Targeting the CD40-costimulation pathway by CFZ533 prevents the cross-talk between follicular T helper cells and B cells

R. Kraaijeveld, C.C. Baan, M.W.F. van den Hoogen

Thursday 15 march 2018

15:35 - 15:40h at Willem Burger Foyer

Categories: Basic, Session (poster)

Parallel session: Poster session 4: Basic/translational research

Aim

Blockade of the costimulatory molecule CD40 by the novel Fc-silent, IgG1 antibody CFZ533 (Novartis Pharma) has shown to inhibit acute alloreactivity in non-human primates and is currently being investigated for prevention of rejection in a CNI-free regimen in kidney transplant patients. CD40 on B cells interacts with its ligand on T cells and serves as a necessary co-stimulatory factor for B cell activation, which promotes B cell proliferation, immunoglobulin class switching and differentiation.

Methods

We examined the effect of CFZ533 on humoral immunity by studying the cross-talk of follicular T helper cells (Tfh) with B cells and subsequent B cell activation parameters. By flow cytometry we determined the blockade of CD40 by CFZ533 on peripheral B cells, and its ability to inhibit their proliferation and differentiation after allogeneic stimulation.

Results

Cell surface expression of CD40 on B cells was blocked by CFZ533 in a dose dependent manner. At a concentration of 1 ug/ml a maximal saturation of >95% was measured. While completely blocked by CFZ533, the presence of CD40 on B cells was confirmed by another antibody (clone HB14) directed against another epitope. Our T and B cell function assays revealed that blockade of CD40 by CFZ533 (100 ng/ml) inhibited alloantigen driven B cell proliferation (up to 70% inhibition). Next, we determined the impact of blockade of CD40 by CFZ533 on the T-B cell cross-talk. In the absence of Tfh cells, plasma blast (PB) differentiation was inhibited when stimulated with polyclonal antigen SEB or alloantigen, while in the presence of these Tfh cells, these stimuli led to B cells differentiation into Ig producing PB’s and exhibition of memory B cell class switching (loss of IgD-expression), confirming T cell dependency of PB formation. CFZ533 inhibited PB differentiation by 80% (mean) and partly inhibited class switching of B cells, SEB vs SEB+CFZ: mean 17% vs 9%, p= 0.02, respectively). Additionally, CFZ533 diminished the expression of other B cell costimulatory markers such as OX40-L (member of the TNFR/TNF superfamily) and CD86 (CD28 superfamily) which both are necessary for T cell activation and survival, was diminished/lower by 42% and 59%, p = 0.05 and p<0.01, respectively, suggesting an inhibition of T-B cell interaction.

Conclusion

Our data show that blockade of CD40 by CFZ533 effectively inhibits the functional Tfh-B cell interaction required for T cell dependent B cell activation and differentiation.