Background High-dose cyclophosphamide (CFA) is used for treatment of patients with severe manifestations of systemic autoimmune diseases. The knowledge about the effect of CFA on circulating cell populations in these diseases is still limited.
Objectives To identify the effect of a single dose of CFA on circulating cell populations in patients with vasculitides and autoimmune systemic diseases.
Methods We immunophenotyped T lymphocytes (CD3, CD4, CD8, CD25, CD127, HLA-DR), B lymphocytes (CD19, CD20, CD27), NK cells (CD3, CD16, CD56, CD69), neutrophils (CD15, CD11b, CD16, CD54, CD62L, CD64), and monocytes (CD11b, CD14, CD16, CD64, HLA-DR) using 6-color cytometer BD FACSCanto II (Becton Dickinson) in peripheral blood of patients with vasculitides (n=6) and systemic disorders of connective tissue (n=6). From each patient, we obtained paired samples before and one month after a single CFA dose. Statistical tests were performed using GraphPad Prism (GraphPad Software, Inc). P-value <0.05 was considered as significant.
Results Single dose of CFA resulted in increase of percentage of CD8+ T lymphocytes (P=0.002), leading to marked decrease of CD4+/CD8+ ratio (P=0.009). Except three patients, overall percentage of neutrophils decreased after the treatment (P=0.01). Although CFA pulse did not influence the percentage of NK cells, the percentage of NK cells carrying stimulatory receptor CD69 increased after CFA (P=0.037). Similarly, CFA enhanced the percentage of co-stimulatory molecule CD27 on B lymphocytes (P=0.048). Among subsets of monocytes, CFA treatment increased percentage of CD14-CD16+ monocytes (P=0.006) and increased expression (MFI) of Fc fragment CD64 (P=0.02). Moreover, increase of HLA-DR was observed on CD14-CD16+ monocytes (P=0.037). The investigations whether the changes in immune cell subpopulations in treated patients have prognostic potential are ongoing.
Conclusions Single dose of CFA resulted in increase of CD8+ T lymphocytes, activation of NK, B lymphocytes and monocytes as well as in some patients decrease in neutrophil counts. Further investigation of selected markers may lead to identification of new prognostic markers and predict the effectiveness of the treatment.
Acknowledgements Grant support: MZ CR VES15–28659A, IGA_LF_2017_009
Disclosure of Interest None declared