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 and characterized the impact of CD38 expression on the activation of mTOR in CD4⁺ T cells.

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. We also CRISPR knocked out (KO) CD38 in these cell lines, leading to six different lines: (1) Rab4A^WT CD38^WT, (2) Rab4A^WT CD38^KO, (3) Rab4A⁺⁺ CD38^WT, (4) Rab4A⁺⁺ CD38^KO, (5) Rab4A^DN CD38^WT, and (6) Rab4A^DN CD38^KO. These cells were cultured with doxycycline and co-stimulated with anti-CD3 mAb (OKT3) and phorbol myristate acetate (PMA) to induce cytokine production⁹. Cell surface markers and cytokines were analyzed by flow cytometry and protein levels by western blot. NAD⁺ levels were measured by LC-MS/MS. To understand the impact of CD38 expression on mTOR activation, we isolated peripheral blood mononuclear cells from SLE patients (n=21) and age, sex, and race matched healthy controls (n=18). The cells were cultured for 24 hours with and without CD3/CD28 co-stimulation and were analyzed by flow cytometry.

Results: In the Rab4A⁺⁺ cells compared to the control, CD38 expression was upregulated (p=2.49×10^-13), intracellular production and secretion of IL-2 significantly decreased (p=1.16×10^-7 and p=0.0401, respectively), NAD⁺ concentration decreased (p=0.0039), while pSTAT3 levels increased (p=0.0318). In the Rab4A⁺⁺ CD38^KO cells compared to the Rab4A⁺⁺ CD38^WT cells, secretion of IL-2 significantly increased (p=0.0145) and pSTAT3 levels decreased. pAkt1 was increased significantly in CD38⁺ CD4⁺ T cells compared to CD38^– CD4⁺ T cells in only the SLE patients (p=0.0004), while p4EBP1 increased significantly in CD38⁺ CD4⁺ T cells compared to CD38^– CD4⁺ T cells for both the SLE patients (p=0.0053) and healthy controls (p=0.0057).

Conclusion: The increased pAkt1 and p4EBP1 in SLE patients’ CD38⁺ CD4⁺ T cells suggests that CD38 activates mTOR via Akt, coinciding with a recent finding of CD38/PI3K/Akt/mTOR axis in cervical cancer¹⁰. CD38 is an NAD⁺ hydrolase, which regulates Sirtuin-1 activity, a NAD⁺-dependent histone deacetylase that suppresses STAT3 activity. STAT3 activation is also known to be regulated by mTOR^11–13. 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, increased CD38, pAkt1, p4EBP1, and pSTAT3, and diminished IL-2 production reflect changes observed in SLE patients. Our results suggest that increased expression of Rab4A and CD38 may underlie the diminished secretion of IL-2 in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/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/