Costimulation blockade alters germinal center responses and prevents antibody-mediated rejection

Abstract

De novo donor-specific antibody (DSA) after organ transplantation promotes antibody-mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti-CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti-CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM(+) B cells, bela- and 2C10R4-added groups maintained a predominant population of IgM(+) B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4(+) CD28(+) CD95(+)) as well as PD-1(hi) CD4(+) T cells were decreased in both bela-added and 2C10R4-added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC-follicular helper T (Tfh) cells, and IL-21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.

Keywords: Antibody-mediated rejection; costimulation blockade; follicular helper T cells; germinal center reaction.

Figure 1. Immunosuppressive regimens, dosing strategy, clinical interventions and outcomes in AMR control and additional costimulation blockade treated rhesus monkeys

Figure 2. Costimulation blockade prevents allograft rejection and alloantibody production

(a) Serum creatinine level of AMR positive control, belatacept-treated, and 2C10R4-treated groups. AMR positive control group (n=5, red line), receiving CD3-immunotoxin, alefacept, and tacrolimus (trough 8–12ng/mL), had early alloantibody production within 4 weeks from transplantation (the first detection of DSA from AMR positive controls is indicated by ▼) and were sacrificed for graft failure. Belatacept-treated (n=4, blue line) and 2C10R4-treated (n=4, purple line) groups received the AMR regimen plus additional belatacept or additional 2C10R4 for 8 weeks (duration and frequency indicated by arrows along the x-axis); these animals failed to demonstrate DSA production and retained graft function, but were sacrificed for non-rejection end-point criteria. Animal survival was equivalent among the three groups (p=0.632). (b) Histological examination of necropsy kidney specimens from healthy control, AMR control, belatacept-added, and 2C10R4-added group. Representative H&E staining portrays evidence of cellular and antibody-mediated rejection in the AMR positive control group, including tubulointerstitial inflammation and transplant glomerulopathy. These features were prevented in animals receiving additional costimulation blockade. Original magnification ×200 (bar scale: 100µm). (c) An electron microscopy image of glomerular basement membranes from healthy control, AMR control, Bela-added, and 2C10R4 added group. AMR control animals showed thickening, lamination and duplication of glomerular basement membranes. (d) De novo DSA production was inhibited in animals receiving belatacept and 2C10R4. *p<0.05 vs. AMR control.

Figure 3. Costimulation blockade prevents B cell repopulation and maturation, and maintains naïve CD4 T cell phenotype in secondary lymphoid organs

(a) Peripheral blood B cells decreased after induction immunosuppression, as reported previously [18]. In contrast to AMR controls, CoB treated animals failed to repopulate B cells. *p<0.05 (b) Memory type B cells (CD20⁺CD27⁺) are decreased and immature phenotype (IgM⁺CD20⁺) B cells are predominant in CoB treated animals. The drop in IgM⁺ B cells among AMR controls coincides with DSA production and IgG⁺B cell proliferation (Supplemental figure 1) c Peripheral blood T cells remain decreased in all groups. The isolated difference at 35 days may represent onset of allograft rejection in AMR controls. *p<0.05 (d Central memory CD4 T cells (CD4⁺CD28⁺CD95⁺) were significantly decreased and CD4 naïve T cells (CD4⁺CD28⁺CD95⁻) increased in bela- (n=4, blue) and 2C10R4-added (^†n=3, purple) animals in the spleen and inguinal lymph nodes compared to AMR positive controls (n=5, red) at time of necropsy (^†One recipient from 2C10R4-added group was censored from this analysis due to its necropsy time point performed at 179d post-transplant; the subject was sacrificed for non-rejection endpoint criteria; supplemental figure 4). NS>0.05.

Figure 4. Post-transplant follicle and GC morphology in secondary lymphoid organs

(a) Representative immunofluorescence image of GC staining with CD3 (Blue), CD20 (Red), and Ki67 (Green) in lymph node and spleen sections from untreated (healthy control) and CD3-immunotoxin treated rhesus monkey (post-transplant day 13). (b) Immunofluorescence analysis for CD20 (red) staining in lymph node sections from healthy control, AMR positive control, additional belatacept, and additional 2C10R4 treated recipients. Original magnification, ×200. (c) Image J (National Institutes of Health) quantitation of the positive fluorescence signals of CD20 in lymph node sections. Follicle size based on CD20⁺ B cells was similar across all groups, including healthy controls. (d) Hoechst nuclear staining in paraffin-embedded lymph node sections. Original magnification ×200. GCs in lymph nodes of rhesus macaques were identified by Hoechst nuclear staining. This less intensely stained GC area was confirmed as corresponding to the same area containing proliferating (Ki67⁺) B cells (Supplemental figure 2). (e) ImageJ quantitation of the less intensely stained Hoechst area. GC size was significantly reduced in Bela- and 2C10R4-added animals compared to AMR positive controls (*p<0.05). These data are representative lymph node sections from healthy controls (n=4), AMR positive controls (n=5), Bela-added (n=4), and 2C10R4-added (n=3) rhesus macaque recipients.

Figure 5. Decreased PD1⁺CD4⁺ T cells in lymph nodes in additional costimulation blockades treated animals

(a) Flow cytometry contour plots show PD-1 expression among CD4 T cells in draining inguinal lymph nodes from healthy controls (n=5) as well as AMR positive controls (n=5), Bela-added (n=4), 2C10R4-added (n=3) rhesus macaque recipients. The frequency of the gated population (PD-1^hiCD4⁺) is shown as a bar graph. *p<0.05, **p<0.01, ***p<0.001, vs. AMR control. (b) Flow cytometry analysis of CD4 T cells based on the expression of CXCR5 and BCL-6 in the lymph nodes of rhesus macaque. Cells were first gated for CD4 T cells and gated based on the differential expression of CXCR5 and BCL-6. High PD-1 expression was shown in CXCR5⁺Bcl-6⁺ CD4 T cell subsets. Numbers in dot plots indicate the percentage of cells in each gate.

Figure 6. Effects of costimulation blockade on B cell clonal expansion, germinal center-Tfh, and IL-21 production

(a) B cell clonal expansion in germinal centers was significantly increased in AMR group (*p<0.05 vs. healthy control). The increased B cell proliferation found in AMR positive controls was significantly suppressed by additional Belatacept and 2C10R4 (*p<0.05 vs. AMR) treatment. Proliferating B cells in germinal centers are identified by CD20, Ki67 and CD3 staining. b Tfh cells were significantly reduced in Bela-added (*p<0.05 vs. AMR) but not in 2C10R4-added (p=0.1429 vs. AMR) rhesus macaques. CD4, CD20, PD-1 were simultaneously analyzed for Tfh cells in the germinal center. c IL-21 expression was significantly reduced in Bela-added and 2C10R4-added animals compared to AMR positive controls (*p<0.05). These data are representative lymph node sections from healthy controls (n=4), AMR positive controls (n=5), Bela-added (n=4), and 2C10R4-added (n=3) rhesus macaque recipients. Original magnification ×200 (scale bar: 50µm).