*Purpose: Difficulty in desensitization in highly sensitized patients remain problematic in organ transplantation. For over 12 years, our laboratory has been carrying out an HLA antibody desensitization program to develop strategies for suppressing donor specific antibodies (DSA).

*Methods: A mouse model of allo-sensitization involving a C57BL mouse as recipient of a skin graft (SG) from a C57BL- tg-HLA.A2 mouse is used to study DSA suppression by either monotherapy or combinational therapies. Drugs tested include (1) anti-CD20 for B-cell depletion; (2) Ibrutinib (Bruton’s tyrosine kinase inhibitor, BTKi); (3) Tofacitinib (Janus Kinase Inhibitor, JAKi); (4) anti-IL6 receptor and anti-IL6 (interleukin 6 pathway manipulation) and (5) CTLA4Ig (abatacept, CD28 pathway blockage).

*Results: DSA suppression was observed in each of the monotherapies studied. Administration of 2 doses of anti-CD20 significantly reduced levels of DSA IgG (p<0.01 vs. control) in association with depletion of >90% B220+/CD5− B-cells. BTK inhibitor ibrutinib and JAK antagonist tofacitinib showed mild but statistically significant reduction in de novo DSA. Anti-IL6 receptor antibody exhibited significant suppression on de novo and recall DSA, which is associated with an increase in Treg cell populations. In comparison, anti-IL6 antibody showed DSA suppression only in a 3-dose regimen, not in an intensified 15-dose treatment group. Serum IL-6 concentrations in the 15-dose group increased by 188-fold (p<0.01) at day 14, indicating that anti-IL-6 antibodies tested in this study cause profound disturbance in IL-6 homeostasis. CTLA4Ig, which interrupts CD28/B7 costimulatory pathway is a potent inhibitor of de novo alloantibody responses and a moderate attenuator of recall alloantibody response. The differential effects of CTLA4Ig in de novo versus recall responses indicate that CTLA4Ig may not only interrupt naïve T cell activation and subsequent T-dependent follicular B cell maturation by blocking CD28/B7 co-stimulation pathway, but also mitigate recall B cell responses by Targeting B7-1 (CD80) expressed on Plasma Cells. A combined therapy using anti-CD20, CTLA4Ig and anti-IL6r resulted in strongest DSA reduction in both de novo and recall responses when compared to monotherapies.

*Conclusions: Our data demonstrate that a maximal suppression can be achieved by combined therapies targeting multiple pathways critical of CD4 T-cell activation, B/plasma cell activation and antibody production.

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