Neutralizing BAFF and APRIL in combination with anti-CD154 Ab treatment and CD8+ T cell depletion induces long term cardiac allograft survival in mice containing memory CD4+ T cells

Sumantha Bhatt¹, Ran Fan¹, Anna Valujskikh¹

¹Department of Immunology, The Cleveland Clinic Foundation, Cleveland, OH, USA

Donor-reactive antibodies initiate acute rejection and chronic pathology of organ transplants. We previously reported that alloreactive memory CD4⁺ T cells can use pathways other than CD40-CD154 to induce IgG alloantibodies (alloAb). Neutralizing B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) abrogates alloAb induction by memory CD4⁺ T cells when the CD40-CD154 pathway is compromised. The goal of this study was to determine whether BAFF/APRIL neutralization in combination with anti-CD154 Ab prolongs graft survival in the presence of donor-specific memory CD4⁺ T cells.

Female B6 (H-2^b) recipients containing male antigen-reactive TCR transgenic memory CD4⁺ T cells (MAR) received male BALB/C (H-2^d) cardiac allografts. MAR T cells recognize male antigen through the indirect pathway and induce donor-reactive Abs.  Groups of recipients were treated with a combination of anti-CD154 Ab (1 mg i.v., d. -1) and either mBAFFR-Fc (neutralizes BAFF alone), mTACI-Fc (neutralizes both BAFF and APRIL), or control mFc4 fusion proteins (ZymoGenetics, Inc) for 2 weeks post transplant. The combination of anti-CD154 Ab and TACI-Fc inhibited anti-donor antibody production in recipients containing donor-reactive memory CD4⁺ T cells.  In contrast, recipients treated with  anti-CD154 Ab and either mFc4 or BAFFR-Fc had high titers of all IgG isotypes. No differences in graft survival were observed between groups, as grafts were rejected through CD8⁺ T cell mediated mechanisms.

Incorporation of anti-CD8 depleting Abs into the treatment regimen resulted in a significant prolongation in graft survival in the mTACI-Fc treated group (MST=69 d. vs. 18 d. in mFc4-treated recipients, n=6-8 per group). mBAFFR-Fc treatment was less efficient in prolonging graft survival after CD8⁺ T cell depletion (MST=42 d, n=7). The priming of anti-donor IFNg-producing splenic CD4⁺ T cells was comparable between groups at the time of rejection and at d. 90 post transplant in non-rejecting mice. Rejecting cardiac allografts in mFc4-treated recipients were heavily infiltrated with mononuclear cells and had diffuse C4d deposition. Rejecting and non-rejecting cardiac allografts in mTACI-Fc and mBAFFR-Fc-treated recipients were characterized by moderate cellular infiltrate and focal C4d staining.

Our results indicate that BAFF and APRIL neutralization in combination with anti-CD154 Ab treatment delays memory CD4⁺ T cell-induced alloAb responses in cardiac allograft recipients. We propose that this delay not only attenuates Ab-mediated tissue injury but also impedes T cell activation and T cell recruitment into the graft, thus prolonging graft survival. These findings may guide the future use of BAFF/APRIL neutralizing agents in sensitized transplant recipients.