Tissue resident lymphocytes in human spinal entheses
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OBJECTIVE: Ankylosing spondylitis (AS) is an inflammatory disorder within the spondyloarthritis family of rheumatic diseases. AS is characterized by chronic inflammation at entheses, the attachment sites of ligaments or tendons to bone, most prominently in the spine. Chronic enthesitis leads to pathological new bone formation, fusion of vertebral bodies and loss of spinal mobility. Studies in mice identified discrete subsets of enthesis-resident lymphocytes that function within the IL-23/IL-17A signaling pathway and are thought to play a critical role in mediating inflammation and new bone formation. Little is known about such enthesis-resident lymphocytes in humans. The goal of this project was therefore to develop methods to isolate lymphocytes from spinal entheses and analyze them in vitro. METHODS: Cell preparation and staining conditions were optimized using peripheral blood mononuclear cells (PBMCs). We compared surface marker staining of PBMCs exposed to collagenase D, Liberase TL, and dispase II. In addition, we compared the staining of multiple antibody clones specific for CD56 and CD4. To measure cytokine expression, cells were stimulated with PMA/ionomycin for 4 hours in the presence of monensin and brefeldin A. Following stimulation, the cells were stained with a 17-color staining panel enabling the identification of 8 lymphocyte subsets, 3 killer immunoglobin-like receptors (KIRs), and 4 cytokines implicated in AS pathology. Cell were analyzed on a 5 laser LSRFortessa flow cytometer. Spine tissue was obtained from laminectomy surgeries performed on patients with degenerative disease of the spine. Interspinous ligament and spinous process bone were cut into 5 mm pieces and cryopreserved in CryoStor CS10, a serum free cryopreservation medium. Samples were stored at -80° C until analysis. After thawing, samples were digested with collagenase D to generate single cell suspensions for analysis by flow cytometry. RESULTS: Incubation of lymphocytes with collagenases resulted in variable signal attenuation for CD56, CD4, and KIR3DL2. Loss of signal for CD56 occurred with all enzymes and anti-CD56 clones tested but correlated with duration of enzyme exposure. For the anti-CD4 clone OKT4, fluorescence remained stable after digestion. KIR3DL2 signal was lost after incubation with Liberase TL but was not affected by collagenase D. Spine tissue samples contained the same lymphocyte subsets present in peripheral blood: natural killer (NK) cells, CD4+ T cells, CD8+ T cells, double negative αβ T cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells including vδ1 and vδ2 cells. PMA/ionomycin-stimulated CD4+ T cells from a spine tissue donor had higher fractions of IL-17A and IL-22 positive cells compared to PBMCs from unmatched donors. Granulocyte-macrophage colony-stimulating factor (GM-CSF) production was also increased in spine tissue compared to unmatched PBMCs. CONCLUSION: Here we describe an optimized method for releasing lymphocytes from cryopreserved spine tissue specimens with minimal impact on surface marker detection. Similar populations of lymphocytes could be detected in spine tissue and peripheral blood. Stimulation of tissue-resident lymphocytes induced them to produce inflammatory cytokines. The methods described will be useful to analyze a larger sample size and further characterize the lymphocytes resident to spinal entheses.
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