After having successfully constructed and expressed the gene of the anti-CD3/anti-CD20 bispecific single-chain antibody (bscCD3 x CD20), here we analyzed its in vitro bioactivity of mediating the lysis of Ramous human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Obvious opoptosis characters were observed by Annexin V/PI(AV/PI) stained and scanning electron microscope. As evaluated by non-radioactive cytotoxity assay, the bscCD3 x CD20 showed potent bioactivity of mediating human B-lymphoma cells lysis in the presence of T-enriched human PBL. The potency of cytotoxicity depended on the ratios of effect cells to target cells (E:T) used. Further, the antibody showed a dose and time-dependent effect on mediating Ramous cells lysis. The specific lysis reached about 87.3% at an antibody concentration of 5microg/mL and E:T used at 10:1. Clear changes in apoptogenes expression profiles were detected by apoptosis gene array after Ramous cells were treated with the antibody and PBL. Among the upregulated apoptogenes, ATM and P53 showed an increase of 187 times and 15 times respectively, which suggested that ATM-p53 pathway may be the main apoptosis way of Ramous cells induced by T cells in the presence of the bscCD3 x CD20.
Antibodies, Bispecific ; immunology ; Antigens, CD20 ; immunology ; Apoptosis ; immunology ; CD3 Complex ; immunology ; Humans ; Lymphoma, B-Cell ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured

OBJECTIVETo investigate the local cellular immune response after injection of superantigen, the highly agglutinative staphylococin (HAS), into the tumor bed after ultrasound-guided percutaneous microwave coagulation therapy (PMCT) in the liver cancer patients.

METHODSNinety-two patients with pathologically proven primary liver cancer were divided into two groups: 45 in group A were treated by PMCT alone and 47 in the group B by combined with ultrasound-guided percutaneous injection of highly agglutinative staphylococin (HAS). Before and after PMCT and HAS treatment, the patients underwent ultrasound-guided percutaneous biopsy from the tumor bed and the samples were examined by pathology and immunohistochemistry. The infiltration of CD3+, CD4+, CD57+ and CD68+ lymphocytes in treatment zone was compared between the two groups. Moreover, the infiltrating immunocytes were observed by transmission electron microscopy.

RESULTSOne week after HAS injection, the densities of CD3+, CD4+, CD57+ and CD68+ cells in the group B were 54.50 +/- 18.44, 38.14 +/- 12.44, 33.38 +/- 10.79 and 45.56 +/- 16.53, respectively. All the above mentioned parameters increased significantly in varying degrees compared with that before PMCT or HAS injection (P < 0.05). Four weeks after HAS injection, the density of CD3+, CD4+, CD57+ and CD68+ cells in the group B were 32.67 +/- 10.42, 23.43 +/- 6.99, 18.63 +/- 7.89 and 30.01 +/- 11.05, respectively, still significantly higher than those before PMCT (P < 0.05). Five weeks after PMCT and HAS injection, the densities of CD3+, CD4+, CD57+ and CD68+ cells in the group B were 54.50 +/- 18.44, 38.14 +/- 12.44, 33.38 +/- 10.79 and 45.56 +/- 16.53, versus 32.03 +/- 8.11, 15.67 +/- 8.32, 15.23 +/- 8.26 and 29.67 +/- 11.98 in the group A, respectively, still with a significant difference between the two groups (P < 0.05). A lot of lysosomes, endoplasmic reticulum and mitochondria in the immune cells after injection of HAS were observed by transmission electron microscopy.

CONCLUSIONThe local cellular immunity in liver cancer treatment area can be significantly improved by ultrasound-guided injection of highly agglutinative staphylococin after percutaneous microwave coagulation therapy.

Adult ; Aged ; Antigens, CD ; immunology ; Antigens, Differentiation, Myelomonocytic ; immunology ; CD3 Complex ; immunology ; CD4 Antigens ; immunology ; CD57 Antigens ; immunology ; Electrocoagulation ; methods ; Female ; Humans ; Liver Neoplasms ; pathology ; therapy ; Male ; Microwaves ; therapeutic use ; Middle Aged ; Superantigens ; therapeutic use ; T-Lymphocytes ; immunology

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.
Antigens, CD/*immunology ; Antigens, CD3/immunology ; Cell Adhesion Molecules/*immunology ; Down-Regulation ; Humans ; Jurkat Cells ; Lymphocyte Activation/*immunology ; Membrane Microdomains/*immunology ; Membrane Proteins/*immunology ; Phosphorylation ; Phosphotyrosine/*metabolism ; Protein Transport ; Receptors, Antigen, T-Cell/*immunology ; T-Lymphocytes/*immunology

In order to study how to activate T cells and their immunological characteristics, the anti-CD3 and anti-CD28 McAbs were used to stimulate PBMNC, then their related immunological changes, such as lymphocyte transformation function, the percentage of CD8(+)CD25(+) cells and TGF-beta expression were deleted by lymphocyte transformation assay, flow cytometry and RT-PCR respectively. The results showed that in costimulation with anti-CD28 antibody stimulation, the activity of anti-CD3 antibody was significantly enhanced, the ratio of CD8(+)CD25(+) cells of T cells was obviously increased, while TGF-beta expression was down-regulated. It was concluded that the anti-CD28 antibody costimulation could provide stimulatory signal II, which make T cells more active, while the expression of TGF-beta significantly down-regulated.
Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; immunology ; CD3 Complex ; immunology ; Down-Regulation ; Humans ; Leukocytes, Mononuclear ; immunology ; metabolism ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; immunology ; Transforming Growth Factor beta ; biosynthesis

Depletion of T and B cells from the graft is prerequisite for haploidentical transplantation to decrease the risk of GVHD and EBV-associated lymphoproliferative disease. This study was aimed to investigate the performance of T-cell and B-cell simultaneous depletion from mobilized peripheral blood stem cells (PBSCs) for the first time in China, using anti-CD3 and anti-CD19 antibodies conjugated to magnetic microbeads by the CliniMACS device. The depletion efficiency of T-cell and B-cells was analyzed by flow cytometry; the function of the stem cells after depletion was evaluated using colony assays. The results indicated that the mononuclear cell count prior to T- and B-cell depletion was 4.88 x 10(10). After depletion, the percentage of T cells was 0.02% with a log (10) depletion of 4.4. The percentage of B cells was less than 0.01% with a log (10) depletion of at least 3.3. The product contained not only CD34(+) stem cells, but also NK cells, monocytes and granulocytes. After T- and B-cell depletion the purity of CD34(+) cells was 0.98%, the number of CD34 cells was 1.84 x 10(8) and their recovery rate was 69.7%. The number of NK cells was 2.54 x 10(9) and the recovery rate of NK cells was 71.7%. In vitro colony assays showed no negative impact on function of the hematopoietic stem cells. In conclusion, the CliniMACS system can be used to efficiently deplete T and B cells from PBSCs simultaneously, without adverse effect on biological function of hematopoietic stem cells. This study provides technical platform for haploidentical hematopoietic stem cell transplantation.
Antigens, CD34 ; immunology ; B-Lymphocytes ; immunology ; CD3 Complex ; immunology ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Lymphocyte Depletion ; methods ; Peripheral Blood Stem Cell Transplantation ; methods ; T-Lymphocytes ; immunology

Antibodies ; immunology ; therapeutic use ; Antigens, CD20 ; immunology ; Antilymphocyte Serum ; therapeutic use ; CD3 Complex ; immunology ; Diabetes Mellitus, Type 1 ; drug therapy ; immunology ; Humans ; Immunotherapy ; methods

OBJECTIVETo explore the diagnostic value of intracellular antibody combination in acute leukemia (AL) expressing cross-lineage cell-surface antigens.

METHODSFlow cytometric immunophenotyping using intracellular antibody combination (cMPO/cCD79alpha/cCD3/CD45) was performed additionally in 60 patients who expressed cross-lineage antigens from 269 previously untreated adult AL.

RESULTSFifty-four of 269 previously untreated adult AL patients who expressed only one kind of intracellular antigen were diagnosed as cross-lineage AL, the percentage of cross-lineage AL in T cell acute lymphoblastic leukemia (T-ALL), B-ALL and acute myeloid leukemia (AML) was 28.6%, 43.6% and 13.4%, respectively. The positive rate of CD7, CD19, CD5 and CD20 in cross-lineage AML was 65.4%, 15.4%, 11.5%, and 7.7%, respectively. The positive rate of CD13, CD33 and CD15 in cross-lineage ALL was 89.3%, 21.4% and 3.6%, respectively. Six (2.3%) patients expressed two-lineage intracellular antigens were diagnosed as biphenotypic AL: 2 of T/B type and 4 B/M (B/myeloid) type.

CONCLUSIONIntracellular antibodies possess lineage specificity and four-color combination flow cytometric immunophenotyping can provide fast and multi-parameter data. To ensure accuracy of the results, CD45/SSC gating and normal cells as internal reference should be used in the immunophenotyping of abnormal cells.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Monoclonal ; Antigens, Surface ; immunology ; CD3 Complex ; immunology ; CD79 Antigens ; immunology ; Cross Reactions ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukocyte Common Antigens ; immunology ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; immunology ; Sensitivity and Specificity

Adult ; Antigens, CD20 ; metabolism ; Brain ; pathology ; Brain Neoplasms ; immunology ; pathology ; CD3 Complex ; metabolism ; Diagnosis, Differential ; Humans ; Leukocyte Common Antigens ; metabolism ; Lymphomatoid Granulomatosis ; immunology ; pathology ; Male

BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2> or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.
Antibodies, Monoclonal/chemistry/*immunology ; Antigens, CD19/chemistry/metabolism ; Antigens, CD3/chemistry/metabolism ; Antigens, CD4/chemistry/metabolism ; Antigens, CD8/chemistry/metabolism ; B-Lymphocyte Subsets/immunology/metabolism ; Color ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/*chemistry ; Humans ; T-Lymphocyte Subsets/immunology/metabolism

Animals ; Antigens, Neoplasm ; biosynthesis ; genetics ; immunology ; Apoptosis ; physiology ; Breast Neoplasms ; immunology ; metabolism ; CD3 Complex ; immunology ; Female ; Humans ; Killer Cells, Natural ; pathology ; Neoplasms ; immunology ; metabolism ; Receptors, Antigen, T-Cell ; immunology ; Stomach Neoplasms ; immunology ; metabolism ; Uterine Cervical Neoplasms ; immunology ; metabolism