Anti-CD20 treatment depletes B-cells in blood and lymphatic tissue of cynomolgus monkeys

Abstract

Introduction: Macaque species offer a valuable model for translational allo-transplantation and tolerance studies. Cardiac allograft vasculopathy in Macaca fascicularis is associated with elaboration of anti-donor antibodies. Since T-independent pathways of B cell activation have been described, and anti-B cell strategies have proven to be a fruitful tolerogenic adjunct in rodent and xenogenic models, here we investigate whether an anti-CD20 antibody (rituximab) would be useful to deplete B-cells in a pre-clinical allo-transplantation setting in macaques. Methods: Three cynomolgus macaques which had previously rejected a cardiac allograft and one with concurrent subacute vascular rejection were treated weekly with rituximab 20 mg/kg IV for 4 and 2 weeks, respectively. B-cell levels (CD19+ cells) were measured by flow cytometry in peripheral blood, spleen, lymph node and bone marrow cells at various intervals after initiation of treatment. B-cells and plasma cells were also analyzed by immunohistochemistry at necropsy in spleen, lymph node, tonsil and thymus tissue sections. Anti-donor antibody titers were measured by flow cytometry. Results: B-cells expressing CD19 were not detectable in the peripheral blood in any animal within 24 h after initial treatment, or over the ensuing month. At necropsy, the germinal centers in spleen and lymph node were completely depleted of CD20+ B-cells in 2 animals, leaving a hypocellular trabecular pattern around preserved plasma cell follicles. Substantial but incomplete depletion of B-cells was demonstrated in the other 2 animals, in each instance immunohistochemical findings in spleen and lymph node exhibiting higher sensitivity for residual B-cells compared to FACS. Anti-donor antibody titers exhibited kinetics similar to untreated animals over this short follow-up. Comment: Treatment with anti-CD20 very efficiently depletes peripheral and tissue B-cells but not plasma cells in this macaque species. Biopsy of lymph node is necessary and may be sufficient to assess B-cell clearance in secondary lymphoid organs in this model.

Introduction

Anti-B-cell therapy using a humanized monoclonal antibody against CD20 is active not only against human tumors expressing this antigen, but also has been associated with clinical improvement in autoimmune symptoms and, anecdotally, with reduced autoantibody titers [1], [2], [3], [4], [5]. In the transplant setting, the T-dependent activation of antigen-specific B-cell clones and maturation to plasma cells is thought to be the principle mechanism mediating the alloantibody response to donor antigens, which in turn is associated with the pathogenesis of both acute vascular and chronic rejection of organ allografts [6], [7], [8]. In addition, T-independent pathways of B-cell activation may also contribute to antibody-driven chronic allograft injury [9]. Further, in rodents either blocking B-cell mediated costimulation [10] or pharmacologic inhibition of B-cells [11] facilitates induction of tolerance in the context of non-depleting costimulation pathway-based regimens [12], [13]. Whether depletion of B-cells can prevent development of alloantibody or is tolerogenic has not yet been evaluated in non-human primate models or in man. To address these questions, a reagent that specifically depletes B-cells in a preclinical, translational primate model would be useful.

The current study was undertaken to evaluate the efficacy of an anti-CD20 agent, rituximab, to deplete B-cells in primate species useful for transplant studies, and to validate an approach for determining efficiency of B-cell clearance from peripheral blood and secondary lymphoid organs.