0666: HRES-1/Rab4 Controls the Overexpression of CD38 and Depletion of IL-2 in CD4⁺ T Cells; Potential Involvement in Proinflammatory Lineage Development in SLE

Seong Hee (Joy) Park¹, Akshay Patel² and Andras Perl³, ¹Upstate Medical University, Syracuse, NY, ²SUNY Upstate Medical University, Syracuse, NY, ³SUNY, Syracuse, NY

Background/Purpose: HRES-1/Rab4 (Rab4A) is a small GTPase that is overexpressed in SLE patient T cells^1,2, mediates the enhanced recycling of CD3 and CD4 cell surface receptors^1,2, and the activation of the mechanistic target of rapamycin (mTOR)³. Recently, increased expression of CD38⁴, mTOR activation⁵, and loss of IL-2 production^6,7 have been implicated in pro-inflammatory T cell development in SLE. In this study, we investigated the impact of Rab4A on the expression of CD38 and the secretion of IL-2.

Methods: To understand the cellular consequences of Rab4A overexpression, our lab has created unique Rab4A-mutant Jurkat cell lines, which contain GFP-expressing vector alone (control), doxycycline-inducible vectors that overexpress Rab4A (Rab4A⁺⁺) or the dominant-negative mutant Rab4A^S27N (Rab4A^DN)⁸. The three Rab4A-modified Jurkat lines were cultured in the presence and absence of 1 µg/mL of doxycycline. They were also separately stimulated with a combination of 1 µg/mL of anti-CD3 mAb (OKT3) and 50 ng/mL of phorbol myristate acetate (PMA) to induce cytokine production⁹. Cells were harvested after 24 hours and analyzed by flow cytometry using fluorochrome-tagged primary antibodies against CD4 and CD38 for surface staining and antibodies against IL-2 for intracellular staining. Mean fluorescent intensity (MFI) was used for quantification. Cell lysates were run for Western Blot analysis, and Rab4A, CD4, CD38, and phosphorylated STAT3 (pSTAT3) (Ser727) were normalized to β-actin. Cell culture supernatant was used to measure cytokine secretion using the cytometric bead array (CBA) kit (BD Biosciences). For statistical significance, t-test was used; p-values < 0.05 were considered significant for hypothesis testing.

Results: CD38 was significantly upregulated in the Rab4A⁺⁺ cells, compared to the control and Rab4A^DN cells (fold change=2.014, p=2.49x10^-13). Intracellular production and secretion of IL-2 were significantly decreased in the Rab4A⁺⁺ cells (fold change=-0.566, p=1.16x10^-7 and fold change=-0.481, p=0.0401, respectively) compared to the control. In the Rab4A^DN cells, IL-2 secretion was significantly increased (fold change=18.091, p=7.513x10^-7, respectively). In Rab4A⁺⁺ cells, pSTAT3 is increased compared to the control (fold change=2.052, p=0.0318).

Conclusion: CD38 is an NAD+ hydrolase¹², which has shown to regulate Sirtuin-1 activity¹³, a NAD+-dependent histone deacetylase that deacetylates and suppresses STAT3 activity¹⁴. We showed that CD38 is increased in the Rab4A⁺⁺ cells, which may explain the increase in pSTAT3 seen in the Rab4A⁺⁺ cells. Phosphorylation of STAT3 is required for STAT3 activity¹⁵. STAT3 binds to Forkhead box, class-O (FoxO)1 promoter, and FoxO1 protein inhibits IL-2 production¹⁶. We showed that IL-2, which inhibits T_H17 differentiation while inducing T_reg differentiation, is decreased in the Rab4A⁺⁺ cells. The overexpression of Rab4A^1,2 and CD38^10,17,18, increased pSTAT3¹⁹, and diminished IL-2 production⁶ reflect changes observed in SLE patients. Our results suggest that increased expression of Rab4A may in fact underlie the overexpression of CD38 and diminished secretion of IL-2 in SLE.
Mechanistic diagram showing the pathway between Rab4A regulating IL-2 production in CD4+ T cells via the CD38/Sirtuin-1/STAT3-axis.

Rab4A controls the surface expression of CD4 and CD38 in Jurkat cells. (A) Flow cytometry and (D) Western Blot results show that Rab4A++ cells have increased CD38 expression compared to the control and Rab4A-DN cells. Rab4A effects on CD4 expression is shown for confirmation. Quantified fluorescent intensity of (B) CD4 and (C) CD38.

Rab4A controls (A) the surface expression of CD38, intracellular expression and (B) production of IL-2 in Jurkat cells. (A) Flow cytometry shows CD38 is increased in Rab4A++ cells compared to the control and Rab4A DN cells. IL-2 expression is decreased both (B) intracellularly and (C) secreted in the cell culture supernatant by the Rab4A++ cells, while increased in Rab4A DN cells. (D) Western Blot results show an increase in pSTAT3 is increased in Rab4A++ cells compared to the control and Rab4A DN cells.
Disclosures: S. Park, None; A. Patel, None; A. Perl, None.