Clinical Relevance Of Lymphocyte Subsets And TCR α/β-chain CDR3 Repertoire Analysis In Pediatric Graves’ Disease

Part I Clinical Relevance of T Lymphocyte Subsets in Pediatric Graves’Disease in Pediatric Graves’ DiseaseObjectives:To study the clinical relevance of lymphocyte subsets in peripheral blood in children with Graves’ disease(GD),and to explore the effects of lymphocyte subsets in the pathogenesis of different GD subgroups and their relationship with pathogenesis.Methods:We studied the lymphocyte subsets in peripheral blood of 133 children with GD,27 boys and 106 girls,with age ranged from 2.4 to 15.1 years old.We collected their clinical characteristics,including onset age,gender,physical signs,with or without Graves ophthalmopathy.We also recorded the results of thyroid function tests,liver function,auto-antibodies,and percentages of lymphocyte subsets,as well as the time period needed for FT4 comes back to normal after treatment.We repeatedly divided the patients into two groups according to different clinical characteristics(the presence or absence of Graves’orbitopathy(GO),abnormal or normal alanine aminotransferase(ALT)levels,TRAb levels,TPOAb levels,ANA levels,and the the time period needed for FT4 comes back to normal after treatment),and compared the lymphocyte subsets measurements between opposite groups.Results:1.We detected higher percentages of CD3-CD19+cells and higher ratio of CD4/CD8 in GO patients.However,the percentages of CD3+CD8+were significantly lower than those in non-GO group(P<0.05).2.We found that the percentages of CD3+CD8+in ALT-abnormal group was significantly higher than that in ALT-normal group,and CD4+/CD8+was significantly lower than that in ALT normal group(P<0.05).3.We found that CD3+CD4+(%)of TPOAb high titer group,middle titer group and low titer group were 37.08±6.55,33.21±6.33 and 34.09±5.66,respectively.The highest level was found in high titer group,and the difference between high titer group and middle titer group was statistically significant(P<0.05).4.No differences of lymphocyte subsets were found between groups with different TRAb or ANA levels,nor between groups with different FT4 recovery time(P>0.05).Conclusion:1.In children with GD,the proportion of B cells and CD4+/CD8+in children with GO is higher than that non-GO group,while the proportion of CD8+T cells is significantly lower than that in non-GO group.2.GD children with liver dysfunction had higher percentage of CD8+T cells,which may suggest that CD8+T cells participate in the pathogenesis of liver dysfunction of GD.The level of serum ALT was related to the proportion of CD8+T lymphocytes,and the correlation was not affected by other factors.3.GD patients with higher TPOAb levels had more severe hyperthyroidism and higher percentage of CD4+T lymphocytes.The unbalance of CD4+T lymphocytes may aggravate GD.Part Ⅱ TCR α/β-chain CDR3 repertoire analysis in Pediatric Graves’DiseaseObjectives:To sequence the TCR α/β-chain CDR3 repertoire via high throughput method in children with Graves’ disease,explore the diversity and specialty of TCR α/β-chain CDR3 repertoire of GD children.The study may clarify the role of T cells in the pathogenesis of GD,and provide basis for future development of immune molecular target therapyMethods:We selected 5 GD girls and five healthy controls,who are all before puberty(6～8 years).We get Peripheral blood mononuclear cell from the subjects,cultured and isolated T lymphocytes.Then we extracted total RNA from the T cells,amplified the CDRs of TCRs using multiplex-PCR,and the sequenced them via NGS method.Results:1.The TRAV genes used in the GD group with highest frequencies are TRAV38-2/DV8,TRAV21 and TRAV13-1,which frequencies are 11.2%,8.7%and 8.5%,respectively.The TRAV gene usages of GD group are quite different from the controls.The TRBV genes used in the GD group with highest frequencies are TRBV5-1,TRBV20-1 and TRBV12-3 which frequencies are 18.0%,8.1%and 6.1%,,respectively.The TRBV gene usages of GD group are also quite dif ferent from the controls.2.The amino acid length of CDR3 region of GD and control groups are similar,and both with normal distribution.3.The fractions of TCR α/β-chain CDR3 region are quite different among subjects,but not various among GD and control groups.4.Totally,151417 TCR α-chain CDR3 sequences have been detected,among with 22 which mean fraction higher than 0.001,and 16 only expressed in GD group.Similarly,171712 TCR β-chain CDR3 sequences have been detected,among which 20 with mean fraction higher than 0.001,and 19 only expressed in GD group.Conclusions:1.GD group had quite different TCR α/β-chain gene usages,which indicates the abnormality of TCR repertoire in GD children.2.The amino acid length of CDR3 region of GD and control groups are similar,which suggests that TCR CDR3 length can’t be used to characterize the diversity of TCR repertoire of GD children.3.There are 16 high fraction CDR3 sequences in TCR α-chain and 19 high fraction CDR3 sequences in TCR β-chain,which do not exist in controls.These sequences can be used to find out the GD specific TCRs and their corresponding epitope.