الفهرس 
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Abstract
RA is a systemic, chronic inflammatory disease mostly affecting the joints, it affects as much as 1% of the population and is thought to be complicated and multifarious, impacted by both genetic and environmental factors. By using GWAS, more than 100 RA susceptibility loci—including HLA-DRB1—have been found to be involved in RA susceptibility. According to the ACR, the diagnostic approach for RA usually entails clinical evaluation (tender and swollen joints), laboratory testing (RF, Anti-CCP, CRP, and ESR), and imaging examinations. A dysregulated immune response is a part of the immunopathogenesis of RA, which results in joint destruction and persistent inflammation. It is distinguished by the generation of pro-inflammatory cytokines including TNF-α, IL-1, and IL-6 as well as the activation of different immune cells such T-cells, B-cells, and macrophages. Different strategies of RA treatments aim to control and reduce inflammation, relieve symptoms, keep the patient in a remission state and prevent any joint and cartilage destructions. It typically involves a combination of pharmacological agents (DMARDs, NSAIDs, glucocorticoids, and biologics) and non-pharmacological interventions (physical therapy and patient education). MHC II tetramers are artificial molecules made up of four MHC II molecules coupled to a certain peptide and tagged to a fluorophore. They help identify and examine T cells that are specific to an antigen in RA patients. Labelling CD4+ T-cells with MHC II tetramers can help in better understanding of the mechanisms underlying the onset, progression, and response to treatment of RA by detecting and quantifying distinct CD4+ T-cell populations involved in the autoimmune response. This technique can also aid in the identification of the specificity of antigens that trigger the disease. Additionally, it offers a new method for developing antigen-tolerizing therapeutic strategies.
In the current study, we have demonstrated citrulline-reactive CD4+ T cells in RA patients and compared them to HVs using a multi-tetramer method (MHC class II) and a multiparameter flow cytometry panel.
Importantly, we have addressed several citrullinated peptides that have been identified as potential autoantigens for RA induction, including vimentin, α-enolase, and CILP. Furthermore, flu peptides were used as a positive control, with two peptides for each antigen loaded on two distinct HLA-DR*04:01 tetramers.
This study included 31 participants, 22 of whom were RA patients and 9 of whom were age- and sex-matched HVs. The serum level of Anti-CCP was measured (by indirect ELISA) using venous blood samples taken from each individual. Illumina performed genotyping on DNA samples to determine HLA-DRB1*04:01 positive samples, and multiparameter flow cytometry was utilised to enumerate, detect and immunologically phenotype citrulline-reactive CD4+ T-cells (after extraction using peptides-MHC II tetramers technology and magnetic bead isolation).
For gathering demographic data, we divided the participants into two age groups: < 50 and ≥ 50. Among the HVs and RA patients, we discovered that most of them were older than 50. Results of the study indicated that, in terms of gender distribution, RA is 3–4 times higher in females than in males.
When comparing the frequencies of each antigen specific CD4+ T-cells detected using MHC II tetramers technology, we found no statistically significant difference between the frequencies of each peptide-specific CD4+ T-cells when compared individually among HVs and RA patients while individual frequencies of each citrulline specific-CD4+ T-cells tended to increase in RA patients. Additionally, the cumulative frequencies of all citrulline specific-CD4+ T-cells were statistically significantly higher in RA patients than HVs.
According to our findings, there was a significant positive correlation between age and the total frequencies of all CD4+ T-cells specific to citrullinated Ags in RA patients aged 50 years or older. Additionally, patients aged ≥ 50 years old had higher frequencies of CD4+ T-cells specific for Flu, CILP, α-enolase, and vimentin than the patients that were < 50 years old.
We also discovered that ACPA positive RA patients had notably greater frequencies of CD4+ T cells specific to citrullinated antigens compared to ACPA negative RA patients. Vimentin+ CD4+ T-cell frequencies and CILP+ T-cell frequencies also showed a positive correlation with anti-CCP titers, with the latter showing a statistical significance.
We managed to identify citrullinated antigens-specific CD4+ T-cells and found that they tended to be memory cells (CD45RO+) while the general CD4+ population in the body was naïve cells (CD45RO- CCR7+). We didn’t find a significant population of Tph and Tfh cells in the PB of our RA patients.
By further analysis, we found that compared to the general CD4+ population, citrullinated antigens-specific CD4+ T-cells are activated cells with different stages of activation. CILP specific CD4+ T-cells seemed to be more at early-stage of activation (HLA-DR+ CD27+) and don’t express CD45RO. While vimentin-specific CD4+ T-cells were more chronically activated (HLA-DR+ CD27-) and seemed to became memory cells (CD45RO+).
In summary we are sure that more data investigation needs to be done on larger number of RA patients with including different markers and more data about different disease stages to be able to determine the exact CD4+ T-cells population that influence pathogenesis of RA and hence could be targeted in the future by new therapeutic approaches.