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OBJECTIVES@#To investigate the diversity of peripheral blood T cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) based on immune repertoire sequencing in neonates with sepsis and the possible pathogenesis of neonatal sepsis.@*METHODS@#A total of 12 neonates with sepsis were enrolled as the case group, and 9 healthy full-term infants, matched for gestational age, birth weight, and age, were enrolled as the control group. Omega nucleic acid purification kit (SQ blood DNA Kit II) was used to extract DNA from peripheral blood samples, TCR β chain CDR3 was amplified by multiplex PCR, and then high-throughput sequencing was performed for the products to analyze the diversity of TCR β chain CDR3 and the difference in expression.@*RESULTS@#The length and type of TCR β chain CDR3 were similar between the case and control groups, and Gaussian distribution was observed in both groups. With D50 and Shannon-Wiener index as the evaluation indices for diversity, the case group had a significantly lower diversity of TCR β chain CDR3 than the control group (@*CONCLUSIONS@#There is a significant change in the diversity of TCR β chain CDR3 in the peripheral blood of neonates with sepsis, suggesting that it might be associated with the immune pathogenesis of neonatal sepsis.
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Humans , Complementarity Determining Regions/genetics , High-Throughput Nucleotide Sequencing , Multiplex Polymerase Chain Reaction , Neonatal Sepsis , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
Objective@#To compare T-cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) in the patients with coexistence of HBsAg and HBsAb and other HBV infected patients. @*Methods@#The clonotype and diversity of CDR3 in blood of group cases (positive HBsAg and HBsAb) (n=11), control 1 (negative HBsAg and positive HBsAb) (n=10) and control 2 (positive HBsAg and negative HBsAb) (n=10) were analyzed by high-throughput TCR sequencing with Illumina HiseqX10. @*Results@#In the case group, the overlap rate of 6.28% (0.25%, 13.10%) was detected between any two samples, which was significantly lower than the overlap rate of 10.49% (6.20%，17.30%) seen in control 1 group (P=0.008). In control 2 group, the overlap rate of 2.60% (0.13%，13.69%) was significantly lower than control 1 group (P=0.001). There was no difference between case group and control 2 group. After pairwise comparison between the three groups, the frequency of clonotype TRBV7-2/TRBD1/TRBJ2-1 in case group was higher than that of control 1 group (P=0.029), the frequency of TRBV7-3/TRBD1/TRBJ2-7 in case group was lower than that of control 1 group (P=0.031). The difference of TRBV5-8 was significant in comparing case group with control 1 group (P=0.047). There were 14 clonotypes which had differences between case group and control 2 group in frequency. TRBV28was significant in comparing case group with control 2 group (P=0.028). For diversity, there was no difference among the three groups. @*Conclusion@#Clonotype TRBV7-2/TRBD1/TRBJ2-1, TRBV7-3/TRBD1/TRBJ2-7 and TRBV5-8 were associated with coexistence of HBsAg and HBsAb, but the diversity was not associated with TCR β chain CDR3.
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@#Periodontitis is a chronic inflammatory and destructive disease of periodontal support tissue initiated by plaque microorganisms, and its pathogenesis and progression are closely related to the immune response of the host, in which T cells play an important role in the periodontitis immune response. This article will start with the T cell immune response and the characteristics of the T cell receptor complementarity-determining region 3 (TCR CDR3) spectrum and will review the relationship between the characteristics of the TCR CDR3 spectrum and the pathogenesis of periodontitis to provide some new ideas for the studies of pathogenesis and the clinical personalized treatment of periodontitis. The study on the TCR CDR3 spectrum of periodontitis by reviewing the literature suggests that there is an oligoclonal accumulation of T cells and biased access of Variable (V) and Joining (J) genes in local lesions of periodontitis; moreover, the repeated use of nucleotide sequences and a conservative amino acid motif were found in the CDR3 region, which suggests that the characteristics of the TCR CDR3 group library play an important role in the immune pathogenesis of periodontitis.
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Objective To analyze the immunogenomic characteristics of antibody repertoire in re-sponse to influenza vaccine in order to provide a theoretical basis for further development of antibody. Meth-ods Based on a time-series immunoglobulin heavy chain ( IGH) repertoire sequencing dataset, we analyzed the immunogenomic characteristics of antibody repertoire in response to trivalent influenza vaccine ( TIV ) from three aspects which included the features in complementarity-determining region 3 ( CDR3 ) , antibody mutation and VDJ usage. Results The frequency of antibody mutation increased significantly upon vaccina-tion. Analysis of the CDR3 region indicated that polar and aromatic amino acids had a higher preference. The length of CDR3 region in naive B cells followed a normal distribution, while specific CDR3 sequences with 15 to 18 amino acids in length occupied a dominant position after vaccination. In addition, the VDJ us-age altered obviously and IGHV3-7-derived antibody had a significant response to the vaccine. Response in-tensity reached the peak on day 7 and gradually weakened over time. Conclusion Antibody repertoire evolves dynamically to express specific antibody upon vaccination and the characteristics of immune responses at sequence level could be used to evaluate their effectiveness.
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Objective:To determine the spectrum drift characteristics of CI4+CD25+Tregs TCR β chain CDR3 in patients with different phases of acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients before and after the entecavir treatment.Methods:Anticoagulation venous blood was collected from 4 normal control subjects,3 AHB patients with acute phase and convalescent phase,and 4 CHB patients before and after the entecavir treatment;and peripheral blood mononuclear cells were isolated;CD4+ CD25+ Tregs were separated by using the magnetic beads,and total RNAs were extracted from CD4+ CD25+ Tregs and used for reverse transcription.The TRBV CDR3 was amplified by polymerase chain reaction (PCR) with forward primers specific for 24 TRBV families and one fluorescence-labeled common reverse primer specific for the BC region.The PCR products were sent out for Genescan,and results were analyzed for the TRBV family CDR3 spectrum characteristics by using the Peak Scanner Software vl.0.Data were analyzed with the comparative t-test to perform the statistical analysis.Results:The CDR3 spectral types of the TRBV family showed drift characteristics in 3 cases of AHB patients with acute and convalescent phases;single/oligo peak spectral type family was observed in most of patients with acute phase;multiple peak spectral type was seen in patients with convalescent phase;and the common spectrum shift of TRBV4,10,14,16,19 families seen in patients with acute phase was changed to multiple peak spectral type.The clonal expansion of TRBV family in the CD4+CD25+Tregs in PBMC from AHB patients with convalescent phase was significantly lower than AHB patients with acute phase (t =9.456,P =0.011).The clonal expansion of Tregs TRBV13.2,15,16,18,20 family seen in C HB patients before treatment may interfere the virus removal through down-regulating the body's immune response;and with the decline of viral load in serum after the antiviral treatment,the clonal expansion of Tregs TRBV1,5.2,6,12,14,24 family may help body induce immune tolerance and result in the HBV persistence.The clonal expansion of TRBV family in the CI4+CD25+Tregs in PBMC from of CHB patients after antiviral treatment was increased (t =-0.666,P =0.553).Conclusion:TRBV4,10,14,16,19 family of spectrum shift seen in AHB patients with acute phase was changed to multiple peak spectral type in patients with convalescent phase,suggesting this transition may be associated with HBsAg and HBeAg turning to negative.The clonal expansion of Tregs TRBV13.2,15,16,18,20 family seen in CHB patients before treatment may interfere the virus removal through down-regulating the body's immune response;and with the decline of viral load in serum after the antiviral treatment,the clonal expansion of Tregs TRBV1,5.2,6,12,14,24 family may help body induce immune tolerance and result in the HBV persistence.
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Objective To analyze the restricted usage of TCR BV,BJ subfamily and their sequences in patients with breast cancer.Methods The total RNA was extracted from tissues of lymph node metastasis,then reverse transcripted.The complete DNA sequence of TCR β-chain was amplified by multi-PCR.The recombinant plasmids were sequenced by using online TCR resources.Results 5 TCR β-chain CDS were obtained in two patients.TCR β-chain presented specific repertoire skewing in metastatic lymph node.There were selected usage of BV 2,BV 14,BV 29S1 of BV subfamily and BJ1S1,BJ2S2,BJ2S3,BJ2S5 of BJ subfamily.The clonal proliferation T cells had different CDR3 amino acid sequences.Conclusions There are selected usages of TCR BV,BJ subfamily in patients with breast cancer.The sequences of CDR3 are mostly different in TCR clone proliferation.
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Objective To analyze the restricted usage of TCRβ V/J subfamily and CDR3 repertoire in patients with peripheral T-cell lymphoma unspecified (U-PTL).Methods The total RNA was respectively extracted from lymph node of U-PTL and reverse transcriptase,then multi-PCR was used to amplify the complete DNA sequence(CDS) of TCR β-chain.The recombinant plasmids were sequenced and sequence was analyzed by using online TCR resources.Results There were 9 TCR β chain CDS obtained from four patients.TCRβ-chain presented specific repertoire skewing in patients with U-PTL.There were restricted usage of BV2,BV4S2,BV14,BV29S1 of BV subfamily and BJ1S4,BJ2S3,BJ2S5,BJ2S7 of BJ subfanily.The clonal proliferation T cells had different CDR3 amino acid sequences.Conclusions There are restricted usage of TCR β V/J subfamily in patients with U-PTL.The sequences of CDR3 in different TCR clone proliferation are mostly different.
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Objective To assess the preferential expressions of peripheral blood T cell receptor beta chain variable region (TRBV) subfamilies in patients with psoriasis vulgaris(PV), and to estimate their role in the pathogenesis of psoriasis. Methods Thirty-three upstream primers were designed to target the human functional TRBV genes, downstream primers to target the common T cell receptor beta constant (TRBC) gene,with T cell receptor alpha constant (TRAC) gene as the internal reference. Total RNA was extracted from the peripheral blood T cells of 10 health human controls and 10 patients with PV, and transcribed into cDNA.Then, TRBV genes were amplified by real-time fluorescence quantitative PCR (RFQ-PCR) and the fluorescence intensity of each samples was detected. The expression levels of TRBV genes in the control group were used to calculate the cut-off values (mean expression levels of TRBV subfamilies in the 10 normal controls + 3 standard deviations). When the expression level of a TRBV subfamily from patients with PV was equal to or higher than the cut-off value, it was considered as the preferentially expressed TRBV subfamily. Results The threshold cycle (Ct) value varied from 21 to 24 for TRAC gene. The difference in the Ct value between TRBV subfamily genes and TRAC gene in patients with PV was 2.98 for TRBV2 gene, 3.24 for TRBV5-7 gene, 2.52 for TRBV6-6/6-9 gene, 2.04 for TRBV 12 gene, 3.56 for TRBV 24 gene, and 4.12 for TRBV 29 gene, and the expression levels of these subfamily genes were significantly higher than those in the normal controls (all P ＜ 0.05). According to the above standard, TRBV6-6/6-9, TRBV12 and TRBV29 were considered to be preferentially expressed subfamilies. Conclusions There is a preferential expression of TRBV gene subfamilies in peripheral blood of patients with psoriasis vulgaris, which may play a vital role in the abnormal T cell-mediated immune responses in psoriasis.
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Objective: To analyze the changes of the complementarity-determining region 3 (CDR3) of T-cell receptor (TCR) beta chain variable region (TCR BV) in peripheral blood mononuclear cells (PBMCs) of patients with pemphigus vulgaris (PV), so as to understand the association of proliferated T cells with autoimmune diseases. Methods: Immunoscope spectratyping technique was used to analyze the distribution of TCR β chain CDR3 in 6 normal blood donors and the dominant CDR3 in the PBMCs in 6 PV patients. Results: The spectratypes of TCR BV subfamily CDR3 region was in a Gaussian distribution manner in all the 6 normal blood donors. The 6 PV patients, however, displayed abnormal proliferation, and oligoclonal expansion of the T cells was observed in TCR BV families with different CDR3 sequences. Conclusion: The abnormal spectratypes of TCR BV subfamily CDR3 region in the peripheral blood of PV patients might be related to the pathogenesis of PV.
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Humanized monoclonal antibodies(mAbs) are increasingly widely used in targeted therapy for cancer and some other major diseases.Complementarity-determining region(CDR) grafting makes quantities of humanized mAbs available.Herein,we provide an overview on the strategy and progress of CDR grafting.
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Objective:To establish the technique for real-time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-PCR)by DNA melting curve analysis for detecting the CDR3 shewing of TCR alpha gene repertoire in human peripheral blood.Methods:Total RNA of peripheral blood mononuclear cell(PBMC)from 4 healthy donors and 2 patients with lymphomatous leukemia were transcripted reversely into cDNA.The cDNA of 32 TRAV gene family CDR3 was amplified by FQ-PCR.Analysis of the monoclonal/oligoclonal/polyclonal CDR3 spectratyping with DNA melting curve.Results:The FQ-PCR products of 32 TRAV family CDR3 were showedas a blur land at the predicted of products size in healthy donors and parts of TRAV family CDR3 products disappeared in patients on 1.5% agarose gel by Gold-View staining.The 32 TRAV family CDR3 were showed with different frequencies by relative fluorescence quantitative in healthy donors and the patients.The CDR3 spetratyping for 32 TRAV families was showed as polyclonal peak(Gaussian distribution)in healthy donors but showed as different monoclonal/oligoclonal/polyclonal peak in the patients with lymphomatous leukemia with DNA melting curve analysis(we called "melting curve spectratyping of CDR3")Conclusion:The study suggests that the technique of "FQ-PCR with DNA melting curve analysis be convenience and celerity for detecting the CDR3 skewing of TCR alpha gene repertoire in human peripheral blood.
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Objective:To establish a method of multi-PCR to amplify the complete DNA sequence (CDS) of TCR ? and ? chain of the antigen-specific T lymphocytes in local pathologic specimen of active pulmonary tuberculosis patients, and to analyze ?/? T cell receptor gene rearrangement and CDR3 repertoire.Methods:The lymphocytes in bronchoalveolar lavage (BAL) of active pulmonary tuberculosis patients were separated. Following total RNA extraction, cDNA synthesis, Multi-PCR, recombinant clones construction, and sequencing, the CDS of TCR ? and ? chains from these lymphocytes were analyzed by using software of DNAstar and internet TCR resources.Results:24 of ? chain CDS and 13 of ? chain CDS from 3 samples of BAL were obtained. As for TCR ? chain, AV1S2 (54%), AV12S3 (41%), and AV12S2(5%) appeared frequently. BV2(38%), BV29S1(46%), BV14(3%), and BV4S2(3%) in TCR ? chain appeared more often. There were CDR3 diversities between samples and even in the same sample by amino acid sequence analysis, but there were a few identical or similar amino acid sequences. There was the same amino acid sequence of SVGTGTLHQETQY in CDR3 region of ? chain of BAL sample No.1 and No.2; The sequence of AVRDWAGNMLT appeared in two ? chains of BAL sample No.2 and No.3; Moreover, the sequence of AV…DNN…RLM appeared in ? chains of BAL sample No.2 and No.3.Conclusion:A method of Multi-PCR is used to amplify TCR ? and ? chain CDS of tuberculosis patients. There are characteristic T cell clones to proliferate,with TCR ? and ? chain repertiore skewing in local infective focus. The sequences of CDR3 in different TCR clones are mostly different but there are a few identical or similar sequences in the same patient or even between different patients. The identical amino acid sequences of CDR3 are possibly specific for recognizing MTB polypeptide.