Reversing donor-specific antibody responses and antibody-mediated rejection with bortezomib and belatacept in mice and kidney transplant recipients

Abstract

Active antibody-mediated rejection (AMR) is a potentially devastating complication and consistently effective treatment remains elusive. We hypothesized that the reversal of acute AMR requires rapid elimination of antibody-secreting plasma cells (PC) with a proteasome inhibitor, bortezomib, followed by the sustained inhibition of PC generation with CTLA4-Ig or belatacept (B/B). We show in mice that B/B therapy selectively depleted mature PC producing donor-specific antibodies (DSA) and reduced DSA, when administered after primary and secondary DSA responses had been established. A pilot investigation was initiated to treat six consecutive patients with active AMR with B/B. Compassionate use of this regimen was initiated for the first patient who developed early, severe acute AMR that did not respond to steroids, plasmapheresis, and intravenous immunoglobulin after his third kidney transplant. B/B treatment resulted in a rapid reversal of AMR, leading us to treat five additional patients who also resolved their acute AMR episode and had sustained disappearance of circulating DSA for ≤30 months. This study provides a proof-of-principle demonstration that mouse models can identify mechanistically rational therapies for the clinic. Follow-up investigations with a more stringent clinical design are warranted to test whether B/B improves on the standard of care for the treatment of acute AMR.

Keywords: B cell biology; alloantibody; costimulation; desensitization; immunobiology; immunosuppression/immune modulation; kidney transplantation/nephrology; translational research/science.

Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure-1.. Bortezomib preferentially depletes mature plasma cells (PC) from the bone marrow and spleen of naïve mice.

(A) Gating strategy used to identify total and subsets of plasma cells, namely, plasmablast, early PC and mature PC. (B) Quantification of total number of plasma cell subsets retrieved from the bone marrow (Left panel on B) and spleen (Right panel on B) of a naïve C57BL/6 WT mouse (N=28/group). (C) Experimental design. Two days post-treatment with Bortezomib or CTLA4-Ig, naïve C57BL/6 WT mouse were sacrificed. (D) Quantification of PC subsets in bone marrow (Top) and spleen (Bottom) harvested from C57BL/6 mice after Bortezomib (Btz) or CTLA-Ig (C4-Ig) treatment (N=9–16/group). Y-axis represents total cells retrieved per mouse from naïve or mice receiving Bortezomib or CTLA4-Ig. Data are pooled from ≥ two independent experiments and presented as Mean ± SEM. Statistically significant differences were assessed by one way ANOVA. (**P <0.01) (***P <0.001) (****P <0.0001).

Figure-2:. Delayed treatment with CTLA4-Ig plus Bortezomib inhibits established primary humoral responses in mice immunized with donor spleen cells.

(A) Experimental design. C57BL/6 mice were immunized with BALB/c donor splenocytes (DST) and received no treatment (None), CTLA4-Ig (C4-Ig; from D5), Bortezomib (Btz; D5, D6) or CTLA4-Ig and Bortezomib (Both; from D5). (B) Quantification of plasma cell (PC) subsets in bone marrow; (C) and spleen harvested from naïve or D10 post-DST. (D) Total IgG secreting cells in the bone marrow (Left) and spleen (Right), harvested from naïve or D10 post-DST. Y-axis represents total cells retrieved per mouse, not receiving (None) or receiving CTLA4-Ig (POD 5+) or Bortezomib (D5, 6) or Both (N=5–8/group). (E) Anti-BALB/c IgG were assessed from D14–28 after DST immunization (Left panel) (N=6–25/group), and effectiveness of mono or combination therapy were compared on D28 post-DST (Right panel) (N=8–25/group). Each dot represents an individual mouse, pooled from >2 independent experiments. Data are presented as Mean ± SEM and statistically significant differences assessed by one way ANOVA. (*P <0.05) (**P <0.01) (***P <0.001) (****P <0.0001).

Figure-3:. Delayed treatment with CTLA4-Ig plus Bortezomib inhibits established primary humoral responses in mice immunized with skin transplant.

(A) Experimental design. C57BL/6 mice were immunized with BALB/c skin allografts and received no treatment (None), CTLA4-Ig (C4-Ig; from D14), Bortezomib (Btz; D14, D15) or CTLA4-Ig and Bortezomib (Both; from D14 or D35). (B) Quantification of plasma cell (PC) subsets in bone marrow; (C) and total IgG secreting cells in the bone marrow (Left) and spleen (Right) harvested from naïve or D28 post-skin transplant. Y-axis represents total cells retrieved per mouse from naïve, skin transplanted mice not receiving (None) or receiving CTLA4-Ig (POD 14+) or Bortezomib (D14, 15) or Both (from POD14) (N=5–8/group). (D) Anti-BALB/c IgG were assessed from D14–28 post-skin transplant (N=4–6/group). (E) Anti- BALB/c IgG from C57BL/6 WT mice post-skin transplant and received no treatment (None) or CTLA4-Ig and Bortezomib (Both) from POD35+. Repeated Bortezomib treatment on D63 and D77. (N=4–6/group). Each dot represents an individual mouse, pooled from >2 independent experiments. Data are presented as Mean ± SEM and statistically significant differences assessed by one way ANOVA. (*P <0.05) (**P <0.01) (***P <0.001) (****P <0.0001).

Figure-4:. Delayed treatment with CTLA4-Ig plus Bortezomib controls established humoral responses in sensitized mice.

(A) Experimental design. C57BL/6 WT recipients were immunized with BALB/c donor splenocytes (DST) on D0 and D28. Mice received no treatment (None), CTLA4-Ig (C4-Ig; POD14+), Bortezomib (Btz; D14, D15) or CTLA4-Ig and Bortezomib (Both). (B) Anti-BALB/c IgG were assessed from D0–42 post-DST (N=4/group). (C) Experimental design. Mice received BALB/c skin transplants on POD-70 and POD0, and treated from POD14, as indicated. (D) Anti-BALB/c IgG from –D70 to D70 (N=4/group). (E) Quantification of PC subsets in bone marrow (N=4–36) and (F) total IgG secreting cells (Left) and anti Kd-IgG secreting cells (Right) (N=4–5) in the bone marrow of naïve or post-2° skin transplantation, on POD28. Experimental groups are: not receiving (None) or receiving CTLA4-Ig and Bortezomib both (Both; from POD14). Y axis represents total cells retrieved per mouse from at least two independent experiments. Data are presented as Mean ± SEM and statistically significant differences assessed by one way ANOVA. (*P <0.05) (**P <0.01) (***P<0.001).

Figure-5

Clinical sequelae for Case 1