BLOCKING OF CD103+ TISSUE RESIDENT MEMORY T CELLS (TRM) AS A THERAPEUTIC STRATEGY IN SJOGREN’S SYNDROME

Abstract

Background: Tissue-resident memory T cells (TRM), are a recently identified T cells population featuring tissue localization and expression of markers of tissue homing, CD69 and CD103. Recently, the expansion of CD8+ TRMs and their involvement in the sialadenitis was described in a murine model of SS. However, CD4+ and CD8+ TRM’s functional relevance in pSS is still not fully understood, and the TRM therapeutic targeting unexplored. Objectives: The study aimed to address the role of CD4+ and CD8+ TRMs in the pathogenesis of pSS and to explore the therapeutic targeting of the tissue residency marker of TRM CD103. Methods: An animal model of experimental (ESS) obtained by immunization of female C57BL/6 mice (n=10) with salivary glands (SG) protein extract and Freund’s complete adjuvant used to investigate the dynamic of infiltration of SG by CD4+ and CD8+ TRMs, their frequency, and the impact of CD103 blockade. For the therapeutic intervention, at 10-weeks post-immunization, the salivary gland was cannulated via Wharton’s duct, and an anti-CD103 neutralizing antibody or vehicle-injected. The mice’s saliva flow rate was assessed, and SGs were analyzed by Flow-cytometry and immunohistochemistry (IHC). The frequency and localization of TRMs was analyzed in minor SG of sicca syndrome (nSS) and pSS patients (n=39) by flow cytometry and IHC. The expression of genes involved in the tissue retention of TRMs was assessed in SG by RT-PCR. Results: Upon the ESS progression, a significant progressive increase in CD45+CD103+ cells frequency was observed from 5wk to 20wk post-immunization (p<0.001), where the CD8+ were the most abundant, followed by CD4+. Consistently, CD103+CD8+ T cells were detected within the lymphocytic infiltration of SG from ESS mice. Sorted purified SG CD10+CD3+CD8+ T cells showed higher Granzyme B, TNF-alpha expression compared to CD103-CD3+CD8+ at both mRNA and protein levels. Notably, ESS mice treated with anti-CD103 showed improvement in salivary function (p<0.05) and reduced lymphocytic infiltrations measured as focus score (FS) (p<0.01) and area-fraction (p<0.01). Consistently, anti-CD103 treatment consistently reduced CD103+ cells and IFN-gamma+, Granzyme B+, and TNFa+ CD8+ cells. We next performed phenotypic analysis of CD45+CD103+ immune cells in the SG of pSS patients observing an increase in both with CD8+CD103+CD69+ and CD4+CD103+CD69+ (p<0.05). Finally, IHC showed that the expansion of TRMs in pSS salivary glands was accompanied by a down-regulation of E-cadherin glandular expression and their migration outside the epithelium in the context of inflammatory infiltrates. SG of patients with pSS showed a significant up-regulation of BLIMP1, KFL-2, and S1PR1 and down-regulation of ITGB2. CXCL9 and CXCL10, and IL-15 involved in the tissue recruitment and long-term survival of TRMs were significantly modulated in pSS salivary glands. Conclusion: TRM are expanded and activated in the SG of pSS and ESS, participating in the organization of tissue inflammation. Although the mechanisms behind this expansion are still not fully understood, CD103 could be a valuable novel therapeutic target to prevent lymphocytic infiltrations and glandular destruction in Sjogren syndrome.