Analysis of CD4+ T cell populations in male NOD mice highlights the potential of local administration of cyclosporine A in the treatment of Sjögren’s syndrome

Abstract

Purpose : We recently reported an elastin-like polypeptide (ELP) fusion protein termed CA192 to bind, solubilize, and systemically deliver cyclosporine A (CsA). This formulation reduces CsA systemic toxicity and mitigates autoimmune dacryoadenitis in non-obese diabetic (NOD) mice, which model Sjögren’s syndrome (SS). To understand the pathogenesis of SS and optimize CsA dosing, we isolated CD4+ T cells from lacrimal gland (LG) and other sites within NOD mice to explore cytokine expression.

Methods : CD4+ T cells were isolated from the LG and spleen (SP) using negative selection from male NOD mice and BALB/c mice at different ages. mRNA extracts from isolated T cells were analyzed by qPCR for gene expression levels of IL-17a and IL-2, the representative cytokines of Th17 and Th1 T cells.

Results : In LG-isolated CD4+ T cells, IL-17a was highly expressed in NOD mice; however, IL17a was not detectable in BALB/c mice (n=3). IL-17a gene expression levels in CD4+ T cells from LG in NOD mice were not correlated with age. IL-2 gene expression in CD4+ T cells from LG did not differ between BALB/c and NOD mice when normalized to cell protein (n=3). However, at least 5-fold more lymphocytes were isolated from NOD mouse LGs, suggesting significantly higher IL-2 expression in these LG relative to BALB/c LG. In splenic CD4+ T cells, IL-17a gene expression was comparable in NOD and BALB/c mice while IL-2 gene expression was significantly reduced by 2.1-3.3 fold (p=0.002, n=3). These findings implicate local expression and accumulation of IL-17a and IL-2 as possible factors in SS-mediated dacryoadenitis. CA192-CsA potently inhibits IL-2 protein expression and secretion from Jurkat cells. In Th17 cells induced by polarization and differentiation in vitro from mouse splenocytes, the inhibitory effect of CA192-CsA on IL-17a was qualitatively confirmed by both ELISA and flow cytometry as the IL-17a positive population was reduced by 8.1-fold.

Conclusions : Our findings underscore the potential utility of local administration of CA192-CsA to the LG in the treatment of SS due to its integrated inhibitory effects both on locally-elevated IL-2 and IL-17a.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

IL-17a gene expression in isolated CD4+ T cells from LG. IL-17a was highly expressed in isolated CD4+ T cells from male NOD mice LG; however, these transcripts were indetectable in LG of control BALB/c mice.