Elevated double negative T cells in pediatric autoimmunity

Abstract

Purpose: Autoimmune diseases are thought to be caused by a loss of self-tolerance of the immune system. One candidate marker of immune dysregulation in autoimmune disease is the presence of increased double negative T cells (DNTs) in the periphery. DNTs are characteristically elevated in autoimmune lymphoproliferative syndrome, a systemic autoimmune disease caused by defective lymphocyte apoptosis due to Fas pathway defects. DNTs have also been found in the peripheral blood of adult patients with systemic lupus erythematosus (SLE), where they may be pathogenic. DNTs in children with autoimmune disease have not been investigated.

Methods: We evaluated DNTs in pediatric patients with SLE, mixed connective tissue disease (MCTD), juvenile idiopathic arthritis (JIA), or elevated antinuclear antibody (ANA) but no systemic disease. DNTs (CD3(+)CD56(-)TCRαβ(+)CD4(-)CD8(-)) from peripheral blood mononuclear cells were analyzed by flow cytometry from 54 pediatric patients including: 23 SLE, 15 JIA, 11 ANA and 5 MCTD compared to 28 healthy controls.

Results: Sixteen cases (29.6 %) had elevated DNTs (≥2.5 % of CD3(+)CD56(-)TCRαβ(+) cells) compared to 1 (3.6 %) control. Medication usage including cytotoxic drugs and absolute lymphocyte count were not associated with DNT levels, and percentages of DNTs were stable over time. Analysis of multiple phenotypic and activation markers showed increased CD45RA expression on DNTs from patients with autoimmune disease compared to controls.

Conclusion: DNTs are elevated in a subset of pediatric patients with autoimmune disease and additional investigations are required to determine their precise role in autoimmunity.

Fig 1. Frequency of DNTs in pediatric patients

(A) Percentage of DNTs from flow cytometric analysis of PBMC from 54 patients (circles) and 28 healthy controls (squares). Percentages shown represent the percent of subjects with DNTs greater than or equal to 2.5%. (B) DNT percentages by disease category. (C) Association between medication use at study entry and DNTs. Nineteen patients were taking no cytotoxic medications; 17 were treated with cytotoxic agents including 6-mercaptopurine, mycophenolate mofetil, cyclophosphamide, methotrexate, and leflunomide; 3 received steroids without cytotoxic drugs; and 15 were taking steroids plus cytotoxic agent(s). There was no difference among groups by ANOVA or percentage of patients with DNT values greater or less than 2.5% between groups by Chi-square. (D) DNT values were determined at multiple time points for an average of 8 months. Three of 16 patients had DNT percentages cross 2.5% on subsequent visits (gray line). (E) Comparison of DNTs to absolute lymphocyte count (ALC) in patients with autoimmune disease (n=49). There was no association of DNTs to ALC (r=−0.11, p=0.45, Spearman's correlation). Abbreviations: Mixed connective tissue disease (MCTD), anti-nuclear antibody (ANA), juvenile idiopathic arthritis (JIA).

Fig 2. Expression of phenotypic and activation markers by DNTs, CD4 and CD8 cells in patients with autoimmune disease and healthy controls

Five cases with DNTs ≥2.5% (DNT^hi, black), 5 cases with DNTs <2.5% (DNT^lo, gray) and 5 healthy controls (white) were evaluated for phenotypic and activation markers. Each case group included 3 SLE, one MCTD, and one patient with an elevated ANA. The percentages of various extracellular and intracellular markers were determined in the following cell types: DNTs, CD3⁺TCRα/β⁺CD4⁺, and CD3⁺TCRα/β⁺CD8⁺ T cells. The following markers were evaluated: A) CD25, B) FoxP3, C) CD45RA, D) CD45RO, E) CD69, F) HLA-DR, G) intracellular granzyme B and H) intracellular perforin. In Figure 6C, DNTs from DNT^hi cases showed significantly increased CD45RA percentages compared to healthy controls (*, p=0.008, Mann-Whitney test). Abbreviations: Granzyme B (GzmB), perforin (PRF).