Objective: 1) To further characterize specific surface and intracellular receptors on cNK cells in untreated JDM with: a) low, or b) normal cNK (age-normed) at diagnosis and after clinical response to therapy, compared with healthy pediatric controls; 2) Evaluate NK localization in inflamed, untreated JDM muscle
Method: Definite/probable JDM and healthy pediatric controls (IRB# 2008-13457) were enrolled. We tested frozen peripheral blood mononuclear cells (PBMCs) from untreated white JDM females with low (Group 1, n=6) and normal cNK (Group 2, n=5) at diagnosis; in both groups the cNK normalized after institution of immunosuppressive therapy (19.92 ± 11.46 mo after diagnosis). Controls for the two groups (n= 7 and 8 respectively) were age matched. Marker expressions on viable CD3-CD56+ NK cell was measured by flow cytometry using FlowJo software. Results were expressed as the percentage (%) NK positive for a specific marker and median fluorescence intensity (MFI) of the specific marker. Results were analyzed using the paired t-test for the two JDM groups and the student’s t-test for comparing the JDM patients and controls (p<0.05 as significant). Using dual stain immunohistochemistry, NK cells were identified in 27 untreated JDM muscle tissue as CD16+/CD56+ cells (n=16, low cNK; n=11, normal cNK; n=7, controls) Wilcoxon Rank Sum test was used for comparisons (p<0.05 as significant)
Results: In Group 1 JDM (low cNK at diagnosis), 7 out of 17 NK receptors analyzed at diagnosis were significantly reduced compared to the levels observed when the cNK normalized, and to controls. The density (MFI) of 5 of these receptors (CD16, CD2, NKG2D, CD11b, and CD94) was also significantly reduced, (p<0.05). After cNK levels normalized following therapy, the % of NK cells expressing NKp46 increased, but % of NK cells expressing CD158b was significantly lower than controls, (p<0.05). The DNAM1+ cNK cells were decreased to 50% normal range at diagnosis, and remained depressed (78% of normal) after response to therapy. In contrast, in Group 2 JDM children (normal cNK at diagnosis), only CD2 was below the healthy control group (both % and MFI), but increased to normal ranges after therapy. This group also had a higher % of cNK cells expressing perforin at diagnosis (92.06 ± 5.81 vs 84.90 ± 4.16, p=<0.05) compared to the control group. In the diagnostic muscle biopsies, the NK cell counts from JDM with either low or normal cNKs were significantly higher than controls (p=0.012, p=0.044) respectively
Conclusion: At diagnosis, the percentage of NK cells expressing specific receptors and the density of these receptors expressed on the cell surface differed from levels observed post therapy and from healthy control age-matched children. Irrespective of the absolute count of cNK cells, we found a significant increase in NK cells localized in inflamed muscle tissue of untreated children with JDM compared to healthy controls. These data suggest NK cells may be involved in the pathogenesis of muscle damage and disease in JDM
Support by CureJM, NIHR01AR48289
Disclosure: S. Shrestha, None; M. O'Gorman, None; J. Orange, None; C. Tessler-Verville, None; K. Snow, None; G. Morgan, None; D. Wang, None; L. M. Pachman, None.