Regulatory T cells (also referred to as suppressor T cells) constitute a cellular subset within the helper T cell group and described as an important components of the homeostasis of the immune system. Naturally occurring CD4+CD25+ regulatory T cells (nTregs) which represent 5-10% from the total human lymphocytes count have received attention due to their immunosuppressive properties both in vitro and in vivo, but in several instances it has been shown that CD4+CD25– T cell populations also contain potent regulatory activity. CD4+CD25+ nTreg cells have the phenotype CD4+CD25+Foxp3+, and express other markers such as CD25, LAG-3, CTLA-4, GITR, CD127 and others.These cells have been shown to play an important role in containing inappropriate and excessive immune activation.Their suppressive effects are mediated by cell-to-cell contact, APC-contact, cytokine dependent, and through CD37-adenosine pathway.The aim of this study is to study the role of CD4+CD25+ nTreg cells (controlling by the transcriptional factor Foxp3 and the cell surface marker LAG-3) in reversing the autoimmunity in rheumatic heart disease (RHD) which occurred as a result of acute post-streptococcal rheumatic fever (RF). Also, this study try to determine the role of CD4+ T cells, and TNF-α cytokine in enhancing the heart damage, and the role of IL-4 in preventing or reducing the autoimmune heart tissue damage and the relationship of TNF-α and IL-4 with the development and function of CD4+CD25+ nTreg cells. In addition to that, we study the role of streptococcal M protein in the development and function of nTregs and CD4+ T cells in rheumatic carditis. Two groups of patients were included in this study; the first one was composed of 48 patients with a clinical and physical diagnosis by the specialists of chronic rheumatic heart disease (CRHD) all of them were selected from Ibn-Al-Bitar for cardiac surgery hospital as the first basic group, and the second one composed of 20 patients diagnosed with acute tonsillitis were selected from Al-Kadhimya teaching hospital for the isolation
of group A Streptococcus pyogenes bacteria to extract the M protein. Immunofluorescent test (IF) was used for the detection the number of peripheral blood CD4+CD25+ nTreg cells and CD4+ T cells by using antihuman CD4 (FITC), CD25 (PE) proteins, (dual color). Whereas, In Situ hybridization test was used for the detection of TNF-α protein expressed on the surface of mononuclear cells which infiltrates the mitral valve of CRHD patients were includes in this study. In addition to that, Immunohistochemical staining test was applied on mitral valve tissue sections for the detection of IL-4 and the transcriptional factor Foxp3 which known as a master switch for CD4+CD25+ nTreg cells function and development. Also, this study include functional part in addition to the detection tests above, this part included 7 patients were selected from the total number of CRHD patients. This part study the role of CD4+CD25+ nTreg cells in streptococcal M protein-activated CD4+ T cells suppression. Also, this part study the role of the lymphocyte activation gene 3 (LAG-3) in the function and development of CD4+CD25+ nTreg cells by using anti-LAG-3 blocker
in a special cell culture system. NTregs and CD4+ T cells were isolated from the peripheral blood of CRHD patients by using Magnetic Cell Separation System-MACS technology. Streptococcal M protein was extracted from Streptococcus pyogenes bacteria by using nitric oxide method. Culture systems were supplemented with IL-2 to activate the proliferation of these cells. Cellular proliferation was detected by IF test, whereas, ELISA test was used for the determination of TNF-α and IL-4 cytokine in culture supernatant. Statistical analysis was performed by using SPSS 10.01 system and Excel-2003. The study samples were included 25 females and 23 males with a mean age of 18 years old. From the total lymphocyte count, our results were displayed lower predominance of CD4+CD25+ nTreg cells in the peripheral blood of CRHD patients and increasing the number of CD4+ T cells especially in high risk group patients without continuous medication [(1.45%) and (65.89%)] respectively and high significant negative association was found between the mean percentage of nTregs and CD4+ T cells in all study groups (p < 0.05). There was no correlation between CD4+CD25+ nTreg cells number and the clinical picture of disease. Histopathological studies were displayed high percentage of TNF-α positive cells especially in groups without continuous medication [single attack- (61.33%), and high risk-(80.33%)] and lower predominance of IL-4 cytokine
production in all groups under study. Foxp3 detection results showed that there was different degree of immunostaining signals (weak positive, positive, and strong positive signals), and there were very low numbers of strong positive Foxp3 signal especially in high risk patients without medication (2.38%) which represents the naturally occurring CD4+CD25+ nTregs, whereas weak positive and positive Foxp3 cells were found in high numbers which represent the activated non-suppressor CD4+ T cells which infiltrate the mitral tissue. High significant negative association was found between the mean percentage of peripheral blood CD4+CD25+ nTreg cells and TNF-α positive cells in tissue sections [(χ 2 = 13.694), (p < 0.05)].
Culture system results were displayed that both CD4+ T cells and CD4+CD25+ nTreg cells were stimulated by streptococcal M protein and IL- 2 displayed increasing in the proliferation of these cells. Also, there was
found that nTreg cells has the ability to suppress the proliferation and TNF-α cytokine production in mixed culture system of both nTregs and CD4+ T cells, but there was found that the massive expansion of nTregs due to the addition of M protein lead to inhibit the suppressive function of these cell against CD4+ T cells which led to increase TNF-α production, but there was found that TNF-α has inhibitory effect on CD4+CD25+ nTreg cells function. Culture systems results were displayed that there was no or very little
production of IL-4 (<4 pg/ml) from CD4+ T cells in all culture system of all cases. Anti-LAG-3 monoclonal antibody was found to inhibit the suppressive function of CD4+CD25+ nTreg cells, therefore, high TNF-α cytokine production was recorded as a result of increasing the proliferation of CD4+ T cells in most of cultures were supplemented with LAG-3 blocker. In conclusion, results were obtained from this study revealed the important role of CD4+CD25+ nTreg cells in suppress the proliferation and cytokine production of CD4+ T cells in CRHD patients and that the function of these regulatory cells were affected to a large degree by the presence or absence of LAG-3 protein expression on the surface of nTreg cells. Also, this
study was displayed the important role of TNF-α in enhancing the heart damage in an opposite picture for the role of IL-4 cytokine, and the role of TNF-α in inhibiting the suppressive function of CD4+CD25+ nTreg cells. In addition to that streptococcal M protein was found to play a crucial role in the immunopathogeneic processes underlining RHD. For better understanding to the role of CD4 + CD25+ nTreg cells in reversing the autoimmunity, furthered studies may answer many of questions which may still remains without full explanations.