Humoral Compensation after Bortezomib Treatment of Allosensitized Recipients

Abstract

The efficacy of bortezomib monotherapy in desensitizing kidney transplant candidates with preformed donor-specific antibodies remains unclear. We evaluated the effect of bortezomib on preformed antibodies and upstream components of the B cell response in a primate model sensitized by fully mismatched allogeneic skin transplants to provide mechanistic insights regarding the use of bortezomib as a means of desensitization. Bortezomib treatment given intravenously twice weekly for 1 month (1.3 mg/m² per dose) clearly reduced the numbers of antibody-producing cells and CD38⁺CD19⁺CD20^- plasma cells in the bone marrow (P<0.05), but donor-specific alloantibody levels did not decrease. We observed a rapid but transient induction of circulating IgG⁺ B cells and an increased number of proliferating B cells in the lymph nodes after 1 month of treatment. Notably, bortezomib treatment induced germinal center B cell and follicular helper T cell expansion in the lymph nodes. These data suggest that bortezomib-induced plasma cell depletion triggers humoral compensation.

Keywords: Bortezomib; Desensitization; alloantibody; antibody mediated rejection; nonhuman primate.

Figure 1.

Bortezomib treatment promotes bone marrow PC reduction in the skin graft of sensitized rhesus macaque. (A) Skin sensitization. Recipient animals were transplanted with abdominal skin from recipient (left panel) and donor (right panel) with no treatment. Syngeneic skin grafts (left panel) were fully accepted, whereas allogeneic skin grafts (right panel) were rejected with a mean survival time of <2 weeks. (B) Dosing strategy for desensitization. Rhesus macaques after skin grafting were treated with desensitization regimens for 4 weeks after their DSA levels stabilized. (C) Absolute white blood cells (WBCs), lymphocytes, and T and B cell counts before and after bortezomib treatment. There was no significant difference in absolute counts of WBCs, lymphocytes, and CD3 and CD20 cells between pre- and postbortezomib treatment. (D) Representative flow plot showing IgD⁻CD20⁻ cells in bone marrow aspirates by flow cytometry. The frequency of the gated population (CD19⁺CD38⁺) is shown as a bar graph. *P<0.05. (E) Representative total IgG-specific ELISPOT assays from pre- and postbortezomib treatment. The frequency of IgG-producing cells in pre- and postbortezomib treatment in bone marrow was evaluated. Lower frequency of ASCs was seen after bortezomib treatment. Data are expressed as the mean±SEM of four independent experiments. BM, bone marrow; BTZ, bortezomib; IV, intravenous; MFI, mean fluorescence intensity; POD, post-transplant day.

Figure 2.

Bortezomib did not alter donor-specific alloantibody levels but increased B cell proliferation, GC B cells, Tfh cells, and GC activation in the sensitized rhesus macaques. (A) DSA level was evaluated by T cell crossmatch. Serum was assayed for IgG alloantibody level pre- and postbortezomib treatment at 4 and 8 weeks. Shown is the mean fluorescence intensity (MFI) of the entire population of the 1:100 serum dilution expressed as the mean±SEM. NS indicates >0.05. (B) Frequency of circulating IgG⁺CD20⁺ B cells at 7 days post-treatment was increased. Error bar indicates SEM. (C) B cell proliferation was evaluated with Ki67 intracellular staining. There was a trend of increased total B cells, naïve (CD27⁺IgD⁺) B cells, and memory B cells (CD27⁺IgD⁻) after bortezomib treatment compared with pretreated time points. (D) More GC-related B cells were identified with BCL-6⁺ intracellular staining in the lymph nodes. BCL-6⁺CD20⁺ cells in the lymph nodes increased 4 weeks after bortezomib treatment. P<0.05. (E) Flow cytometry contour plots show the ICOS⁺PD-1^hi cell population among CXCR5⁺CD4⁺ T cells in draining lymph nodes before and after bortezomib treatment. PD-1⁺ICOS⁺ CXCR5⁺CD4 T cells in the lymph node showed an increasing trend after bortezomib treatment. (F) In situ GC staining with CD20, Ki67, and CD3 staining. B cell clonal expansion in GC was increased after bortezomib treatment. Original magnification, ×200. (G) The serum levels of IL-6, IL-21, and BAFF were measured with ELISA. Serum B-cell activating factor (BAFF) level was significantly increased after bortezomib treatment. Increased BAFF level after PC depletion could be a mediator for the humoral compensation. BTZ, bortezomib; LN, lymph node.