Background The balance between pro- and anti-apoptotic BCL-2 family proteins plays a critical role in regulation of apoptosis in lymphocytes. Navitoclax is a potent and orally bioavailable BH3 mimetic which inhibits the anti-apoptotic activity of BCL-2 and BCL-xL. Inhibitors of BCL-2 family proteins have shown efficacy in animal models of autoimmune diseases¹ and navitoclax shown efficacy in animal models of systemic lupus erythematosus (SLE)². However, there are no published reports quantifying the expression of BCL-2 family members in healthy human peripheral blood mononuclear cell (PBMC) subsets.

Objectives To identify a BCL-2 family expression profile from healthy PBMC samples using quantitative, multi-parameter flow cytometry and examine the relation with navitoclax response.

Methods Blood samples from healthy donors were incubated with navitoclax overnight at 37^oC. T cell, B cell and NK cell enumeration was performed using a BD Multitest kit. IC50 values for each lymphocyte subset were then calculated. For BCL-2 family profiling, healthy donor PBMC were isolated by Ficoll gradient and stained with surface markers for various subsets of T cells, B cells and NK cells. Cells were fixed and permeabilized for intracellular staining of Bim, BCL-2, BCL-xL and MCL-1. Cells were then interrogated using a five laser, 18 parameter, BD LSRFortessa flow cytometer and data analyzed using FlowJo software. A two-tailed Pearson correlation analysis was performed using graphpad prism software.

Results Expression of the pro-apoptotic BCL-2 family member Bim, and the anti-apoptotic BCL-2 family members BCL-2, BCL-xL and MCL-1 varied between human PBMC cell types examined. T cells expressed similar amounts of all three anti-apoptotic proteins examined. NK cells expressed more BCL-xL than either BCL-2 or MCL-1. B cells expressed lower levels of BCL-2 than either T cells or NK cells, but higher levels of the pro-apoptotic protein Bim.

When assessing apoptotic sensitivity of PBMC subsets to navitoclax, we found that the B cells and NK cells were more sensitive than T cells. Expression of any single BCL-2 family member alone did not correlate with apoptosis induced by navitoclax; however, the BCL-2:Bim ratio significantly correlated with sensitivity to navitoclax (r>0.9, p<0.0005). There was no correlation between BCL-xL:Bim ratios and sensitivity to navitoclax (r<0.1, p>0.6).

Conclusions Profiling of selected BCL-2 family members Bim, BCL-2, BCL-xL and MCL-1 in healthy human PBMC samples revealed differential regulation of these proteins in human T cell, B cells, and NK cells. In addition, B cells and NK cells are more sensitive to navitoclax than T cells, which coincides with a lower BCL-2:Bim ratio observed in B cells and NK cells compared to T cells. Therefore the BCL-2:Bim ratio may be a useful biomarker for predicting PBMC subtypes that will be sensitive to BCL-2 family inhibitors.

References

1. Bardwell et al. J Immunol. 2009 Jun 15;182(12):7482-9.

2. Wang et al; Arthritis Rheum 2013;65(Suppl 10):555.

Disclosure of Interest K. Grebe Shareholder of: AbbVie, Employee of: AbbVie, A. Souers Shareholder of: AbbVie, Employee of: AbbVie, P. Bansal-Pakala Shareholder of: AbbVie, Employee of: AbbVie, L. C. Wang Shareholder of: AbbVie, Employee of: AbbVie

DOI 10.1136/annrheumdis-2014-eular.3368