Desensitizing highly sensitized heart transplant candidates with the combination of belatacept and proteasome inhibition

Abstract

HLA antibodies pose a significant barrier to transplantation and current strategies to reduce allosensitization are limited. We hypothesized that augmenting proteasome inhibitor (PI) based desensitization with costimulation blockade (belatacept) to mitigate germinal center (GC) responses might increase efficacy and prevent rebound. Four highly sensitized (calculated panel reactive antibody [cPRA] class I and/or II >99%, complement-dependent cytotoxicity panel reactive antibody [CDC PRA+], C1q+) heart transplant candidates were treated with the combination of belatacept and PI therapy, which significantly reduced both class I and II HLA antibodies and increased the likelihood of identifying an acceptable donor. Three negative CDC crossmatches were achieved against 3, 6, and 8 donor-specific antibodies (DSA), including those that were historically C1q+ binding. Posttransplant, sustained suppression of 3 of 3, 4 of 6, and 8 of 8 DSA (cases 1-3) was achieved. Analysis of peripheral blood mononuclear cells before and after desensitization in one case revealed a decrease in naïve and memory B cells and a reduction in T follicular helper cells with a phenotype suggesting recent GC activity (CD38, PD1, and ICOS). Furthermore, a shift in the natural killer cell phenotype was observed with features suggestive of activation. Our findings support synergism between PI based desensitization and belatacept facilitating transplantation with a negative CDC crossmatch against historically strong, C1q binding antibodies.

Keywords: costimulation; desensitization; heart transplantation/cardiology; histocompatibility; immunosuppressant-fusion proteins and monoclonal antibodies: belatacept; immunosuppression/immune modulation; natural killer (NK) cells/NK receptors; sensitization; translational research/science.

Figure 1.

Approach to desensitization. a) All candidates (n=4) were treated with multiple cycles of PI therapy and belatacept. Plasmapheresis was added in cases 3 (cycle 2, 3) and 4. (b) Detailed protocol for a representative case (case 3). Bela, belatacept; Bort, bortezomib; Carfilz, carfilzomib; Dex, dexamethasone; IVIG, intravenous immunoglobulin; Pheresis, plasmapheresis; Txp, transplant. *Case 4 not transplanted

Figure 2.

Effect of desensitization on class I & II HLA antibodies. (a) Mean MFI for all class I HLA antibodies before and after desensitization in each case (n=4). Mean MFI was calculated as the average of the MFIs of all antibodies against HLA class I. Diluted serum was used when MFI was >15,000 to keep values within the linear portion of the curve. HLA antibodies were reassessed at >1 month after PI therapy except in case 3 (n=3 days due to anticipated transplant). (b) Mean MFI for all class II HLA antibodies before and after desensitization in each case. Case 1 did not have class II antibodies (N/A). (c) Heatmap illustrating the response of class I HLA antibodies to desensitization with sequential cycles of treatment (case 3). Cycle 1: bortezomib/belatacept (no plasmapheresis); Cycle 2: bortezomib/plasmapheresis, belatacept continued; Cycle 3: carfilzomib/plasmapheresis, belatacept continued. (d) Heatmap illustrating the response of class II HLA antibodies to desensitization with sequential cycles of treatment as above (case 3; results shown in 1:8 dilution). MFI, mean fluorescence intensity.

Figure 3.

Desensitization with proteasome inhibition and belatacept results in a clinically relevant response. (a) Strong class I antibodies prior to desensitization were reduced to low levels or suppressed below the threshold for positivity after desensitization (case 3). Desensitization reduced the MFI of all class I HLA antibodies to <6000 (except A3, MFI 6200). (b) Reduction in MFI with treatment of HLA antibodies specific to the donor in cases 1–3 (CDC crossmatch negative in all cases). Reduction calculated using (MFI_post – MFI_pre)/MFI_pre. Pre, before desensitization; post, at transplant.

Figure 4.

Post-transplant course. (a) Case 1 underwent heart/liver transplant (DSA=3, class I). Uncomplicated post-transplant course with no rejection and no DSA at last follow-up. (b) Case 2 underwent heart transplant (DSA=6, class II). Belatacept continued post-transplant. DSA (4/6) suppressed. Graft dysfunction (grade 1R/1B ACR, no AMR) in the setting of medication nonadherence. Treated with IV corticosteroids and thymoglobulin. Developed several viral infections. LVEF improved to 50% and the remaining 2/6 DSA have MFI< 5000 at last follow-up. (c) Case 3 underwent heart transplant (DSA=8, class I and II). Belatacept was continued post-transplant. All 8 DSA suppressed. Mild (grade 1R/1B) ACR treated with outpatient oral prednisone pulse (normal graft function, no AMR). Mild cardiac allograft vasculopathy on 6-month surveillance angiography. Graft function remains normal (>55%). ACR, acute cellular rejection; AMR, antibody mediated rejection.

Figure 5.

Effect of desensitization on the peripheral blood immune cell repertoire in case 3. (a) Analysis of PBMCs from case 3 before and after desensitization. Individual tSNE plots of the lineage defining surface markers are shown to complement the overall density plots which provide a visual overview of the changes in major immune cell subsets after desensitization. Analyses were performed using equal sampling (n=100,000). (b) Quantification of major PBMC subsets before and after treatment. Peripheral blood CD19+ B cells were reduced (7.2% to 1.0%) and CD14+ monocytes increased (16.6% to 27.6%) with treatment. (c) Unsupervised cluster analysis of CD4+ T cells using self-organizing maps (flowSOM) and consensus hierarchical clustering identified two metaclusters with an activated T follicular helper cell (Tfh) phenotype (M5, ICOS+CD38+PD1+CXCR5^dim and M9, CXCR5^brightCD38⁺ICOS^dim). Median surface marker expression is normalized to the cluster with the minimum expression in each row. (d) Changes in the frequency of CD4+ T cells in each metacluster. Tfh (M5 and M9) were amongst the populations most reduced after desensitization. (e) Analysis of CD3-CD19-CD14-CD56+NK cells before and after desensitization. Marked shifts in the size of major metaclusters (top) are seen which visually correspond to increased CD38 and CD45RA expression (bottom). (f) Pie charts illustrating the changes in metacluster frequency (left) and a heatmap providing an overview of the surface markers defining each metacluster (right). Metaclusters are arranged from left to right in order of the change in their frequency after desensitization (M5,7,3,10,6,9 increased; M1, no change; M2,8,4 decreased). Median surface marker expression is normalized to the cluster with the minimum expression in each row.