PF4 Modulates Proliferation and Suppressive Activity of CD4⁺CD25⁺ Regulatory T Cells: Gene Expression Analysis Reveals Involvement of the ERK1 and Bcl-2 Pathway.

Heparin induced thrombocytopenia associated with thromboembolism (HIT/T) is an important cause of morbidity and mortality in patients treated with this otherwise useful anticoagulant. The recognition that patients with HIT/T almost invariably have antibodies specific for complexes of platelet factor 4 (PF4; CXCL4), a component of platelet α granules, and heparin, has led to improved diagnosis. However, knowledge of the molecular nature of the immune-response is far from complete. Moreover, no observations have been reported that would explain why a high percentage of patients given heparin in conjunction with coronary artery bypass grafting develop this antibody response. We have recently reported that human PF4 reverts the anergic CD4⁺CD25⁺ T regulatory (Tr) cells phenotype, and impairs their suppressive activity, suggesting a previously unrecognized role of PF4 in the regulation of immune responsiveness. How PF4 modulates CD25⁺CD4⁺ Tr cell-mediated suppression and proliferation remains to be determined. Understanding of CD4⁺CD25⁺ Tr cells function has relied principally on phenotypic features and cell-to-cell interaction studies. Molecular characterization is a necessary prelude to deeper understanding of the accompanying biological process. We employed cDNA microarray technology to define the gene expression profiles in CD4⁺CD25⁺ Tr cells stimulated by anti-CD3 mAb and exposed to PF4. CD4⁺CD25⁺ Tr cells were isolated from normal donor’s peripheral blood mononuclear cells by positive selection on magnetic beads (Miltenyi Biotec, Auburn, CA). Total RNA was extracted from freshly isolated CD4⁺CD25⁺ Tr cells, stimulated with anti-CD3 mAb in the presence or the absence of PF4 (at 6, 12, and 24 h time point). Using “focused” cDNA microarray (SuperArray, Frederick, MD) and a customized 6K human expression cDNA microarray developed by the Microarray and Genomics Core Facility of Roswell Park Cancer Institute (Buffalo, NY - see web link for genes represented: http://microarrays.roswellpark.org/Services/Arrays/HIC.xls) we have identified a relatively small number of genes that are differentially expressed, in the presence of PF4, in CD4⁺CD25⁺ Tr cells following activation with anti-CD3 mAb. As reported on the Table below, one gene encodes a cell surface receptor (IL-2Ra) that appears to be involved in the generation of suppressor/effector function and several genes encode factors that may be related to the proliferative effects of PF4 on CD4⁺CD25⁺ Tr cells, among these we identified: IL-4, interferon-γ (INF-γ), lymphotoxin α (LTA; TNF superfamily member 1), the mitogen-activated protein kinase 3 (MAPK3; ERK1), the cyclin-dependent kinase (CDK) CDK4, the CDK inhibitors CDKN1B (p27, kip1) and CDKN2A (p16, INK4), and the antiapoptotic proteins Bcl-2 and Bcl-xL. Our findings suggest a mechanism whereby PF4 promotes CD4⁺CD25⁺ Tr cell proliferation and survival by activating the ERK1 pathway and the apoptosis regulator Bcl-2, hence stimulates cell cycle by downregulating CDKN1B (p27, kip1) and CDKN2A (p16, INK4) which, in turn, sustains CDK4 activity and, thus, leading to cell proliferation.

Gene differentially expressed in CD4+CD25+ Tr cells (stimulated with anti-CD3 mAb) exposed to PF4