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 37oC. 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.
Bardwell et al. J Immunol. 2009 Jun 15;182(12):7482-9.
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