Control of Humoral Response in Renal Transplantation by Belatacept Depends on a Direct Effect on B Cells and Impaired T Follicular Helper-B Cell Crosstalk

Abstract

Generation of de novo donor-specific antibodies (dnDSAs) after renal transplant is recognized as the leading cause of late transplant failure. Hence, the optimal immunosuppressive strategies to limit dnDSA development need to be defined. Recent clinical trials using the novel costimulatory blockade agent CTLA4-Ig (Belatacept) have shown that kidney transplant recipients (KTRs) treated with Belatacept have better graft survival and function and a lower proportion of dnDSAs than control-treated KTRs. Mechanisms involved in the control of humoral responses by Belatacept remain to be investigated. Here, we analyzed the effect of Belatacept on different steps of the B cell-mediated response in humans. In vitro, Belatacept reduced plasmablast differentiation, Ig production, and the expression of the major transcription factor involved in plasma cell function, Blimp-1, in a T cell-independent manner. Moreover, Belatacept induced activation of the STAT3 transcription factor in stimulated B cells and reduced the expression of CD86. Additionally, Belatacept blocked CD28-mediated activation of T follicular helper cells (Tfhs) in an autologous Tfh-memory B cells model. We then validated these observations in KTRs treated with Belatacept, who had a reduced proportion of blood effector B cells and activated Tfh (PD1+ICOS+) compared with control-treated KTRs. Our in vitro and in vivo results suggest that Belatacept modulates B cell function directly and at the level of B cell-Tfh interaction. These mechanisms likely account for the optimal control of humoral responses observed in KTRs treated with Belatacept.

Keywords: B cells; Belatacept (CTLA4-Ig); T follicular helper cells; kidney transplantation.

Copyright © 2018 by the American Society of Nephrology.

Figures

Graphical abstract

Figure 1.

Belatacept alters differentiation of plasmablasts and function of stimulated B cells in vitro. B cells were cultured with CD40L and IL-21 stimulation for 10 days. (A) Analysis of plasmablast (CD19loCD27hiCD38hi) differentiation on day 5 using the indicated gating strategy. (B and C) Proportion of plasmablasts (B) before and after stimulation (n=18) and (C) with Belatacept (Bela) or without Belatacept (w/o Bela; n=25). (D–F) Quantification of (D) IgG2 (n=14), (E) IgG4 (n=8), and (F) IgM (n=6) after 10 days of culture. (G) Expression of Blimp-1 in B cells lysate by Western blot on day 5. Tubulin was used as a loading control. One representative of eight immunoblots is shown. (H) Ratio of Blimp-1 signal on tubulin signal (n=8). (I) Proportion of IFNγ-producing plasmablasts on day 5 with or without Belatacept. Data are given as mean±SEM for each group. *P<0.05 versus Belatacept+ (Wilcoxon matched pairs signed rank test).

Figure 2.

Belatacept induces STAT3 phosphorylation in stimulated B cells. B cells were cultured with or without CD40L and IL-21 stimulation for 15 and 30 minutes in the presence or absence of Belatacept. (A) Expression of phospho-Akt (Ser473), total Akt, phospho-STAT3 (Tyr705), and STAT3. Tubulin was used as a loading control. One representative of five immunoblots is shown. (B) Ratio of p-Akt (Ser473) signal on Akt total signal (n=5). (C) Ratio of p-STAT3 (Tyr705) signal on STAT3 total signal (n=5). Data are given as mean±SEM for each group. *P<0.05 without Belatacept (w/o Bela) versus Belatacept (Bela); #P<0.05 unstimulated versus stimulated (two-way ANOVA test).

Figure 3.

Belatacept modifies the pattern of expression of costimulatory molecules on the surface of B cells were cultured with CD40L and IL-21 stimulation in the presence or absence of Belatacept for 5 days (n=10). (A) Proportion and (B) MFI of CD86 in total B cells, memory B cells, and plasmablasts. (C) Proportion and (D) MFI of CD80 in B cells subsets described in Figure 1A. (E) Expression of CD86 in B cells lysates on day 5 by Western blot. Tubulin was used as a loading control. One representative of eight immunoblots is displayed. (F) Ratio of CD86 signal on Tubulin signal (n=8). (G) Expression of CD80 in B cells lysates on day 5 by Western blot. Tubulin was used as a loading control. One representative of five immunoblots is displayed. (H) Ratio of CD80 signal on Tubulin signal (n=8). (I) Proportion of CD28+ B cells after Belatacept treatment analyzed by flow cytometry (n=5). (J) Proportion of PDL1+ B cells after Belatacept treatment analyzed by flow cytometry (n=7). Data are given as mean±SEM for each group. Bela, Belatacept; w/o Bela, without Belatacept. *P<0.05 versus Belatacept (Wilcoxon matched pairs signed rank test).

Figure 4.

Stimulated B cells generated with Belatacept decrease proliferation of CD4+ cells and Tfhs. B cells were cultured with CD40L and IL-21 stimulation in the presence or absence of Belatacept for 5 days (n=6). After 5 days, stimulated B cells generated with or without Belatacept were washed and cocultured with autologous CD4 cells labeled with cell proliferation die (C.P.D.) at a 1:1 ratio (5×104) in the presence of soluble anti-CD3 (2 μg/ml) for 4 days. Proliferation and ICOS proportion were analyzed by flow cytometry. (A) Dot plots of a representative donor showing gating strategies for proliferative CD4 cells. (B) Proportion of proliferative CD4 cells cocultured with stimulated B cells generated with or without Belatacept for each donor. (C) Dot plots showing the gating strategy for Tfhs, proliferative Tfhs, and ICOS+ Tfhs. (D) Proportion of Tfhs in CD4+ cells. (E) Proportion of proliferative Tfhs in Tfhs. (F) Proportion of ICOS+ Tfhs in Tfhs. Bela, Belatacept; w/o Bela, without Belatacept. *P<0.05 versus Belatacept (Wilcoxon matched pairs signed rank test).

Figure 5.

Tfhs-memory B cells crosstalk is altered in the presence of Belatacept. Purified blood memory B cells (CD19+IgD−CD38dimCD27±) were cocultured with Tfhs (CD4+CD45RA−CXCR5+PD1+) at a 1:1 ratio (1–3×104) in the presence of the superantigen staphylococcal enterotoxin B (SEB) for 5 days with or without Belatacept. (A) Dot plot of ICOS in CD4+CXCR5+ cells for a representative donor with or without Belatacept or stimulation by SEB. Notably, the expressions of ICOS (left panel) and PD1 (data not shown) were negative without SEB stimulation. (B and C) Proportions of (B) ICOS+ and (C) ICOS+PD1+ Tfhs were analyzed by flow cytometry. (D) MFI of ICOS+ Tfhs. (E) Plasmablasts (CD19+CD38hi) proportion. Data are given as mean±SEM for each group. Bela, Belatacept; w/o Bela, without Belatacept.

Figure 6.

KTRs treated with Belatacept display lower proportion of circulating effector B cells. Blood B cells immunophenotyping of HBDs (n=12) or KTRs treated with CNI (n=12) or Belatacept (n=10). (A) Representative dots plots showing gating strategies for switched and unswitched memory B cells (upper panel) and memory cells (CD24+CD38−), plasmablasts (CD24−CD38hi), and transitional (CD24hiCD38hi) B cells. (B) Proportion and (C) number of memory (CD27+) B cells among CD19 cells. (D) Proportion and (E) number of switched memory (CD19+CD27+IgD−) B cells in CD19+ cells. (F) Proportion and (G) number of unswitched memory (CD19+CD27+IgD+) B cells in CD19+ cells. (H) Proportion of plasmablasts (CD19+CD24−CD38hi) in CD19+ cells. Data are given as mean±SEM for each group. Bela, Belatacept. *P<0.05 versus CNI (Kruskal–Wallis test [B, D, F, and H] or Mann–Whitney U test [C, E, and G]); #P<0.05 versus HBD (Kruskal–Wallis test [B, F, and H]).

Figure 7.

CD80 and CD86 expression varies according to the maturation stages of circulating B cells and KTRs treated with Belatacept display decreased CD80 expression on B cells, particularly on memory B cells. (A) Staining of CD19+ B cells with CD80 and CD86 antibodies (upper panel) and isotype control (lower panel). (B and C) Proportions of (B) CD80+ and (C) CD86+ B cells of HBDs were evaluated according to B cell subsets described in Figure 6A (n=10). Data are given as mean±SEM for each group. *P<0.05 memory versus mature B cells (Kruskal–Wallis test); #P<0.05 memory versus transitional B cells (Kruskal–Wallis test); &P<0.05 transitional versus mature B cells (Kruskal–Wallis test). (D) CD80 proportion and (E) MFI in B cells of HBDs (n=10) or KTRs treated with CNI (n=12) or Belatacept (n=10) described in Table 1. (F) CD80 proportion and (G) MFI in memory B cells of patients described above. Data are given as mean±SEM for each group. Bela, Belatacept. *P<0.05 versus CNI (Kruskal–Wallis test); #P<0.05 versus HBD (Kruskal–Wallis test).

Figure 8.

KTRs treated with Belatacept display decreased proportion of circulating Tfh. (A) Representative dot plot showing gating strategy of memory (CD4+CD45RA−) T cells and Tfhs (CD4+CD45RA−CXCR5+) (upper panel) as well as Tfh subsets: Tfh17 (CCR6+CXCR3), Tfh2 (CCR6−CXCR3−), and Tfh1 (CCR6−CXCR3+; lower panel). (B) Proportion and (C) number of memory CD4 cells (CD45RA− cells) in CD4+ cells. (D) Proportion and (E) number of Tfhs (CD45RA−CXCR5+ cells) in CD4+ cells. (F–H) Proportions of (F) Tfh1 (CCR6−CXCR3+ cells), (G) Tfh2 (CCR6−CXCR3−), and (H) Tfh17 (CCR6+CXCR3−) in CD4+ cells. Data are given as mean±SEM for each group (Belatacept, n=10; CNI, n=8; HBD, n=10). Bela, Belatacept. *P<0.05 versus CNI (Kruskal–Wallis test [D and F–H] or Mann–Whitney U test [C and E]); #P<0.05 versus HBD (Kruskal–Wallis test [B, D, and F–H]).

Figure 9.

The circulating Tfh of KTRs treated with Belatacept display lower levels of markers of activation. PD1, ICOS, and CD28 expressions were studied on gated Tfhs (CD4+CD45RA−CXCR5+) by flow cytometry. (A and B) Dot plots are shown for Fluorescence Minus One, one representative of KTRs treated with CNI, and another with Belatacept. (C) Proportion and (D) number of activated ICOS+ Tfhs in CD4+ cells. (E) Proportion and (F) number of activated PD1+ICOS+ Tfhs in CD4+ cells. (G and H) Proportions of (G) ICOS+ Tfhs or (H) PD1+ICOS+ Tfhs in Tfhs. Data are given as mean±SEM for each group (Belatacept, n=6; CNI, n=6; HBD, n=6). Bela, Belatacept. *P<0.05 versus CNI (Kruskal–Wallis test [D, F, and G] or Mann–Whitney U test [C, E, and H]); #P<0.05 versus HBD (Mann–Whitney U test [C, E, and H]).