The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors (TCRs) or chimeric antigen receptors (CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cells to target B-cell malignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin's lymphoma.
B-Lymphocytes ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Humans ; Immunotherapy, Adoptive ; Leukemia ; Lymphoma, B-Cell ; therapy ; Lymphoma, Non-Hodgkin ; therapy ; Neoplasms ; Receptors, Antigen ; Receptors, Antigen, T-Cell ; Receptors, CCR1 ; T-Cell Antigen Receptor Specificity ; T-Lymphocytes

Minimal residual disease (MRD) is the principal root of relapsed leukemia. Application of specific immunotherapy is effective in eradication of MRD and one of the immunotherapeutic strategies is induction and re-transfusion of leukemia-specific cytotoxic T lymphocytes (CTLs). This study was aimed to investigate the possibility of ex vivo induction and generation of CD8 positive CTLs of umbilical cord blood cell origin and to explore their potential of specific anti-leukemia cytotoxicity so as to evaluate the feasibility of application of cord blood cell derived lymphocytes to specific immunotherapy. Dendritic cells (DCs) were induced and generated ex vivo from cord blood mononuclear cells (MNC) with a cytokine cocktail, and loaded with frozen and thawed U937 leukemia antigen. The matured DCs were used to stimulate T lymphocytes derived from the same cord blood cell sample into CTLs. CD8 positive CTLs were then isolated by magnetic activated cell sorting (MACS). Inverted microscopy, scanning electronic microscopy and flow cytometry were used to detect DCs and methyl thiazolyl tetrazolium (MTT) cytotoxicity method was used to assay the killing activity. The results showed that DCs with typical morphology and mature function were cultured from 10 human cord blood cell samples. The cytotoxicities of CD8 positive CTLs, CD8 negative CTLs and T lymphocytes (TLs) to U937 cells were (66.36 +/- 12.43)%, (34.47 +/- 8.19)% and (15.79 +/- 4.64)% respectively under the same effector target ratio (40:1). Among them, the anti-leukemia cytotoxicity of the CD8 positive group was highest. At effctor target ratio of 40 to 1, the cytotoxicity of CD8 positive CTLs to U937 cells (66.36%) was higher than that to K562 cells (41.97%) (P < 0.05), whereas the cytotoxicity of CD8 negative CTLs to U937 cells was not significantly different from that to K562 cells (P > 0.05). It is concluded that specific CD8 positive CTLs can be generated from cord blood lymphocytes by induction of mature cord blood DCs loaded with U937 leukemia antigen. The cytotoxicity of CD8 positive CTLs against U937 cell is more potent than CD8 negative CTLs, and is strain specific.
Antibody Specificity ; Antigens, Neoplasm ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Dendritic Cells ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; Immunotherapy ; T-Cell Antigen Receptor Specificity ; T-Lymphocytes, Cytotoxic ; immunology ; U937 Cells

OBJECTIVE: To explore the better freezing protocol to preserve peripheral blood monuclear cells (PBMCs), islets antigen-specific T cells responses compared with freshly isolated samples in type 1 diabetic (T1D) patients. METHODS: The T cell Workshop Committee of the Immunology of Diabetes Society (IDS-TCW) organized the Freezing Study I and we were one of the 9 centers in the world to participate in the study. According to the two standardized T cell freezing protocols (warm and cold) to freshly isolated PBMCs in terms of recovery, viability, cell subset composition (FACS) and performance in Enzyme-linked immunospot (ELISPOT) assays, we chose 5 newly onset T1D patients and 5 age and sex matched healthy controls. Besides the protocols, all the freezing reagents and antigens were also centralized. The antigens used in ELISPOT were labeled blindedly. RESULTS: 1) Although warm frozen-thawed (W) samples had a slightly higher recovery rate (61.2% vs 60.1%, P>0.05) and viability (77.5% vs 74.9%, P>0.05) as compared with the cold frozen ones (C), the difference was not significant. 2) Both protocols led to a relative loss in monocytes as compared with the fresh samples (F) [3.2±1.1% (C) and 3.0±0.9% (W) vs 7.0±1.1% (F), both P<0.05], while other subsets including CD4+T, CD8+T, B cells, NK cells and NKT cells didn't. 3) Freezing and fresh samples showed similar IFN-γ secretion responses to polystimuli in ELISPOT. Irrespective of the freezing protocol, recall antigen Pediacel and islet antigen-reactive responses were both lower in the frozen cells compared with fresh PBMCs. The stimuli index (SI) of GADspecific T cell response in the fresh samples from T1D patients was 5.1, higher than that of frozen samples with either cold protocol (1.3) or warm one (1.4) (both P<0.05). Only fresh samples from T1D showed significantly higher GAD-specific T cell responses than the healthy controls no matter in SI (5.1 vs 0.9, P<0.05) or spot forming cells (8.1 vs 0.1, P<0.05), whereas the frozen samples did not show such difference. CONCLUSION More studies are needed to verify a freezing method to bring comparable islets antigen specific T cell responses in T1D patients to fresh PBMCs.
Adult ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cryopreservation ; Diabetes Mellitus, Type 1 ; blood ; immunology ; Female ; Humans ; Islets of Langerhans ; immunology ; Leukocytes, Mononuclear ; cytology ; Male ; T-Cell Antigen Receptor Specificity ; immunology ; T-Lymphocytes ; cytology ; immunology ; Young Adult

Major histocompatibility complex (MHC) tetramer technology offers a powerful means to study specific T cell populations of interest. To investigate the immune response of H-2Db-restricted CD8+ T cells in immunotherapy, we prepared the H-2Db tetramer and verified its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV). First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2Db-BSP, i.e. the ectodomain of H-2Db fused to a BirA substrate peptide (BSP), was constructed and overexpressed in E. coli BL21(DE3). Then, the denatured H-2Db-BSP was refolded in the presence of human beta2-microglobulin as well as the GP33-41 peptide (KAVYNFATC, KAV) of LCMV. The biotinylated H-2Db/KAV molecules were purified, then bound to streptavidin-PE and tetramerized. Finally, the prepared H-2Db KAV tetramer reagent was verified by detecting the CD8+ T cells specific for HCMV in KAV peptide vaccinated C57BL/6 mouse, with a mouse receiving subcutaneous injection of only adjuvant as negative control. The results showed that the tetramer positive rates were 0.27%, 0.11%, and 0.24% within the CD8+ T cell populations in the peripheral blood, draining lymph nodes, and spleen of vaccinated mouse, respectively. There was only very low background staining (< or = 0.01%) of those samples from the control mouse. Beside, the best results were achieved in the staining of the peripheral blood sample. In conclusion, the established procedure of preparing H-2Db tetramer will facilitate the study of the immune responses of antigen-specific CD8+ T cells in the experimental immunotherapy on the mice with H-2Db allele background.
Animals ; Antibody Specificity ; CD8-Positive T-Lymphocytes ; immunology ; H-2 Antigens ; genetics ; Histocompatibility Antigens Class I ; genetics ; Immunologic Techniques ; Lymphocytic choriomeningitis virus ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; T-Cell Antigen Receptor Specificity ; immunology

INTRODUCTIONPolymerase chain reaction (PCR)-based molecular techniques are useful adjunctive tools in the diagnosis of cutaneous T-cell lymphomas (CTCL). This study compares the sensitivity of PCR analysis of the T-cell receptor-gamma (TCR-gamma) gene rearrangements using conventional polyacrylamide gel electrophoresis (PCR-PAGE) and fluorescent capillary electrophoresis (PCR-FCE).

MATERIALS AND METHODSA total of 22 paraffin blocks were analysed using PCR-PAGE and PCR-FCE. There were 17 cases of mycosis fungoides (MF), 4 cases of non-MF CTCL and 1 case of lymphoblastic leukaemia.

RESULTSComplete agreement was obtained between PCR-PAGE and PCR-FCE in 19 of the 22 cases, giving a concordance rate of 86.4%. PCR-FCE had a higher sensitivity of 77.3%, compared to 63.6% for PCR-PAGE, allowing the detection of 3 additional cases of clonal T-cell rearrangements, which had equivocal or polyclonal bands on PAGE. Two of these 3 cases were in erythrodermic MF patients. PCR-FCE also allowed the detection of matching clones in serial specimens taken from different sites and at different time intervals in patients with MF. However, matching clones from different specimens can be achieved qualitatively in PCR-PAGE by running and comparing these on the same polyacrylamide gel block.

CONCLUSIONSBoth PCR-PAGE and PCR-FCE are useful in detecting T-cell clones in CTCL, with both methods being comparable in sensitivity and showing a high concordance rate of 86.4%. PCR-FCE has the added advantage of exhibiting semiquantitative properties, which may be important in early or erythrodermic MF cases, but the requirement for sophisticated and costly machinery limits its availability to high-capacity laboratories. The well-established PCR-PAGE method is a suitable alternative in routine clinical applications.

Base Sequence ; Electrophoresis, Agar Gel ; Electrophoresis, Capillary ; methods ; Electrophoresis, Polyacrylamide Gel ; Fluorescence ; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor ; Humans ; Lymphoma, T-Cell ; diagnosis ; Mycosis Fungoides ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Adult ; CD8-Positive T-Lymphocytes ; immunology ; Case-Control Studies ; Female ; Hepatitis B, Chronic ; immunology ; metabolism ; pathology ; Humans ; Liver ; immunology ; pathology ; Male ; Middle Aged ; T-Cell Antigen Receptor Specificity ; Young Adult

BACKGROUND/AIMS: The protective role of HBV-specific CD8+ cells is dependent on their ability to efficiently migrate to the infected liver, where they may exert an effector function. The migratory behavior of CD8+ cells is influenced by their expression of different chemokine receptors. This study was intended to analyse the pattern of chemokine receptor expression of HBV specific CD 8+ cells in chronic B viral infection. METHODS: We analysed the CCR5 and CCR3 profile of HBV-specific CD8+ cells isolated from the blood and liver of patients with different patterns of HBV infection. Purified T cells were stained directly ex vivo, or after antigen-specific stimulation, using HBV peptide-specific HLA tetramers and monoclonal antibodies to CD8, CCR5 and CCR3, with analysis by flow cytometry. RESULTS: In patients with chronic hepatitis B characterised by low levels of virus (serum HBV DNA <0.5pg/mL) and minimal liver inflammation, analysis of circulating and intrahepatic CD8+ cells demonstrated that liver infiltrating Tc18-27-specific cells were preferentially CCR5+ (up to 80% of HBV-specific CD8+ cells), in contrast to cells of the same specificity within the circulating compartment (up to 35% of HBV-specific CD8+ cells). Furthermore, CCR3 was expressed by about 10% of Tc18-27+ cells infiltrating the liver, but was absent from circulating cells. Following HBV-specific stimulation in vitro the CCR5 expression of circulating Tc18-27-specific cells was up-regulated, to levels found in liver infiltrating cells, whereas CCR3 expression was unchanged. CONCLUSIONS: The chemokine receptor profile of HBV-specific CD8+ cells is influenced by the anatomical site of these cells, and the clinical pattern of disease. The ability of circulating HBV-specific CD8+ cells of patients with low replicating virus to upregulate CCR5 suggests that these cells may respond to increases in virus replication by efficiently migrating into the infected liver.
CD8-Positive T-Lymphocytes/immunology/*metabolism ; English Abstract ; Hepatitis B Virus/immunology ; Hepatitis B, Chronic/*immunology/pathology ; Human ; Liver/pathology ; Receptors, CCR5/metabolism ; Receptors, Chemokine/*metabolism ; T-Cell Antigen Receptor Specificity

OBJECTIVETo investigate the early changes of some immunological function of T-cell in chromate workers.

METHODSA total of 115 workers exposed to different levels of soluble chromate were enrolled in exposed group; while 90 non-exposure workers who lived far away from the chromate plant were enrolled as control. The air concentration of soluble chromate was determined by atomic absorption spectrometry. CD3(+), CD3(+)CD4(+), CD3(+)CD8(+) and CD4(+)/CD8(+) of T-cell were determined by flow cytometry analysis.

RESULTSThe individual air chromate concentration in the exposed group was (27.51 +/- 33.25) microg/m(3), and the control group was (0.16 +/- 0.15) microg/m(3). The significant difference between the two groups was observed (z = 8.045, P < 0.01). The levels of the lymphocyte subsets (CD3(+), CD3(+)CD4(+), CD3(+)CD8(+) and CD4(+)/CD8(+)) in exposed group were (30.08 +/- 17.75)%, (1.04 +/- 1.73)%, (11.94 +/- 9.78)%, 0.10 +/- 0.14. While, those of control group were (63.00 +/- 13.57)%, (30.51 +/- 5.16)%, (14.82 +/- 4.59)%, 2.17 +/- 0.53, higher than that of the exposed group (z values were 4.484, 5.227, 1.976, -5.218, respectively, P < 0.05).

CONCLUSIONOn the basis of individual air monitoring, the cellular immune function affected by soluble chromate is mainly based on T lymphocyte inhibition. The indicators CD3(+)CD4(+) mentioned above may be considered as efficient biomarkers in further research.

Adult ; CD4-CD8 Ratio ; Case-Control Studies ; Chromates ; adverse effects ; Female ; Humans ; Male ; Middle Aged ; Occupational Exposure ; T-Cell Antigen Receptor Specificity ; drug effects ; immunology ; T-Lymphocytes ; drug effects ; immunology

OBJECTIVETo explore the characteristics of T cell receptor beta (TCRbeta) gene rearrangements in children with T-cell acute lymphoblastic leukemia (T-ALL) and establish a system of quantitative detection of MRD with real-time quantitative (RQ-PCR) targeted at TCRbeta gene rearrangement.

METHODSMultiplex polymerase chain reaction (PCR) designed by BIOMED-2 was used to detect TCRbeta gene rearrangements in the bone marrow samples of 26 children with T-ALL. Sequence of junction region were then compared and analyzed in IMGT database. Allele specific oligonucleotide (ASO) upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRbeta gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells (MNC) from a pool of 20 healthy donors to generate the patient specific standard curves. Subsequently, a TCRbeta RQ-PCR assay to quantify MRD with germline Jbeta primer/probe combinations was applied in six patients. To check the quantity and quality of DNA, the investigators used RQ-PCR analysis for the N-ras gene.

RESULTSClonal rearrangements were identified in 92.3% childhood T-ALL (Vbeta-Dbeta-Jbeta rearrangements in 84.6%, Dbeta-Jbeta rearrangements in 50%). Comparative sequence analysis of 42 TCRbeta recombinations revealed two downstream Vbeta families (BV5, BV6) were preferentially used. The segment Jbeta2.7 in childhood T-ALL was preferentially used. Jbeta1.3, Jbeta2.4, and Jbeta2.6 were not found to be used. The slope of the standard curves was from -3.54 to -3.37 and the intercepts were from 19.35 to 20.51. The correlation coefficients of all 6 standard curves were excellent (> or = 0.98). All the RQ-PCR quantitative range reached 10(-4). MRD analysis of follow up samples showed that MRD increased before relapse.

CONCLUSIONRQ-PCR analysis of TCRbeta gene rearrangements was highly sensitive and specific, it will be of high value for future T-ALL MRD studies. And quantitative and serial study of MRD may be of prognostic importance.

Adolescent ; Base Sequence ; Child ; Child, Preschool ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; genetics ; Humans ; Infant ; Male ; Molecular Sequence Data ; Neoplasm, Residual ; diagnosis ; genetics ; Polymerase Chain Reaction ; methods ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA