Background/Purpose: Recently, T follicular helper cells (Tfh cells) have been discovered to be the main CD4⁺ T cells assisting B cells to produce antibody. Tfh cells are over activated in patients with systemic lupus erythematosus (SLE), consequently lead to excessive immunity. Hematopoietic progenitor kinase 1 (HPK1) negatively regulates T cell-mediated immune responses and TCR signal. This study aims to investigate the roles of HPK1 and the mechanisms that regulate HPK1 expression in SLE Tfh cells.

Methods: Naive CD4⁺ T cells and B cells were isolated from 30 normal controls and 30 SLE patients. Subsequently naive CD4⁺ T cells were induced to differentiate into Tfh cells by culturing with anti-CD3 antibody, anti-CD28 antibody, IL-6, IL-12, IL-21, and TGF-β. HPK1 mRNA and protein levels in Tfh cells were measured by quantitative PCR (qPCR) and western blot analysis respectively. The productions of IL-21, BAFF, IFNγ, IL-17A, IgM, IgG1, IgG2, and IgG3 were analyzed using ELISA. Tfh cells proliferation was evaluated with MTT assay. Numbers of histone H3 lysine 27 trimethylation (H3K27me3), H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2), H3K27 demethylase jumonji domain containing 3 (JMJD3), and DNA methyltransferase 3a (DNMT3a) within the HPK1 promoter were determined by chromatin immunoprecipitation (ChIP) combined with qPCR. DNA methylation abundance at the HPK1 promoter was assessed by methylated DNA immunoprecipitation (MeDIP) combined with qPCR.

Results: HPK1 mRNA and protein levels were significantly reduced in SLE Tfh cells, and HPK1 mRNA level negatively correlated with SLE Disease Activity Index (SLEDAI). Knocking down HPK1 with siRNA in normal Tfh cells significantly elevated Tfh cells proliferation and secretions of IL-21, BAFF, IFNγ, IgG1, IgG2, and IgG3. There were no marked alters in IL-17A and IgM productions. The opposite effects were observed in SLE Tfh cells transfected with HPK1 overexpressing plasmid: Tfh cells proliferation and productions of IL-21, BAFF, IFNγ, IgG1, IgG2, and IgG3 were all alleviated. And there were no significant changes in IL-17A and IgM levels. H3K27me3, DNA methylation, and DNMT3a amounts sharply increased at the HPK1 promoter in SLE Tfh cells compared to controls. Moreover, a striking decrease was detected in JMJD3 enrichment, but no marked change in EZH2 number at the HPK1 promoter of Tfh cells from patients with SLE.

Conclusion: Our results suggest for the first time that at the HPK1 promote region of SLE Tfh cells, decreased JMJD3 binding up-regulates H3K27me3 amount, and increased DNMT3a enrichment elevates DNA methylation level. All these factors inhibit expression of HPK1 in SLE Tfh cells, leading to Tfh cells overactivation and B cells overstimulation, subsequently, the onset and progression of SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/down-regulation-of-hematopoietic-progenitor-kinase-1-by-aberrant-epigenetic-alterations-in-t-follicular-helper-cells-accounts-for-excessive-immunity-in-systemic-lupus-erythematosus/