Postgraduate Institute of Medical Education and Research CHANDIGARH, Chandigarh, India
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AASTHA KHULLAR1, Varun Dhir2, Leishangthem Bidyalaxmi1, Chandra Bhushan Prasad3, Sankar Jayaprakash1, Siddharth Jain4, Biman Saikia1, Aman Sharma5, Shefali Sharma6, Ashok Kumar Yadav1, Shankar Naidu1 and Sanjay Jain7, 1PGIMER, Chandigarh, India, 2PGIMER, CHD, INDIA, Chandigarh, India, 3Healthway Hospital, Goa, Zuarinagar, India, 4AIIMS, Delhi, India, 5PGIMER, Chandigarh, India, Chandigarh, India, 6PGIMER< Chandigarh, Chandigarh, India, 7Post Graduate Institute of Medical Education and Research, Chandigarh, India
Background/Purpose: Background: GMCSF producing T-cells may be implicated in the pathogenesis of autoimmune diseases like rheumatoid arthritis (RA), multiple sclerosis (MS) and psoriatic arthritis. Few studies reported higher frequencies of CD4+GMCSF+ T-cells in the MS lesions and RA synovial fluid; however, there is no data on CD8+GMCSF+ T-cells. Thus, we planned to assess their frequencies in blood and synovial fluid, and characterize based on polyfunctionality, phenotype, cytotoxic ability and the effect of methotrexate on them.
Methods: The frequency of GMCSF producing CD8 and CD4 T-cells was assessed by stimulation with PMA/Ionomycin, followed by immunostaining and flowcytometry. Subsequently, polyfunctionality for other cytokines (TNFα, IFNγ and IL-17), memory/naïve phenotype and perforin/granzyme expression was also assessed by flowcytometry
Results: Enrichment of GMCSF producing T-cells was found in the synovial fluid, with elevated frequencies of both CD8+GMCSF+ T-cells (5.8, 3.9%, p= 0.0045) and CD4+GMCSF+ T-cells (8.5, 4.5%, p=0.0008) compared to peripheral blood. These cells were characterised as extremely polyfunctional, with higher frequencies of triple cytokine positive GMCSF+TNFα+IFNγ positive CD8 T-cells (81, 36%, p=0.049) and CD4+GMCSF+TNFα+IFNγ+ (48, 32%, p=0.01)) in synovial fluid compared to blood. Majority of GMCSF producing T-cells had an effector memory (EM) phenotype, besides CD8+GMCSF+ T-cells also having increased expression of granzyme B. After MTX treatment, there was a significant reduction in circulating CD4+ T-cells producing GMCSF (4.6% to 2.9%, p= 0.0014) and a similar trend was seen in the CD8 T (3.7% to 2.9% p= 0.068) cells.
Conclusion: Synovial fluid enrichment with GMCSF producing CD8 T-cells (and CD4 T-cells) was observed, demonstrating elevated polyfunctionality and granzyme B expression with majority belonging to EM phenotype. Upon treatment with methotrexate a reduction in the frequencies of these cells was found.
A. KHULLAR: None; V. Dhir: None; L. Bidyalaxmi: None; C. Prasad: None; S. Jayaprakash: None; S. Jain: None; B. Saikia: None; A. Sharma: None; S. Sharma: None; A. Yadav: None; S. Naidu: None; S. Jain: None.
