Circulating T Follicular Helper Cells in Pediatric Immune Cytopenias

Introduction: Immune cytopenias, including immune thrombocytopenia (ITP), autoimmune hemolytic anemia (AIHA), and multiple immune cytopenias known as Evans syndrome (ES), are heterogeneous autoimmune disorders. Some cases of immune cytopenia isolated to a single cell type will resolve, whereas others can over time involve autoimmune destruction of multiple cell types and autoimmune complications of other organ systems. There is currently no test to predict disease type or measure disease activity, both of which would be helpful to guide clinical management. Circulating T follicular helper cell (cTfh) measurement is a clinically available CLIA-approved test for multi-system autoimmunity, but the utility of cTfh measurement in pediatric immune cytopenias is not well understood. The objective of this study is to evaluate cTfh percentages in children with immune cytopenias.

Methods: This IRB-approved single center cohort study included 142 pediatric patients with immune cytopenias and 52 healthy controls. Collected clinical data included demographic features, clinical diagnoses, blood counts, and treatments. cTfh cells were defined as CD4⁺ CXCR5⁺PD1⁺ cells measured as percentage of CD4⁺ cells by peripheral blood flow cytometry. Outcomes were summarized using descriptive statistics. Statistical analysis was performed using Graphpad. Mann-Whitney tests were performed for comparisons of two variables. Kruskal-Wallis tests and Dunn's multiple comparisons tests were used for comparisons of three or more variables. ROC curve was used to determine sensitivity and specificity.

Results: The median age was 12.8 years (range: 4 months-32 years) at the time of cTfh measurement with 49.3% (n=70) female. There were 75 patients with ITP, 24 with warm AIHA, and 43 with ES. Patients with ES had significantly higher cTfh than those with ITP, AIHA, or controls (mean 14.4% in ES vs 6.8% in AIHA vs 6.7% in ITP vs 5.9% in controls, p<0.0001). There were not significant differences in cTfh between patients with ITP, AIHA, and controls (all p>0.9999). ROC curve demonstrated that cTfh >9% had sensitivity 76.6% and specificity 79.8% for distinguishing ES from non-ES immune cytopenias (AUC 0.8445). Patients who had immune cytopenias in addition to autoimmunity affecting another organ system had significantly higher cTfh than those with immune cytopenias alone (mean 11.2% vs 8.5%, p=0.0239). ANA testing was performed in 62% (88/142) of patients, of whom 20.5% (18/88) had ANA titer ≥1:160. There was no significant difference in cTfh between those who were ANA positive versus ANA negative.

Among patients with ES, 42/43 had genetic testing and 47.6% (n=20/42) had an identified genetic immune disorder. cTfh was higher in patients with ES with a genetic diagnosis than in those with ES without a genetic diagnosis (mean 16.9% vs 12.3%, p=0.0326). cTfh was higher in patients with ES who had active cytopenias at the time of testing as compared to those without active cytopenias (mean 17.2% vs 11.3%, p=0.0084). Mean cTfh was highest in those who had active cytopenia present who were not on treatment (22.1%), as compared to 15.8% in those with active cytopenia who were on treatment, 12.4% in those without active cytopenia who were on treatment, 9.6% in those without active cytopenia who were not on treatment (p=0.0319).

Conclusions: Elevated cTfh was associated with diagnosis of ES and presence of multi-system autoimmunity. Among those with ES, cTfh was associated with disease activity and treatment and was higher in those with genetic diagnoses. Further study is needed to validate the diagnostic and prognostic utility of cTfh in ES and to elucidate the mechanisms underlying cTfh expansion in these disorders.

Grace:Agios, Sobi, Novartis: Research Funding; Agios, Sanofi, Sobi: Consultancy.