Distribution Of Peripheral And Minor Salivary Gland CD3~+CD56~+NKT-like Cells And Its Possible Pathogenesis In Primary Sjogren’s Syndrome

Objectives:To investigate the distribution of CD3+CD56+NKT-like cells in peripheral blood and minor salivary gland of pSS patients.To explore how CD3+CD56+NKT-like cells were involved in primary Sjogren’s syndrome pathogenesis.Methods:CD3+CD56+NKT-like cells were identified as CD3,CD56 double positive cells.The proportion of peripheral NKT-like cells and the expression of CXCR5,CX3CR1 and FasL on NKT-like cell surface were evaluated by flow cytometry.Annexin V/7AAD and CFSE staining were used to explore the apoptosis state and proliferation ability of NKT-like cells respectively.To define the cytokine secretion profile of NKT-like cells,intracellular cytokine stimulation(ICCS)was performed.We applied enzyme-linked immunosorbent assay(ELISA)to identify the serum CXCL13 level and cytometric Bead array(CBA)to measure the circulating CX3CL 1 level.In vitro migration assay was performed to investigate whether NKT-like cells had chemotactic response to recombinant human(rh)chemokine CXCL13 and CX3CL1.While in the minor salivary gland tissue,we used immunofluorescence(IF)to clarify the distribution of NKT-like cells and CX3CR1 expression,and immunohistochemistry(IHC)to identify the expression of CX3CL1.Results:Compared with healthy controls,pSS patients had significantly reduced proportion of peripheral CD3+CD56+NKT-like cells,of which the apoptosis state and proliferation ability were similar.Increased NKT-like cells could be noticed around acini of pSS labial gland.Circulating CXCL13 and CX3CL1 were both elevated in pSS patients significantly.In addition,CX3CL1 was positively correlated with serum IgG level.NKT-like cells rarely express CXCR5,but commonly express CX3CR1.The expression of either chemokine receptor was not significantly different between pSS patients and healthy controls.RhCX3CLl,but not rhCXCL13,had obvious chemotactic ability toward NKT-like cells which was concentration dependent.The epithelium in minor salivary gland strongly expressed CX3CL1 while the relatively healthy controls did not.CX3CR1 positive lymphocytes were observed around acini.The majority of circulating NKT-like cells secreted TNF-α and IFN-γ while a quite small proportion secreted IL-4.Scarce NKT-like cells expressed IL-17.The cytokine secretion profile of pSS patients was not significantly different from healthy controls.Conclusions:In pSS,CD3+CD56+NKT-like cells may be recruited by high concentration of CX3CL1 in the minor salivary gland expressed by acinar epithelium,resulting in reduced circulating NKT-like cells.Increased number of NKT-like cells may destroy the minor salivary gland via secretion of inflammatory cytokines such as TNF-α and IFN-γ.