Project Details
Early Career
Status: Funded - Open
Developing a Diagnostic Test for Pediatric Immune Cytopenias
Emily Harris, MD
Summary
BACKGROUND: Evans syndrome (ES) is a life-threatening disorder resulting in autoimmune destruction of at least two hematopoietic cell lineages and a mortality rate of 36%. Other immune cytopenias, such as autoimmune hemolytic anemia (AIHA), are limited to a single lineage. There is no way to know whether an immune cytopenia will progress to ES. GAP: There are no available tests distinguish ES from other immune cytopenias, necessitating empiric treatment and a ‘watch and wait’ approach for a disease with 36% mortality. HYPOTHESIS: This study aimed to test the hypothesis that dual expression of CXCR3 and CCR6 on CD4+CXCR5+ T cells differentiates ES from AIHA, and to test the hypothesis that cTfm percentages correlate with disease activity. METHODS: Patients with ES, AIHA, ITP, and controls were enrolled and blood samples were collected for research evaluations at time of routine clinical care. Clinical data was collected from electronic medical records including patient demographics, disease course, clinical blood counts and immunophenotyping, treatments and clinical response. cTfh (as % of CD4+ T cells) and cTfm cells (as % of CD4+CXCR5+ T cells) were measured using flow cytometry of whole blood. We used single-cell RNA-sequencing (scRNAseq) to profile cTfh cells from patients with ES as compared to those from patients with ITP, wAIHA, and healthy controls. RESULTS: Pathway analysis of differentially expressed genes (>1.5 fold change, adjusted p<0.05) revealed a robust interferon gamma (IFN-𝛾) transcriptional signature in cTfh, cTfm, and effector CD4+ T cells from patients with active ES, compared to cells from those with wAIHA, ITP, and controls. Patients with active or inactive ES on treatment had suppressed T cell activation, the Th1 pathway, and IFN- pathways in cTfh cells consistent with treatment effects. cTfh cells from patients with active ES on treatment also demonstrated upregulation of the vitamin D receptor/retinoic acid receptor axis, known to inhibit genes important for the differentiation of Tfh and cTfh cells. Patients with inactive ES off treatment had cTfh cells with transcriptional signatures similar to controls, consistent with clinical remission. CXCL9, a chemokine induced by IFN-𝛾, was significantly elevated in patients with ES. CXCL9 levels in plasma significantly correlated with cTfh levels in patient samples and was highest in those with active disease off treatment. CXCL9 levels were lowest in patients in remission off therapy. IMPACT: These findings uncover a novel transcriptional signature in patients with active ES that is a potential future therapeutic target. Additionally, these results identify CXCL9 as a promising clinically orderable biomarker in ES.
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