Cytokine Response and Genotype Frequencies in ITP of Childhood.
Authors: Imfeld Karen , Nakagawa Peggy , Tai LeiQian , Williams Shirley , Kunicki Thomas , Nugent Diane 
Published: October 2019

Pediatric idiopathic thrombocytopenic purpura (ITP) is a bleeding disorder characterized by immune-mediated platelet destruction. Current evidence suggests that childhood ITP is associated with an imbalance within the T cell regulatory network following an external immune trigger resulting in an excessive or sustained proinflammatory T helper cytokine response. In order to understand why certain children are at risk for sustained thrombocytopenia or chronic ITP, we investigated cytokine responses to peptides from a common platelet membrane autoantigen, glycoprotein (GP) Ib, and the relationship to the genotype frequencies of single nucleotide polymorphisms (SNPs) from genes associated with immune regulation. Methods: PBMC and DNA were collected from 114 children with ITP, (80 acute, 34 chronic) and 25 controls. PBMC were stimulated with Phytohemaglutinin-P (PHA) or GP Ib peptides and the resulting Interferon gamma (IFNγ), Interleukin 1 beta (IL-1β), and Interleukin 4 (IL-4) cytokine production was measured by ELISA. DNA was isolated and genotypes determined for inflammatory cytokine IL-1α (−889), Fcγ receptor FcγRIIIA (F158V), and Complement C1qc (exon 1, codon 42). Results: Previously, we have shown a large shift in the allele frequencies of the FcγRIIIA receptor SNP (F158V) in ITP patients (F=.36, V=.64 vs F=.70, V=.30; p<0.001) and IL-1α −889 SNP (C=.67, T=.33 vs C=.84, T=.16; p=0.005) compared to controls. Data from the current analysis is shown in table below. ITP patients produce significantly less IL-4 (54%; p=0.0001) and IL-1β (80%; p=0.0041) than controls when stimulated with the mitogen PHA. Conversely, when stimulated with GP Ib peptides, ITP patients produce more IFNγ (252%; p=0.0272) than controls. In particular, ITP patients with genotype FcγRIIIA V/V, or IL-1α C/C, or C1qc C/T produce more IFNγ than controls when stimulated with autoantigenic peptides. In addition to increased IFNγ production, only ITP patients with FcγRIIIA V/V genotype produce significantly more IL-1β and IL-4 than controls. These same patients were shown to have 100% response rate to IVIG in a previous report.

Differential Cytokine Production from ITP Patients’ PBMC Compared to Controls Cytokine Comparison PHA GP Ib peptides Fc γRIIIA V/V IL-1α C/C 1qc C/T P values in ( ), from nonparametric ANOVA, P<0.05 considered significant IFNγ 90% (.4977) 252% (.0272) 775% (.0324) 584% (.0002) 319% (.0079) IL-4 54% (.0001) 92% (.3674) 908% (.0048) 118% (.2451) 112% (.6266) IL-1β 80% (.0041) 74% (.7114) 272% (.0168) 97% (.0134) 52% (.0163)

Conclusions: Our results show that:

ITP patients’ T cells have an abnormal response to non- specific immune stimulation (PHA) resulting in a lack of production of IL-4, which normally aids in extinguishing an autoimmune response, and these same T cells are hyper responsive to the platelet autoantigen GP Ib peptides, based on increased levels of the proinflammatory cytokine, IFNγ.

This polarization of ITP patients towards a proinflammatory immune response may result in sustained stimulation of antigen primed B cells leading to platelet destruction. In addition, to fully understand the etiology of childhood ITP, robust statistical analysis of cytokine abnormalities should be performed in the context of immune receptor polymorphisms, as these genetic differences appear to modulate the impact of the proinflammatory trigger in these patients.