAbs (N = 7) (closed downward triangle) but rejection by B6 recipients treated with anti-CD154/DST and anti-K^d mAbs (N = 15) (open upward triangle). B6 recipients treated with anti-CD154/DST (N = 11) (closed circle) also accepted BALB/c skin grafts.