Phenotypic and functional analysis of T-cell recovery after anti-CD3 immunotoxin treatment for tolerance induction in rhesus macaques

Abstract

T-cell reduction utilizing specific antibody has been widely used in human transplantation, and is a cornerstone of several tolerance induction strategies in nonhuman primates. We have established a population of long-term tolerant rhesus macaques induced with an anti-CD3epsilon immunotoxin (IT). This treatment effects transient, specific and profound ablation of T cells in blood and lymphoid tissues. In most instances the IT was used in combination with the NF-kappaB inhibitor, 15-Deoxyspergualin. This 2-week long protocol produces a "window of opportunity" for tolerization in which the animal exhibits an enduring quiescent state of unresponsiveness to the allograft, all accomplished without maintenance immunosuppressive drugs. During this induction period, the treated immune system bears some resemblance to that of the neonate, in that T cell numbers are abnormally low and antigen presentation by dendritic cells is precluded by an arrest in their NF-kappaB dependant maturation. In addition, IL-4 production is prominent during and after the tolerance induction interval. For this study we focused on measuring the monkey's ability to repopulate T cells with particular emphasis on the memory T-cell phenotype. Three "memory" phenotypes were utilized; CD3(+)CD45RO(+), CD3(+)CRTH2(+), and CD3(+)CD4(+)CD8(+). All three phenotypes exhibited different patterns of recovery, all of which included transient bursts in their numbers during repopulation. We also estimated thymic activity after T-cell ablation with the use of a newly-described RTE or recent thymic émigré phenotype (a naïve CD8(+)CD103(+) T cell). This marker revealed production of RTE cells including supranormal levels at approximately 6 months post-transplant, implicating thymic function in the repopulation of T-cells. Finally, we measured antibody responses to a panel of antigens (vaccines, environmental antigen, and foreign proteins) that indicated there was no apparent loss of immunologic function during or after the tolerance induction period. Results of studies of T-cell receptor repertoire expression suggest preservation of the pretreatment repertoire, which is consistent with rapid recovery of immune competence to the test antigens. Taken together, these results suggest that while aggressive, this tolerance induction protocol does not appear to incur a prolonged immunologically-compromised state, if at all.