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Objective:To investigate the diversity of T cell receptor repertoire in patients with Takayasu arteritis and analyze the side chain gene expression and distribution pattern of V、J gene rearrangement of T cell receptors.Methods:The peripheral blood samples of 8 patients with Takayasu arteritis and 4 healthy controls were collected. After constructing the library, high-throughput sequencing was performed with Illumina hiseq X10 sequencer. Bioinformatics analysis was conducted to obtain the sequences and compared with the reference sequences. the frequency information of V/D/J genes, and extraction of CDR region sequenceswere compared. The diversity of the TCR repertoire was also evaluated, and the comparative analysis and cluster analysis between groups and within samples were carried out. The data were analyzed by R language statistical software. Comparisons between two groups were analyzed by Mann-Whitney U test. Results:There was no significant difference in D50 index and Shannon entropy of chain CDR3 between Takayasu arteritis group and healthy control group. There was no significant difference in high-frequency cloning between the two groups. However, a total of 21 gene rearrangement fragments were different between the two groups. The expression of 14 V/J gene rearrangement fragments such as TRBV15-TRBJ2-3 [0.31 (0.27, 0.70) ], TRBV26-TRBJ2-6[0.30 (0.23, 0.57) ], TRBV28-TRBJ1-4[179 (139, 412) ], TRBV28-TRBJ1-6[362 (253, 419) ] in the patient group was significantly higher than that in the control group ( Z score were 2.65, 2.08, 2.27, 2.27, 2.27, 2.08, 2.65, 2.08, 2.27, 2.27, 2.08, 2.08, 2.46 and 2.22 respectively, P<0.05). The expression of seven V/J gene rearrangement fragments such as TRBV10-1-TRBJ1-2 [7.49 (4.9, 12.1) ],TRBV29-1-TRBJ2-2[10.5 (4.0, 12.8) ], TRBV-4-2-TRBJ2-6 [3.31 (1.8, 5.8) ] in the patients with Takayasu arteritis group was significantly lower than that in the control group ( Z score were -2.08, -2.27, -2.08, -2.08, -2.27, -2.08 and -2.29, P<0.05). Conclusion:Although there is no significant decrease in the diversity of peripheral blood TCR repertoire in patients with Takayasu arteritis, there are differences in the expression of chain V and J genes of TCR genes, and there is unique V/J rearrangement clones.
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Chimeric antigen receptor T cell (CAR-T) therapy, a novel immunotherapy, shows great potential in the treatment of hematological tumors to conventional therapies. Great progress has been made over the past few decades in the treatment of relapsed/refractory acute/chronic lymphocytic leukemia. However, due to the lack of real-time monitoring methods, it is impossible to predict and assess the therapeutic effect during the treatment of blood tumors, and we cannot learn more about the complications and risks. Many challenges exist in the clinical transformation of CAR-T therapy. As a non-invasive method, molecular imaging shows promise on real-time visualization of the biological behavior of CAR-T in vivo. Tracking CAR-T by directly labeling or indirectly evaluation by reporter gene methods has achieved breakthroughs. This article reviews the current situation of monitoring systems of CAR-T therapy and future expectations for each of the methods presented.
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Objective:To investigate the characteristics of changes in peripheral blood regulatory T lymphocyte (Treg) levels in patients with B-cell lymphoma who received chimeric antigen receptor (CAR)-T cell immunotherapy, and the relationship between Treg levels and optimal efficacy and treatment response.Methods:The data of 23 patients with relapsed/refractory B-cell malignancies who received CD19/CD22 CAR-T cell immunotherapy in Wuhan Tongji Hospital from 2019 to 2021 were retrospectively studied. The enrolled patients were divided into complete remission (CR) group (8 cases), partial remission (PR) group (7 cases) and no response(NR) group (8 cases) according to Lugano′s revised lymphoma efficacy evaluation criteria. A total of 16 patients with B-cell lymphoma who did not receive CAR-T cell immunotherapy during the same period in Wuhan Tongji Hospital were collected as the control group.In different periods during CAR-T cell immunotherapy, multicolor flow cytometry(MFC) was used to dynamically detect peripheral blood the proportion of Treg in CD4 +T cells (Treg/CD4 +T), the proportion of lymphocytes (Treg/Lym), the proportion of Treg in white blood cells (Treg/WBC), and the absolute number of Treg (Treg#). The trend of Treg levels over time, as well as the differences in Treg levels in patients with different prognosis groups in different periods were analyzed.According to the proportion of Treg and the median level of absolute number within 1 to 15 days after CAR-T cell infusion, the patients were divided into a low-level group with 11 cases and a high-level group with 12 cases. The statistical differences in the peak value of CAR-T copy, iron protein, and IL-6 were compared between various groups. Independent samples t test, Mann-Whitney U test, Cox-Stuart trend existence test and one-way analysis of variance was used in statistical analysis. Results:In the 23 patients who received CAR-T cell immunotherapy, the mean values of Treg/CD4 +T and Treg/Lym before CAR-T cell infusion were (20.42±7.96)% and (13.61±7.13)%, respectively, which were significantly higher than that of the control group [(7.33±3.61)%, t=5.893, P<0.001; (1.91±0.90)%, t=6.53, P<0.001]. The number of Treg in the meantime was significantly lower [(1.81±1.52)/μl<(13.66±9.89)/μl, t=4.261, P<0.001]. After infusion, Treg/CD4 +T and Treg/Lym all remarkably decreased ( P<0.001),Treg/WBC increased significantly( P=0.01). The mean values of Treg/CD4 +T (12.87±1.93)%, Treg/Lym (6.35±2.84)%, and Treg/WBC (0.05±0.05)% in the patients with CR as the best response group were lower than those in the PR group [(29.68±5.49)%( P<0.01), (21.85±2.1)%( P<0.01), 0.50±0.69( P<0.05)] before CAR-T cell immunotherapy. Patients with lower mean Treg/CD4 +T within 1 to 15 days after reinfusion of CAR-T cells had higher peak CAR-T copy number ( P<0.05). Conclusion:Treg/CD4 +T and Treg/Lym were increased and then decreased during CAR-T treatment in B cell malignancies. The patients with lower proportions of Treg before infusion have favorable treatment efficacy. Besides, patients with lower Treg/CD4 +T after infusion have better CAR-T cell expansion. In the process of CAR-T cell immunotherapy, the use of MFC to dynamically monitor the proportion of Treg has certain clinical significance for the prediction of the optimal efficacy of immunotherapy and the prediction of treatment response.
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Objective:To explore an assay that can concisely, rapidly, and accurately quantify the amount of chimeric antigen receptor (CAR)-T cells in the bone marrow or peripheral blood of patients after CAR-T cell immunotherapy by morphological analysis and flow cytometry assay, providing timely and accurate feedback for clinical treatment.Methods:We analyzed the CAR-T cell detection results in peripheral blood and bone marrow of 256 patients who received CAR-T cell immunotherapy in the Department of Hematology, Affiliated Hospital of Xuzhou Medical University from August 2016 to August 2021. All 256 patients survived more than one month after CAR-T cell infusion. Among them, there were 118 patients with multiple myeloma, 68 patients with acute lymphoblastic leukemia, and 70 patients with lymphoma. The morphological characteristics, positive rate and detection rate of CAR-T cell in peripheral blood and bone marrow were analyzed by morphological methods. The positive rate and detection rate of CAR-T in peripheral blood and bone marrow were analyzed by flow cytometry protein L detection. χ 2 test was used to comprehensively analyze the difference between the detection rate of the combined analysis of the two methods and the detection rate of the single method. Results:CAR-T cells have significant morphological characteristics, and there are obvious morphological differences from normal lymphocytes. The detection rates of CAR-T cells in peripheral blood or bone marrow by morphological methods and flow cytometry were 88.28%(226/256) and 79.29% (203/256), respectively. When the two methods were combined, the detection rate of CAR-T cells can reach 99.22%, with statistically significant difference comparing to that of single method( P<0.05). Through the analysis of the detection results of peripheral blood at different time points, it was found that the average detection rates of morphology and flow cytometry in 118 patients with multiple myeloma were 9.50% and 10.23% on the 7th day, and 13.50% and 15.19% respectively on the 15th day. On the 21st day, the average detection rates of morphology and flow cytometry were 8.00% and 10.07%, respectively. The average detection rates of morphology and flow cytometry in 68 patients with acute lymphoblastic leukemia were 12.00% and 11.22% on the 7th day, and 21.00% and 23.10% respectively on the 15th day. On the 21st day, the average detection rates of morphology and flow cytometry were 13.50% and 10.91%, respectively. The average detection rates of morphology and flow cytometry in 70 lymphoma patients were 7.50% and 10.35% on the 7th day, and 9.00% and 10.35% respectively on the 15th day. The average detection rates of morphology and flow cytometry at 21 days were 6.50% and 5.69%, respectively. The number of CAR-T cells in samples from patients with different diseases reached a peak around the 15th day. Conclusion:The detection rate of CAR-T cells from peripheral blood or bone marrow was significantly higher with the combination of the 2 methods compared to the single method.
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CD19 chimeric antigen receptor T cells (CAR-T) therapy is a new immunotherapy for B-cell hematological tumors, and has good efficacy. With the increase of its use, the incidence of immune effector cell-associated cytokine release syndrome is increasing, and further clinical research on its precise mechanism and treatment is urgently needed. This review summarizes the mechanisms, clinical manifestations, grading systems, treatments and management strategies of cytokine release syndrome.
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Chronic lymphocytic leukemia (CLL) is a heterogeneous, mature B-cell clonal malignancy that mainly affects the senior. The immunotherapies such as chimeric antigen receptor T cell therapy, bi/tri-specific cell binding agent, immune check point therapy, etc., pave several new avenues for CLL treatment, especially the combined applications of new and existing therapies have shown improved efficacy and safety. This article attempts to review the immunotherapies and their combinatorial applications in CLL.
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Objective To evaluate therapeutic effect of the sphingosine-1-phosphate receptor antagonist FTY720 on the imiquimod-induced psoriasis-like mouse model,and to explore its molecular mechanism.Methods A total of 8 specific pathogen-free (SPF) female C57BL/6 mice were used in this study,and divided into experimental group (n =3) and control group(n =5):each mouse ear was topically treated with 10 mg/d imiquimod for 7 consecutive days,and the experimental group and control group were intraperitoneally injected with FTY720 and phosphate buffer saline (PBS) respectively on days 2,4 and 6.Ear skin thickness was measured every day.These mice were sacrificed on day 8,and histopathological changes of the ear skin were observed.The ear tissues and draining cervical lymph node cells were obtained.Flow cytometry was performed to analyze changes in the proportion of interleukin (IL)-17+ γδT cells in the mouse skin and draining lymph node tissues between the two above groups,and to determine the expression of sphingosine-1-phosphate receptor 1 (S 1P 1) and cutaneous lymphocyte antigen (CLA) on the surface of the γδT cells.The means of two independent samples were compared by using t test.Results After the 2-day topical application of imiquimod,the mouse ear thickness was significantly lower in the experimental group (0.217 mm± 0.003 mm) than in the control group (0.232 mm ± 0.002 mm,t =4.23,P ＜ 0.01) on day 3,and the significant difference existed till day 8 (all P ＜ 0.01).Histopathological examination of the ear skin revealed that the epidermal thickness was significantly lower in the experimental group (18.62 μm ± 0.19 μm) than in the control group (27.79 μm ± 1.58 μm,t =4.35,P ＜ 0.01).Immunofluorescence staining showed that the degree of neutrophil infiltration in the mouse ear tissue was lower in the experimental group than in the control group.As flow cytometry showed,the proportion of neutrophils was significantly lower in the experimental group (1.57％ ± 0.12％) than in the control group (3.03％ ± 0.33％,t =3.31,P =0.016).In the ear tissues,the experimental group showed significantly decreased proportion of γδT cells or IL-17+ γδT cells (4.88％ ± 0.42％,40.53％ ± 1.76％ respectively) compared with the control group (9.45％ ± 1.22％,56.56％ ± 0.66％ respectively;t =2.75,10.27 respectively,both P ＜ 0.05).In the draining lymph nodes,the experimental group showed significantly increased proportion of γδT cells or IL-17+ γδT cells compared with the control group (t =5.781,4.140 respectively,both P ＜ 0.05),and the fluorescence intensity of S1P1 and CLA in the γδT cells was significantly lower in the experimental group than in the control group (P ＜ 0.05).Conclusion FTY720 can alleviate imiquimod-induced psoriasis-like manifestations in mouse models,likely by down-regulating the expression of S1P1 and CLA in γδT cells,increasing the proportion of γδT cells in the draining lymph nodes followed by the decrease of their proportion in the skin,and decreasing the production of IL-17 in skin tissues.
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Objective To evaluate the immune status of acute rejection recipients,and to improve the short-term and long-term survival rate of renal transplant recipients and grafts,and to investigate dynamically the changes in the immune repertoire of patients with acute rejection.Methods Combined multiplex PCR amplification technique and high throughput sequencing technique,the TCR β chain complementarity determining region 3(CDR3)diversity and repertoire characteristics at different time points during renal transplantation were analyzed,in order to reveal the immunological characteristics of T lymphocytes in patients with acute rejection.Results The diversity of TCR CDR3 in acute rejection patients was reduced to the lowest one day after surgery.The diversity of TCR CDR3 before acute rejection was higher than before.The acute rejection-related upregulated TCR CDR3 amino acid sequences were screened out.In addition,TCR beta chain V and J subfamily showed the phenomenon of advantage usage in pre-acute rejection,which may be due to T cell recognition of transplanted kidney antigens in vivo.Conclusions The immune diversity of patients with acute rejection is significantly lower.In addition,TCR beta chain V and J subfamily show the phenomenon of advantage usage.
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Chimeric antigen receptor (CAR) T cell shows its significant therapeutic effect in part of leukemia and lymphomas, which indicates that transgenic adoptive cellular immunotherapy may be one of the breakthroughs in treatment of solid tumors and blood diseases. The functional inhibition of CAR endogenous cells in the tumor microenvironment , molecular biology function of molecules, difficulties of CAR T cells in clinical application and the solutions , how to improve the clinical efficacy and to reduce toxic side effects for future cancer immunotherapy in clinic are reviewed in this paper.
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T-cell-mediated drug hypersensitivity represents a significant proportion of immune mediated drug hypersensitivity reactions. In the recent years, there has been an increase in understanding the immune mechanisms behind T-cell-mediated drug hypersensitivity. According to hapten mechanism, drug specific T-cell response is stimulated by drug-protein conjugate presented on major histocompatibility complex (MHC) as it is presented as a new antigenic determinant. On the other hand, p-i concept suggests that a drug can stimulate T cells via noncovalent direct interaction with T-cell receptor and/or peptide-MHC. The drug binding site is quite variable and this leads to several different mechanisms within p-i concept. Altered peptide repertoire can be regarded as an 'atypical' subset of p-i concept since the mode of the drug binding and the binding site are essentially identical to p-i concept. However, the intracellular binding of abacavir to HLA-B*57:01 additionally results in alteration in peptide repertoire. Furthermore the T-cell response to altered peptide repertoire model is only shown for abacavir and HLA-B*57:01 and therefore it may not be generalised to other drug hypersensitivity. Danger hypothesis has been postulated to play an important role in drug hypersensitivity by providing signal 2 but its experimental data is lacking at this point in time. Furthermore, the recently described allo-immune response suggests that danger signal may be unnecessary. Finally, in view of these new understanding, the classification and the definition of type B adverse drug reaction should be revised.
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Binding Sites , Classification , Drug Hypersensitivity , Drug-Related Side Effects and Adverse Reactions , Hand , Haptens , HLA Antigens , Major Histocompatibility Complex , Receptors, Antigen, T-Cell , T-LymphocytesABSTRACT
ObjectiveTo investigate the effect of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor PD98059 on the proliferation and killing function of γδT cells cultured in vitro. MethodsMononuclear cells were separated from peripheral blood in healthy subjects and placed in RPMI 1640 complete medium containing zoledronic acid and interleukin-2 to obtain γδT cells through induction and culture. The ERK1/2 specific inhibitor PD98059 was used to block the ERK1/2 signaling pathway in γδT cells, and the proliferation of γδT cells was measured by cell counting, and the killing function of γδT cells was measured by CCK-8 method. Flow cytometry was used to measure the expression of granzyme B and perforin in γδT cells. The t-test was used for comparison of continuous data between groups. ResultsAfter 10 days of culture, the purity of γδT cells reached 87.94%±2.36%. The number of γδT cells treated with PD98059 was significantly lower than that of control cells ［(6.74±0.36)×105/ml vs (9.42±0.31)×105/ml, t=-12.708, P＜0.001］. Compared with the control cells, those treated with PD98059 had significantly higher positive expression rates of granzyme B and perforin (granzyme B: 48.89%±1.31% vs 41.58%±1.58%, t=7.582, P＜0.001; perforin: 65.92%±3.29% vs 33.49%±2.83%, t=15.478, P＜0001) and a significantly higher killing rate of HepG2 cells (69.28%±4.96% vs 48.34%±3.01%, t=11.201, P＜0.001). ConclusionThe ERK1/2 specific inhibitor PD98059 can inhibit the proliferation of γδT cells, but it can enhance the in vitro killing function of γδT cells.
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Objective To investigate the expression and clinical significance of T-cell receptor (TCR) Ⅴβ subfamily in hepatitis B virus (HBV)-related acute-on-chronic liver failure (HBV-ACLF) patients.Methods Twenty-eight patients with HBV-ACLF (HBV-ACLF group) and 32 patients with chronic hepatitis B flare (CHB-F group),who were treated in The Second People's Hospital from Oct.2010 to Mar.2012,and 20 healthy controls (HC group) were included in the study.Reverse transcriptase-polymerase chain reaction was used to detect the levels of TCR Ⅴβ subfamily and enzymelinked immunosorbent assay was used to detect the levels of serum cytokines [interleukin (IL)-2,IL-4,IL-6,IL-10,interferon (IFN)-γ and tumor necrosis factor (TNF)-α)] in the three groups.The comparison among three groups was done by one-way analysis of variance and the comparison between two groups was done by LSD-t test or rank sum test.Results The three groups had similar gender and age distribution (all P＞0.05).The HBV-ACLF group had significant different profiles of total bilirubin,albumin,prothrombin activity,international normalized ratio and cholesterol tatol compared with the CHB-F group (all P＜0.05).For patients in the HBV-ACLF group,the serum IL-2,IL-4,and IL-10 levels were lower(all P=0.000),and the IL-6 and IFN γ levels were higher than those of the HC group (all P=0.000).The IL-4,IL-10,and TNF-α levels in the CHB-F group were also significantly lower than those of the HC group (all P=0.000).Compared with the CHB-F group,the HBV-ACLF group had significantly lower IL-2,IL-10,and TNF-α levels (P=0.003,0.002,0.004),and higher IL-6 and IFN-γ levels (P=0.015,0.006).By one-way analysis of variance,there were significantly differences of △Ct1,△Ct5,△Ct7,△Ct12,△Ct15,△Ct20,△Ct22,and △Ct23 among the three groups (H=20.368,14.368,19.500,31.532,19.985,19.116,41.752 and 20.649,all P＜0.05).Conclusion The expression levels of TCR Ⅴβ subfamily and cytokines are changed in HBV-ACLF patients.
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Inflammatory bowel diseases(IBD), including Crohn's disease and ulcerative colitis, are chronic inflammatory states of the intestinal tract. While the exact mechanisms inducing chronic inflammation are still unclear, it is hypothesized that the inflammation is caused in part by an inappropriate immune response to the intestinal microflora. Although inflammatory diseases are not directly linked to patient survival, symptoms of these diseases significantly decrease quality of life. The incidence rate is higher in western people than eastern people, but the incidence rate of IBD in eastern people, including Korean, is increasing. Recently, it has been reported that IL-17 is an important factor that appears to be involved in IBD induction and progression. This report reviews many recent papers reporting the relationship between IBD and IL-17, which may provide an understanding leading to new means of prevention and treatment for IBD.
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Humans , Colitis , Colitis, Ulcerative , Crohn Disease , Incidence , Inflammation , Inflammatory Bowel Diseases , Interleukin-17 , Quality of Life , Receptors, Antigen, T-Cell, gamma-delta , Th17 CellsABSTRACT
Objective To study the T cells lineage polymorphism of TCR BV CDR3 in the peripheral blood of ankylosing spondylitis (AS) patients,in order to provide experimental basis for the immunological patho-genesis study of AS.Methods Twenty-six subfamilies of CDR3 T cells of TCR BV in the PBMC of AS patients were amplified by RT-PCR method,then TCR BV CDR3 lineages polymorphism were analyzed by immunization scanning spectrum.Results TCR BV CDR3 scanning spectrum of 20 active AS patients showed abnormal distribution peak,including monoclonal,oligoclonal/oligoclonal trend,skewing peak and irregular abnormal peak.Among them,some subfamilies of 18 patients showed oligoclonal/oligoclonal trend expansion,BV16 and BV18 two subfamilies of one case showed monoclonal expansion.Most spectral type of PBMC TCR BV CDR3 in five normal controls showed Gauss distribution.Conclusion TCR BV CDR3 lineage have significant characteristic polymorphism and spectrum drift characteristics in the peripheral blood of AS patients,which further indicate that T cells has plaied an important role in the immunological pathogenesis of AS.Monoclonal/oligoclonal expansion of T cells may be autoreactive T cells in nature and they may be involved in the pathogenesis of AS.
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Objective To investigate the relationship between TGFβ1-509 C/T, TCRCα-575 A/G SNPs and primary AAV using a transmission disequilibrium theory based pedigree analysis Methods Genotypes of 264 individuals from 88 AAV families include patients, their parents, brothers and sisters were determined by PCR-RFLP and direct sequencing. Transmission disequilibrium test(TDT) and HRR were employed for the data analysis to observe the transmission disequilibrium of TGF31-509 C/T and TCRCα -575 A/G polymorphisms. Results No transmission disequilibrium from heterozygous parents onto the patients was found in the trios analyzed by TDT for either TGFβ1-509 C/T (observed C/T = 36/28, expected C/T =33. 5/30. 5, x2 =0.51, P＞0.05) or TCRCo-575 A/G ( observed A/G = 29/39, expected A/G = 33.5/34. 5, x2 = 1. 59, P ＞ 0. 05 ). The genotype-based HRR and haplotype-based HRR showed there was no increased risk of AAV in the observed trios for either -509 C/T polymorphism of TGFβ1 (transmitted genotype CC/CT/TT =12/20/6, allele C/T = 44/32; nontransmitted genotype CC/CT/TT = 10/19/9,allele C/T =39/37, genotype-based HRR x2 =0.81, P ＞0. 05, haplotype-based HRR x2 =0. 66, P＞0. 05,HRR = 1.30) or -575 A/G polymorphism of TCRCα ( transmitted genotype AA/AG/GG = 9/18/12, allel A/G = 36/42; nontransmitted genotype AA/AG/GG = 15/15/9, allel A/G = 35/33, genotype-based HRR x2=2. 20, P ＞0. 05. Haplotype-based HRR x2 =0. 41, P ＞0. 05, HRR =0. 81 ). The deviation of HRR coefficient was not excessive(1.00). Conclusion TGFβ1-509 C/T and TCRCo-575 A/G polymorphism may not be associated with the genetic susceptibility of primary AAV in Guangxi Han population.
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Objective To analyse the spectral patterns of complementarity determining region 3 (CDR3) length distribution of T lymphocyte receptor beta chain variable (TRBV) gene families in infiltrating T cells of the liver tissues and the peripheral blood samples of patients with chronic hepatitis B (CHB) in order to evaluate the characteristics of T cell clonal expansion. Methods The spectral patterns drift of TRBV gene families (the monoclonal/oligoclonal TCR β T cells) in the peripheral blood and hepatic tissues from 11 cases of CHB patients were analyzed by the real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ-PCR) with DNA melting curve analysis, and abnormal rates of TRBV gene families were compared between CHB patients and healthy control. The comparison of rates was done by chi square test. Results The gene melting spectral pattern of 26 TRBV families of the 11 CHB patients, no matter in the peripheral blood or hepatic tissue, showed either a single peak or prominent melting peaks, even disappeared for certain TRBV families. The abnormal rate of TRBV gene families in the hepatic tissues was significantly higher than that in the peripheral blood samples (x2 = 23. 246, P＜0. 01). What is more interesting was that some parts of TRBV families were identical in both the peripheral blood and the hepatic tissue in certain patients. TCR BV13.1, TCR BV17 and TCR BV22 fragments were found to be restricted used in both the peripheral blood and hepatic tissue by some CHB patients. Conclusions T cells in the peripheral blood and the hepatic tissues of CHB patients can develop clonal expansion to some extent.Parts of TRBV families are restricted used in the peripheral blood and hepatic tissue in some CHB patients, which offers a foundation for further studying the common specific spectral drift patterns of TRBV CDR3 gene in CHB patients.
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Objective To investigate the optimized parameters of dye (SYBR Green Ⅰ) realtime fluorescent polymerase chain reaction (RF-PCR) for detecting αβT lymphocyte clones in the peripheral blood and its application in monitoring specific T cell clone in the peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis B (CHB). Methods The total RNA was extracted from the PBMC of six healthy donors, and was reversely transcripted into cDNA. Then the cDNA was amplified using RF-PCR with the primers specific for T cell receptor β viable region (TCRBV) gene families as upstream primers and the primer for T cell receptor (TCR) β constant region (TCRBC) as downstream primer. The annealing temperature,concentration of primers and the total number of cycles were comparatively analyzed. The optimized PCR was performed to investigate the 24 TCRBV gene families from 12 patients with CHB, and the PCR products were monitored by melting curve analysis, and the clone expansion of peripheral blood T cell was detected by peak-motif of melting curve analysis. Results The optimized annealing temperature, final premier concentration,the number of cycles were 60.6 ℃, 0.5 μmol/L and 40 cycles, respectively. The begin temperature for melting curve analysis was better as 80 ℃ compared to 75 ℃. There was mono-peak on melting peak chart for TCRBV gene families in PBMC from patients with CHB, and PCR products of the single peak were determined as monoclonal T cell by sequencing. Conclusions The optimized reaction parameters of RF-PCR for monitoring 24 TCRBV gene families are determined. The melting peak chart could be used to monitor the clone expansion of the peripheral lymphocytes and to detect the clone-specific T cells in the peripheral blood from patients with CHB.
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γδT lymphocytes,a numerically small subset of T lymphocytes,have been shown to directly recognize protein antigens without restriction by polymorphic MHC class I or class Ⅱ molecules and their asso-ciated peptide ligands,secrete cytokines,kill tumor cells and display cytolytie activity against various tumors.In osteosarconla patients,γδT lymphocytes can recognize antigens expressed on the surface of osteosarcoma cells,lyse and kill tumor cells.Combined with converntional chemotherapy and surgery,the new γδT lympho-cytes-based cell immunotherapy of osteosarcoma can increase the survival rate greatly.
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We investigated the co-stimulatory role of a cell-surface protein, CD99. Co-ligation of CD99 and suboptimal CD3 induced T-cell activation to a level comparable to that obtained with optimal CD3 or CD3+CD28. We also noted concomitant enhancement of the earliest T-cell receptor (TCR) signaling events. In addition, co-ligation of CD99 and CD3 led to translocation of TCR complexes into the lipid raft, without concomitant migration of CD99 to the raft, and consequent enhancement of TCR zeta-mediated signal 1. These data demonstrate the unique properties of CD99 co-stimulation that distinguish this molecule from CD28 and other raft-resident co-stimulatory factors.
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Humans , Antigens, CD/immunology , CD3 Complex/immunology , Cell Adhesion Molecules/immunology , Down-Regulation , Jurkat Cells , Lymphocyte Activation/immunology , Membrane Microdomains/immunology , Membrane Proteins/immunology , Phosphorylation , Phosphotyrosine/metabolism , Protein Transport , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunologyABSTRACT
Intradermal gene administration was found to induce a more profound immune response than direct intramusclular gene injection. We performed intradermal vaccination of B10.PL mice with DNA encoding for the V 8.2 region of the T-cell receptors (TCR). Three weeks later, these mice were immunized with rat myelin basic protein (MBP). Daily mean clinical scores and mortality rate were lower in this group compared with controls. The proliferative responses of lymph node cells to rat MBP were slightly less in the vaccination groups than in the control groups (p<0.05). However, we detected no differences between the two groups with regard to the production of MBP-specific IgG, IgG1, & IgG2a antibodies. The levels of cytokine mRNA expression in the vaccination groups were observed higher than in the control groups without antigen-specific stimulation, but all of cytokine expressions between the vaccination and control groups after antigen-specific stimulation were identical. These results demonstrate that intradermal DNA vaccines encoding for TCR might prove to be useful in the control of autoimmune disease.