Rheumatoid arthritis: early thymic involution or increased proliferation rate?

Abstract

Event Abstract Back to Event Rheumatoid arthritis: early thymic involution or increased proliferation rate? Elena A. Blinova1*, Vladimir S. Kozhevnikov1, Alexey E. Sizikov2 and Vladimir A. Kozlov1 1 Research Institute of Clinical Immunology Russian academy of medical science Siberian Branch, Clinical department, Russia 2 Immunology Clinic of Research Institute of Clinical immunology Russian academy of medical science Siberian Branch, department of Rheumatology, Russia It is often observed impaired thymic function in adults with immunopathology, particularly with rheumatoid arthritis (RA). Loss of naive and memory T cell diversity, oligoclonal proliferation, restricted T cell receptor repertoire, emergence of CD28null T cells, and erosion of telomeres in circulating T cells are typical features of the immune disfunction in the eldery and although in RA patients [1,2]. Therefore, it was hypothesized that patient with RA have premature immunosenescence, beginning with early thymic involution. One of the way to asses recent thymic emigrants is a direct analysis of T cell receptor excision circles or TREC level [3]. But TREC number can be influenced by peripheral expansion or cell death. Recently, CD31 has been proposed as a cell surface marker expressed preferentially by naive TREC-rich T cells that have undegone low number of cell divisions [4]. In this study, we determined 2 markers of thymic functional activity (TREC number and amount of CD31+ T cells) to establish the mechanisms for replenishment of the peripheral T cell pool in RA. Absolute and relative TREC number in CD4+ and CD8+ cells was measured in 30 patients with RA (mean age 50±8,2) and 15 healthy age-matched controls using Real-time PCR. Reaction mixture in volume 25μl contained 5μl eluted DNA, 200μM dNTPs, 200 nM each primers and fluorescent probe, and 2 units HotStart Taq DNA polymerase. In each experiment, serial dilutions of a cloned TREC-PCR product were used as a standard for absolute quantification of TREC level. Before DNA purification mononuclear cells from peripheral blood were sorted to CD4+ and CD8+ subsets by cell sorter FACSAria. Reanalysis of the isolated subsets showed that the purities were at least 95%. Proportion of CD4+CD31+ and CD8+CD31+ cells was quantified in 11 patients with RA (mean age 43±14,3) and 5 healthy donors (mean age 43±5,5) using flow cytometry. Statistical analysis was performed using Wilcoxon/Mann-Whitney test for unrelated pairs. A p value of <0,05 was considered significant. Results are shown as median with 25th and 75th percentiles in parenthesis. Relative TREC number was reduced about 2 times in RA compared with control: in CD4+ cells it came to 1650 (range 976-2800) vs 3700 (range 1050-5800) and in CD8+ cells - 1500 (range 838-2400) vs 4200 (range 719-8500). Absolute TREC number in CD4+ population did not differ in patients with RA from donors, whereas it was decreased in CD8+ population in RA. Patients with RA have low levels of serum IL-7, which leads to competition between CD4+ and CD8+ lymphocytes and perhaps, asymmetry in the absolute TREC number in this populations. To determine whether the reduction of TREC number in RA due to insufficient thymic function or increased proliferation rate, it was quantified proportion of CD31+ T cells. Some authors have reported that the proportion of CD31-positive CD4+ and CD8+ cells decreased with age and correlated with thymus volume [5, 6]. Moreover, CD31+CD45RA-CD45RO+ cells, comprising less then 5% CD4+ cells, did not contain TREC. All this makes CD31 as a marker of thymic naive T cells. There were no significant differences in the amount of thymic naïve T cells between patients with RA and healthy donors. CD4+31+ value in RA group was 26 (range 21,6-32,2) vs 27,8 (range 24,9-40,1) in control group, and CD8+CD31+ value – 62 (49,7-70,8) vs 53,8 (50,8-66,5) respectively. Obtained data suggest that replenishment of the peripheral T cell pool in adults with RA occurs more due to the cell proliferation, thymic function is maintained within the normal limits. Acknowledgements The authors are grateful to Evgeniy Khrapov for help in the development of a standard for quantitive measuring TREC.