Successful desensitization with proteasome inhibition and costimulation blockade in sensitized nonhuman primates

Abstract

The detrimental effects of donor-directed antibodies in sensitized transplant patients remain a difficult immunologic barrier to successful organ transplantation. Antibody removal is often followed by rebound. Proteasome inhibitors (PIs) deplete antibody-producing plasma cells (PCs) but have shown marginal benefit for desensitization. In an allosensitized nonhuman primate (NHP) model, we observed increased germinal center (GC) formation after PI monotherapy, suggesting a compensatory PC repopulation mediated via GC activation. Here we show that costimulation blockade (CoB) targets GC follicular helper T (Tfh) cells in allosensitized NHPs. Combined PI and CoB significantly reduces bone marrow PCs (CD19+CD20-CD38+), Tfh cells (CD4+ICOS+PD-1hi), and GC B cells (BCL-6+CD20+); controls the homeostatic GC response to PC depletion; and sustains alloantibody decline. Importantly, dual PC and CoB therapy prolongs rejection-free graft survival in major histocompatibility complex incompatible kidney transplantation without alloantibody rebound. Our study illustrates a translatable desensitization method and provides mechanistic insight into maintenance of alloantibody sensitization.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Graphical abstract

Figure 1.

Dual targeting with proteasome inhibitor and CoB (belatacept and anti-CD40 mAb [2C10]) successfully promoted desensitization. (A) Timing and dosing of bortezomib, belatacept, and anti-CD40 mAb’s for desensitization and biopsy scheme for treated animals. (B) DSA from T-cell flow-cytometric cross-matching of sensitized animals before and after dual targeting treatment. DSA levels are expressed as mean channel fluorescent intensity (MFI) ratio. Serum DSA level was significantly reduced after CoB treatment (pre- vs posttreatment). (C) Visualization of BM PCs. Dual targeting treatment significantly affected CD19+CD20−CD38+ cells in the BM biopsy after dual targeting treatment. Representative flow plot and percentages of the CD19+CD38+ PC population in the BM at the indicated time points (pre- vs posttreatment). (D) Tfh cells were traced with PD-1 and inducible T-cell costimulator (ICOS) from the LN biopsies. LN-Tfh cells showed a significant reduction after dual targeting treatment. Representative flow plot (CD4 gated) and percentage of the ICOS+PD-1high cell population in the LN. (E) A strong trend of reduction of CXCR5+BCL-6+ B cells in the LN biopsy after CoB treatment. Representative flow plot (CD20 gated) and percentage of the CXCR5+BCL-6+ GC B-cell population in the LN. (F) Proliferated isotype switched B cells before and after CoB treatment. Ki67+IgG+IgDloCD20+ B cells in the LNs were greatly reduced after CoB treatment. (G) Immunofluorescent analysis of LN including B-cell follicles and GC staining for Ki67 (green), CD20 (red), and CD3 (blue). Original magnification ×200. Quantification of positive fluorescence signal of CD20 for B-cell follicle, and Ki67/CD20 for proliferating GC. Data represent the mean ± standard deviation (SD) of 4 monkeys per group. NS, nonsignificant.

Figure 2.

The effect of dual targeting with proteasome inhibitor and CoB on renal allograft survival, posttransplant humoral and memory T cell responses in sensitized NHPs. (A) Timing and dosing of desensitization and immunosuppressive regimen with kidney transplantation in the sensitized NHPs. (B) Death censored graft survival shown in days by treatment group. P values were determined by log-rank test comparing sensitized NHPs receiving dual targeting therapy vs without desensitization. NHPs with desensitization had significantly prolonged rejection-free survival compared with sensitized controls without desensitization. (C) Renal allograft histologic examination of necropsy specimens (hematoxylin and eosin) from a control and desensitized macaque. Original magnification ×100. (D) Representative immunohistochemistry for T cells (CD3+), B cells (CD20+) and macrophages (CD68+) of necropsy specimens from controls vs desensitized NHPs. Scanned images were magnified (×10) from the original whole-slide scans. (E) Post–renal transplant serum DSA level from controls vs desensitized NHPs. Values represent mean ± SD and are representative of all separate experiments. (F) Representative post–renal transplant GC response in the LNs from controls vs desensitized NHPs as shown by immunofluorescence for CD3 (blue), CD20 (red), and Ki67 (green). Original magnification ×200. Data represent the mean ± SD of 3 monkeys treated with dual targeting treatment.