Elsevier

Journal of Autoimmunity

Volume 86, January 2018, Pages 19-28
Journal of Autoimmunity

CD4+CD28+KIR+CD11ahi T cells correlate with disease activity and are characterized by a pro-inflammatory epigenetic and transcriptional profile in lupus patients

https://doi.org/10.1016/j.jaut.2017.09.011Get rights and content

Highlights

  • KIR+CD11ahi T cells can be induced using a DNA methylation inhibitor.

  • Lupus CD4+CD28+KIR+CD11ahi T cells are demethylated and pro-inflammatory.

  • African-American lupus patients have a higher genetic risk score for lupus.

  • KIR+CD11ahi T cells correlate with lupus activity independent of genetic risk.

Abstract

Objective

The goal of this study was to comprehensively characterize CD4+CD28+ T cells overexpressing CD11a and KIR genes, and examine the relationship between this T cell subset, genetic risk, and disease activity in lupus.

Methods

The size of the CD4+CD28+KIR+CD11ahi T cell subset was determined by flow cytometry, and total genetic risk for lupus was calculated in 105 female patients using 43 confirmed genetic susceptibility loci. Primary CD4+CD28+KIR+CD11ahi T cells were isolated from lupus patients or were induced from healthy individuals using 5-azacytidine. Genome-wide DNA methylation was analyzed using an array-based approach, and the transcriptome was assessed by RNA sequencing. Transcripts in the CDR3 region were used to assess the TCR repertoire. Chromatin accessibility was determined using ATAC-seq.

Results

A total of 31,019 differentially methylated sites were identified in induced KIR+CD11ahi T cells with >99% being hypomethylated. RNA sequencing revealed a clear pro-inflammatory transcriptional profile. TCR repertoire analysis suggests less clonotype diversity in KIR+CD11ahi compared to autologous KIR-CD11alow T cells. Similarly, primary KIR+CD11ahi T cells isolated from lupus patients were hypomethylated and characterized by a pro-inflammatory chromatin structure. We show that the genetic risk for lupus was significantly higher in African-American compared to European-American lupus patients. The demethylated CD4+CD28+KIR+CD11ahi T cell subset size was a better predictor of disease activity in young (age ≤ 40) European-American patients independent of genetic risk.

Conclusion

CD4+CD28+KIR+CD11ahi T cells are demethylated and characterized by pro-inflammatory epigenetic and transcriptional profiles in lupus. Eliminating these cells or blocking their pro-inflammatory characteristics might present a novel therapeutic approach for lupus.

Introduction

Systemic lupus erythematosus is a chronic multisystem autoimmune disease characterized by periods of disease flares and remission. Genetic susceptibility might explain at least in part familial aggregation of the disease, and plays a role in determining age of disease onset, severity, and disease heterogeneity [1], [2], [3]. Epigenetic aberrancies also play a role in the pathogenesis of lupus [4]. Lupus T cells are characterized by reduced expression and activity of the maintenance DNA methyltransferase DNMT1, resulting in T cell hypomethylation. Several factors might be mechanistically involved in reduced DNMT1 expression and the methylation defect in lupus T cells [5]. These include defective ERK pathway signaling, and increased expression and activity of PP2A, mTOR, and GADD45a [6], [7].

CD4+ T cells from lupus patients are characterized by robust demethylation in interferon-regulated genes [8]. In addition, CD4+ T cells in lupus are characterized by overexpression of several methylation sensitive genes, including CD11a, CD70, Perforin, CD40L, and the KIR gene cluster [9]. Normal CD4+ T cells treated with DNA demethylating agents overexpress these same genes similar to lupus T cells, and become autoreactive in vitro [10]. Further, demethylated T cells or T cells with induced defect in DNMT1 expression can cause autoimmunity in animal models [11], [12], [13].

Using multi-color flow cytometry, a novel CD4+CD28+ T cell subset characterized by cell surface CD11ahi and KIR expression was recently identified in patients with active lupus [14]. This T cell subset also expresses other methylation sensitive genes known to be overexpressed on lupus T cells, including CD70 and CD40L. Indeed, treating T cells from normal healthy individuals with DNA demethylating agents results in expansion of this T cell subset [14]. The goal of this study was to characterize this novel T cell subset to reveal the complete repertoire of genes that identify this subset and therefore understand its functional role upon disease pathogenesis. In addition, we aimed to determine if expansion of this T cell subset interacts with genetic risk to predict disease activity in lupus patients.

Section snippets

Lupus patients

Female participants previously diagnosed with lupus were included in this study. All patients fulfilled the American College of Rheumatology classification criteria for SLE [15]. A Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score was calculated at the clinical visit concurrently with enrollment in the study and blood sampling draw. Patients who had received cyclophosphamide within the past 6 months or who were on methotrexate were excluded from this study, as cyclophosphamide

In vitro generated CD4+CD28+KIR+CD11ahi T cells are demethylated and characterized by a pro-inflammatory transcriptional profile

We previously demonstrated that CD4+CD28+KIR+CD11ahi T cells express proteins of other methylation sensitive genes known to be hypomethylated in lupus T cells, such as CD70 and CD40L, and that treating CD4+ T cells with the DNA methylation inhibitor 5-azacytidine results in the generation of CD4+CD28+KIR+CD11ahi T cells [14]. To assess the extent of DNA demethylation in this T cell subset, we evaluated genome-wide DNA methylation changes in experimentally derived CD4+CD28+KIR+CD11ahi T cells

Discussion

The role of CD4+ T cell DNA hypomethylation in the pathogenesis of lupus has been thoroughly investigated [4]. We described a demethylated T cell subset characterized by KIR and CD11a overexpression in lupus patients. The KIR locus is comprised of 15 KIR genes, which are most commonly expressed on natural killer cells. However, because regulation of the KIR locus is methylation sensitive, KIR genes have previously been shown to be expressed in the context of lupus [14], [34]. Furthermore, KIR

Conclusion

Our findings indicate that the novel CD4+CD28+KIR+CD11ahi T cell subset is demethylated and characterized by pro-inflammatory chromatin accessibility and transcriptional profiles in lupus patients. We identified a number of pathways and specific genes overexpressed in this T cell subset. These data suggest that eliminating CD4+CD28+KIR+CD11ahi T cells or blocking some of the specific pro-inflammatory characteristics we identified might provide novel therapeutic options for lupus.

Conflict of interest

None of the authors has any financial conflict of interest to disclose.

Competing interest

None declared.

Acknowledgements

This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health grants number U19AI110502 and R01AI097134.

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    These two authors equally contributed to this work.

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