Anti-CD28 Antibody and Belatacept Exert Differential Effects on Mechanisms of Renal Allograft Rejection

Abstract

Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation.

Keywords: acute rejection; immunology; immunosuppression; kidney transplantation; lymphocytes.

Figure 1.

FR104 and belatacept equally inhibit in vitro T cell proliferation. FR104 (blue triangles) and belatacept (red squares) dose response of baboon (n=4) PBMC mixed lymphocyte proliferation assays at day 5 assessed by 3H-thyminidine uptake.

Figure 2.

FR104 Prevents Steroid–Resistant Allograft Rejection in contrast to belatacept therapy. (A) Timeline of immunosuppressive regimens, clinical interventions, and outcomes for FR104- and belatacept-treated groups. Highlighted rectangles represent acute renal failure episodes (blue indicates caused by rejection, red indicates caused by infection, and green indicates caused by obstruction). Black triangles represent steroid bolus, and white triangles indicate antibiotics use. (B) IgG DSAs as detected by flow cytometry after transplantation in baboons treated with FR104 (blue triangles) or belatacept (red squares). (C) Kaplan–Meier plot of overall survival for baboons treated with 1 month of low-dose tacrolimus and chronic FR104 (blue line; n=5) or 1 month of low-dose tacrolimus and chronic belatacept (red line; n=5). White squares represent animals still alive 1 year post-transplantation. Deaths by causes other than rejection have been censored, and the black marks represent recipient deaths. Survival time was evaluated by a log rank test, *P<0.05.

Figure 3.

Assessment of TSDR methylation status reveal a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. (A) Flow cytometry contour plot showing gates used to assess percentage of CD4⁺CD25^hiCD127^low Treg cells. (B) Kinetics of Treg cells post-transplantation in FR104-treated recipients (blue triangles; n=5) and belatacept-treated recipients (red squares; n=5). (C) Quantitative real–time PCR measurement of Foxp3 and TGFβ gene expression in 1-month protocol biopsies or earlier biopsies in cases of rejection from animals treated with FR104 (blue triangles; n=5) or belatacept (red squares; n=5). (D) TSDR analysis in 1-month protocol biopsies or earlier biopsies in cases of rejection from animals treated with FR104 ((blue triangles; n=5) or belatacept (red squares; n=5) ; data are means±SEMs. Graph shows gene expression relative to HPRT; data are means±SEMs.

Figure 4.

CD28 negative T cells and the co-inhibitory receptor 2B4 are not differentially regulated between the FR104 group and the belatacept group. (A) Flow cytometry contour plot showing gates used to assess percentage of naïve T cells (T_Ns; CD28⁺CD95⁻), central memory T cells (T_CMs; CD28⁺CD95⁺), and effector memory T cells (T_EMs; CD28⁻CD95⁺) among CD4⁺ and CD8⁺. (B) Kinetics of the CD4⁺ and CD8⁺ compartments of T_EMs post-transplantation in FR104-treated recipients (blue triangles; n=5) and belatacept-treated recipients (red squares; n=5). (C and D) Quantitative real–time PCR measurement of gene expression in 1-month protocol biopsies (or earlier in cases of rejection) from animals treated with FR104 (blue triangles; n=5) or belatacept (red squares; n=5). Graph shows gene expression relative to HPRT; data are means±SEMs.

Figure 5.

The IL-21 gene expression but not the Th17 gene expression signature is increased in belatacept-treated recipients. (A and B) Quantitative PCR measurement of mRNA expression on 1-month protocol biopsies (or earlier in cases of rejection) from animals treated with FR104 (blue triangles; n=5) or belatacept (red squares; n=5). Gene expression is relative to HPRT. Data are means±SEMs. A Mann–Whitney nonparametric test was used, and P values <0.05 were considered significant. (C) Pairwise correlation analysis to determine the direction and strength of the linear relationships between genes. The upper right of the matrix is the color map of correlations. Bright colors indicate the pairs of variables closely related using the Spearman rank correlation coefficient (r), and faded colors encode for decreasing r values. The lower left of the matrix is the color map of P values.

Figure 6.

Tfh–like graft infiltrating lymphocytes were elevated in biopsies from belatacept-treated animals. (A) Immunostaining of a protocol biopsy showing cells with a Tfh-like phenotype (CD4⁺PD1⁺ T cells coexpressing ICOS and IL-21). (B) Representative images of CD4 and PD1 staining in rejection biopsies of FR104-treated animals. (C) Representative pictures of CD4 and PD1 staining in rejection biopsies of FR104-treated animals. (D) Number of CD4⁺PD1⁺ cells per millimeter² in protocol biopsies in FR104- (blue; n=2) and belatacept-treated animals (red; n=1); each point represents an area (five per biopsy), and each symbol is an animal. (E) Number of CD4⁺PD1⁺ cells per millimeter² in rejection biopsies of FR104- (blue; n=2) and belatacept-treated animals (red; n=2), and each point represents an area (five per biopsy) and each symbol is an animal.

Figure 7.

Selective CD28 blockade with FR104 control in vitro Ag primed Tfh responses more effectively than belatacept. (A) Flow cytometry plot of representative of human tonsil cell suspension showing gates used to sort pre-Tfh (CD4⁺CXCR5⁺ICOS⁺) and Tfh (CD4⁺CXCR5^hiICOS^hi). (B) Proliferative responses to autologous B cell plus Staphylococcal enterotoxin B after 8 days presented as percentages of pre-Tfh and Tfh compared with control conditions under either FR104 (blue circles) or belatacept (red circles). Each pair of connected points represents a paired experiment (n=10). The Wilcoxon matched-paired signed rank test was used. *P<0.05.

Figure 8.

Selective CD28 blockade controls Tfh recall responses to KLH immunization in mice more effectively than CTLA-4-Ig. (A) Mice were immunized with KLH and LPS and treated with either CTLA-4-Ig or the Fab-PV1-Peg antibody, an anti-CD28 antagonist effective in mice, either on day 0 to study primary responses or 1 month after a first immunization to study memory responses. (B) Postimmunization draining popliteal LNs were collected, and levels of Tfh cells (CXCR5⁺PD1⁺) among the CD4⁺ or CD4⁺CD44⁺ population were assessed by flow cytometry as represented in the flow cytometry contour plot. (C) Time course under control condition (n=3). Percentage of Tfh cells among CD4⁺CD44⁺ (D) on day 6 postimmunization or (E) 3 days after reimmunization according to conditions indicated on the x axis (n=5 per group). Data are means±SEMs. One-way ANOVA and Kruskal–Wallis tests were used. *P<0.05; **P<0.01.