Tu152 - rab4a Controls the Depletion of IL-2 in CD4+ T Cells via Enhanced CD38 Expression: Potential Involvement in Proinflammatory Lineage Development in Systemic Lupus Erythematosus

Akshay Patel, MS – Upstate Medical University; Daniel Krakko, PhD – Upstate Medical University; Jessica Nolan, MS – Upstate Medical University; Andras Perl, MD, PhD – Upstate Medical University

Abstract Text: Rab4A is a GTPase that is overexpressed in systemic lupus erythematosus (SLE) patients’ T cells and enhances surface receptor recycling. Increased expression of CD38 and loss of IL-2 production have independently been associated with proinflammatory skewing in SLE. We investigated the impact of Rab4A on the expression of CD38 and the production of IL-2.

Our lab created Rab4A-mutant Jurkat cell lines, containing GFP-expressing vector alone (control), doxycycline-inducible vectors that overexpress Rab4A (Rab4Ahi) or the dominant-negative mutant Rab4AS27N (Rab4ADN). Furthermore, we CRISPR knocked out (KO) CD38 in these cell lines. These cells were co-stimulated with anti-CD3 antibody and PMA. Cell surface markers and cytokines were analyzed by flow cytometry and protein levels by western blot. NAD+ levels were measured by LC-MS/MS.

In the Rab4Ahi cells compared to the control, CD38 expression was upregulated (p=2.49x10-13), intracellular production and secretion of IL-2 significantly decreased (p=1.16x10-7 and p=0.0401, respectively), NAD+ concentration decreased (p=0.0039), while pSTAT3 levels increased (p=0.0318). In the Rab4Ahi CD38KO cells compared to the Rab4Ahi CD38WT cells, secretion of IL-2 significantly increased (p=0.0145) and pSTAT3 levels decreased.

CD38 is an NAD+ hydrolase, which regulates Sirtuin-1 activity, a NAD+-dependent histone deacetylase that suppresses STAT3 activity. Elevated pSTAT3 levels may underlie diminished IL-2 production by binding to the promoter of FoxO1, which inhibits IL-2 production. The overexpression of Rab4A and CD38, increased pSTAT3, and diminished IL-2 production reflect changes observed in SLE patients. Our results suggest that increased expression of Rab4A may underlie the overexpression of CD38 and diminished secretion of IL-2 in SLE.