Objective To investigate the differences of type 2 innate lymphocytes (ILC2) and interlukin 9 (IL-9) between chronic lymphocytic leukemia (CLL) patients and healthy controls, and to understand the effects of ILC2 on the function of regulatory T cells (Tregs), CD8⁺ T cells and CLL cells through IL-9. Methods Flow cytometry was used to detect the levels of ILC2 and Tregs in the peripheral blood of 45 newly diagnosed CLL patients and 24 healthy controls, and the expressions of granzyme B and perforin in CD8⁺ T cells in the peripheral blood of 28 patients and 15 healthy controls; ELISA was used to detect the level of IL-9 in the serum. ILC2 of patients and healthy controls was sorted by immunomagnetic beads and cultured separately, and the level of IL-9 in the culture supernatant was measured by ELISA. ILC2 sorted from CLL patients and healthy control-derived peripheral blood mononuclear cells(PBMCs) were co-cultured with the B cell leukemia MEC-1 cells, one group was supplemented with IL-9 antibody and the other group was not. After 72 hours of culture, the ratio of Tregs, programmed death 1 (PD-1), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), cytotoxic T lymphocyte antigen 4 (CTLA-4) on Tregs, granzyme B and perforin in CD8⁺ T cells were measured by flow cytometry, IL-9 level of the culture supernatant was measured by ELISA, the apoptosis of MEC-1 cells was measured by Annexin V-PI. Results Compared with the healthy control group, the levels of ILC2, Tregs and IL-9 in the CLL group increased significantly. The levels of granzyme B and perforin in CD8⁺ T cells were positively correlated in the peripheral blood of CLL patients. Compared with the healthy control group, IL-9 levels in the supernatant of sorted ILC2 from CLL patients increased. In the anti-IL9 antibody group, the level of PD-1 and TIGIT on Tregs decreased, and the level of granzyme B in CD8⁺ T cells increased significantly. The level of IL-9 in the anti-IL9 antibody group decreased statistically. And MEC-1 cells showed increased early apoptotic rate in the anti-IL9 antibody group statistically. Conclusion In CLL, ILC2 affects CD8⁺ T cells and Tregs through IL-9, which weakens the anti-tumor effect of CD8⁺ T cells, enhances the immunosuppressive effect of Tregs, and plays a role in the occurrence and development of CLL disease.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; CD8-Positive T-Lymphocytes/immunology* ; T-Lymphocytes, Regulatory/immunology* ; Middle Aged ; Male ; Female ; Interleukin-9/blood* ; Aged ; Granzymes/metabolism* ; Perforin/metabolism* ; Immunity, Innate ; Adult ; Lymphocytes/immunology*

OBJECTIVE: To investigate the expression of co-inhibitory molecules TIGIT/CD155 and PD-1 on CD4⁺T cells and Treg cells in peripheral blood of patients with chronic lymphocytic leukemia (CLL) and analyze their clinical significance. METHODS: The expression of PD-1 and TIGIT on CD4⁺T cells and Treg cells was detected by flow cytometry in 40 CLL patients and 20 healthy controls. Additionally, the expression of CD155 on peripheral blood B cells and DC cells of the enrolled subjects was detected. RESULTS: The proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells and CD155⁺DC cells in peripheral blood of CLL patients were significantly higher than those of healthy controls ( P < 0.05). The proportions of PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells in CLL patients were significantly higher than those of PD-1⁺TIGIT^-CD4⁺T cells and PD-1⁺TIGIT^-Treg cells, respectively ( P < 0.05). Both PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells were positively correlated with the level of CD155⁺DC cells (r =0.742, r =0.766). With the progression of Binet stage, the proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells, and CD155⁺DC cells gradually increased ( P < 0.05), and the aforementioned three types cells were all increased in patients with CD38≥30%, IGVH unmutated, or poor prognosis due to chromosomal abnormalities ( P < 0.05). CONCLUSION Co-inhibitory molecules PD-1 and TIGIT may be involved in immunodepletion in patients with advanced CLL, which has clinical prognostic value. Dual inhibitor molecular targeted therapy provides a new direction for the individualized treatment of CLL.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; Receptors, Immunologic/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; T-Lymphocytes, Regulatory/metabolism* ; Receptors, Virus/metabolism* ; CD4-Positive T-Lymphocytes/metabolism* ; Male ; Female ; Middle Aged ; Flow Cytometry ; Clinical Relevance

<b>OBJECTIVEb>To explore the immunophenotypic characteristics of CD5⁺ B cell lymphoproliferative disorders (B-LPD) of Chinese patients.

<b>METHODSb>Immunophenotyping of bone marrow and (or) of peripheral blood was performed in patients with B-LPD by four color multiparameter flow cytometry analysis using a panel of monoclonal antibodies, and the patients clinical data were retrospectively analyzed. The difference in immunophenotypes and the related clinical features were retrospectively analyzed. Fluorescence in situ hybridization (FISH) for t(11;14) detection was applied to diagnose or exclude mantle cell lymphoma.

<b>RESULTSb>(1)A total 260 CD5⁺ B-LPD patients were enrolled in this study, including 186 chronic lymphocytic leukemia (CLL), 40 mantle cell lymphoma (MCL), other B-LPD including 5 splenic marginal zone lymphoma (SMZL), 2 B-cell prolymphocytic leukemia (B-PLL), 3 hairy cell leukemia (HCL). The other 26 cases (10%)were not classified and defined as unclassified B-LPD (BLPD-U). MCL patients were all positive for t(11;14) detected by FISH, while all the BLPD-U patients were negative for t(11;14). (2) All patients expressed CD19, CD20 and CD5. According to the immunophenotypic score system, 186 CLL patients scored 4-5, 99.5% of patients with CD23⁺, 11.3% with sIgM⁺, 10.2% with FMC7⁺, 44.1% with CD22⁺ and 51.1% with CD11c⁺. MCL patients scored 2-3, with 50% expressing CD23 and sIgM, 81.6% expressing FMC7, 92.1% expressing CD22 and 5.3% expressing CD11c. In aspect of BLPD-U and other B-LPD, the expression of CD23, sIgM, FMC7, CD22 and CD11c were 73.1% and 50%, 34.6% and 50%, 88.5% and 100%, 92.3% and 90%, 69.2% and 70%, respectively. (3)In comparison of CLL with MCL, there was a significant difference in the expression of CD23, sIgM, FMC7, CD22 and CD11c between the two groups (P<0.01). Between MCL and BLPD-U, similar expression type of CD23, sIgM, FMC7 and CD22 was found except CD11c, which was highly expressed in BLPD-U (P<0.001). The difference of CD11c expression was also statistically significant between MCL and other B-LPD (P<0.01). In comparison of MCL with other B-LPD, there was a significant difference in the expression of CD11c (P<0.01). The expression of CD23 and sIgM in MCL are 7%-21% and 82%-100% respectively in Western population, while the expression of other immunophenotypic markers is similar with our study.

<b>CONCLUSIONb>The significant high incidence of CD23 and low incidence of sIgM compared to the Western population was observed in Chinese patients, and CD11c coud serve as a useful marker to distinguish MCL from CLL and other CD5⁺ B-LPD.

Adult ; Aged ; Aged, 80 and over ; Cell Count ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Leukemia, Lymphocytic, Chronic, B-Cell ; immunology ; Lymphoma, Mantle-Cell ; immunology ; Lymphoproliferative Disorders ; immunology ; Male ; Middle Aged ; Retrospective Studies

As targeted drugs to B-cell malignancies, anti-CD20 monoclonal antibodies have been proved to be important in therapeutic antibody field. With three generations in more than ten years' development, the structures of these drugs have been improved, and many new indications have been found. Nowadays, these kinds of antibodies are not only used in the treatment of lymphoid malignancies, but also been proved to be useful in some autoimmune diseases treatment, and their new indications are still being expanded. With the optimization of their clinical dosage regimens, drug reaction has been increased, thus, therapeutic and side effects of anti-CD20 monoclonal antibody have been further improved as well. However, the exact mechanism of action of their combination therapy with other chemical drugs is still unclear, which remains to be further studied. This article reviewed new development of anti-CD20 therapeutic monoclonal antibodies research in recent years.
Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Antigens, CD20 ; immunology ; Antineoplastic Agents ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; drug therapy ; Lymphoma, Non-Hodgkin ; drug therapy ; Rituximab ; therapeutic use

This study was purposed to evaluate the safety and curative effect of autologous cytokine induced killer cells (CIK) combined with low-dose IL-2 regimen containing immune enhancement of thymic peptide on elderly patients with B-cell chronic lymphocytic leukemia (B-CLL). Thymic peptide α1 was subcutaneously given as the immunoenhancement agent at a dose of 1.6 mg/d, 14 days as one cycle. Peripheral blood mononuclear cells (PBMNC) from 5 patients with B-CLL were isolated once a week to induce ex vivo CIK cells through culture in the context of interferon (IFN)-γ, interleukin (IL)-2 and anti-CD3 monoclonal antibody. The PBMNC were separated from patients before and after 14 days as one cycle of thymic peptide α1 administration. Parameters of amplification ability, effector cells quantity, lymphocyte subgroups percentage and antitumor cytotoxicity were compared before and after thymic peptide administration. The 5 patients were treated with CIK cells combined with low-dose IL-2 regimen immediately after injection of thymic peptide α1. The CIK cells plus low-dose IL-2 regimen containing thymic peptide enhancement was defined as: thymic peptide α1 1.6 mg/d was subcutaneously administered once every other day; (4 - 6) ×10(9) of CIK cells were transfused followed by IL-2 subcutaneous administration of 1 mU/d on days 1-10, 28 days as one cycle. Clinical evaluation parameters including cellular immunity function, CLL related biomarkers, disease state and infectious frequency and degree were investigated before and after CIK cells infusion puls IL-2. The results showed that the amount of amplified CIK cells, the percentage and amplification times of effector cells and antitumor cytotoxicity more significantly increased after thymic peptide α1 treatment than before its use (P < 0.05). The total 46 cycles of CIK cells infusion plus IL-2 were completed in the 5 CLL patients. No adverse reaction was observed. After treatment of CIK cells plus IL-2, the general conditions of 5 CLL patients were to different extent improved. Simultaneously, percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD56(+) cells in peripheral blood remarked by raised (P < 0.05), the serum level of β2 microglobulin was significantly declined (P < 0.05), and the frequency and degree of infection was also decreased (P < 0.05). Following CIK cells plus IL-2 therapy, the transformation of disease state from partial remission (PR) to complete remission was seen in 3 patients, from stable disease (SD) to PR in 1 patient, and from progress of disease to SD in 1 patient. It is concluded that the regimen of autologous CIK cells combined with low-dose IL-2 containing immune enhancement of thymic peptide is safety and effective for the treatment of elderly patients with B-CLL.
Aged ; Aged, 80 and over ; Cytokine-Induced Killer Cells ; immunology ; Humans ; Interleukin-2 ; administration & dosage ; therapeutic use ; Leukemia, Lymphocytic, Chronic, B-Cell ; therapy ; Male ; Thymosin ; immunology

This study was aimed to investigate the immunophenotypic characteristics of 109 cases of B-cell chronic lymphoid leukemia (B-CLL) so as to provide evidences for the diagnosis and therapy of B-CLL, and for the detection of the minimal residual disease and its prognosis. Immunophenotyping was performed in 109 patients of B-CLL by two/three color multiparameter flow cytometry analysis using a panel of monoclonal antibodies. The results showed that in 109 cases of B-CLL, all cases expressed CD19, the positive ratios of other B lineage antigen such as CD20, CD22 and CD23 were 95.40%, 94.50%, 86.20% respectively. None of the B-CLL cases expressed CD10. The expression ratio of FMC-7 and CD38 in 105 cases of B-CLL were 28.60% and 36.20%. Among the B-CLL cases the CD5(+) cells amounted to 86.23%, CD5(-) cells amounted to 13.76%, ZAP-70 protein was expressed in 12 out of 50 patients. It is concluded that immunophenotypic data are very useful for the diagnosis and detection of minimal residual disease of B-CLL, and the relationship between the immunophenotypic characteristics and the prognosis of B-CLL needs further study.
Adult ; Aged ; Aged, 80 and over ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; immunology ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis ; immunology ; Prognosis

The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20+B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFM tips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20+B lymphocytes was much higher than that of B-CLL CD20+B cells. The AFM data show that ultrastructure of B-CLL CD20+B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20+B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20+B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical. Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.
Antigen-Antibody Reactions ; immunology ; Antigens, CD20 ; immunology ; B-Lymphocytes ; immunology ; ultrastructure ; Binding Sites, Antibody ; Cell Membrane ; immunology ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; immunology ; Microscopy, Atomic Force ; Microscopy, Confocal ; Quantum Dots

<b>OBJECTIVEb>To investigate the overrepresentation of specific gene segments of immunoglobulin heavy chain variable region (IgVH) among unmutated and mutated chronic lymphocytic leukemia (CLL) patients and its prognostic implication.

<b>METHODSb>Multiplex PCR was used to identify the expression of IgVH segment and its mutation status in CLL.

<b>RESULTSb>Analyses were successfully performed in 80 of 85 samples. Marked skewed IgVH families were disclosed. The most commonly used VH was VH3 (40.0%), followed by VH4 (30.0%), VHI (13.8%), VH2 (10.0%) and VH5, VH7 (2.5%). Fifty-six patients (70.0%) had mutated VH, 24 (30.0%) unmutated VH. Nine cases (11.3%) were with 100% germline sequence. Fifteen cases (15/24, 62.5%) in VH4, 29 (29/32, 90.7%) in VH3, and 4 (4/11, 36.3%) in VH1 had mutated VH. The most frequently used IgVH gene was VH4-39 (13.8%), and VH4-34 (8.8%). J4 (36/66, 54.5%) and D3 (25/66, 37.8%) were the most frequently used in J and D genes. The progression-free survival (PFS) was 82 and 17 months (P = 0.000), and the overall survival (OS) was 90 and 41 months (P = 0.009), respectively, for mutated and unmutated cases. Recurrent CDR3 sequences were found in our patients and 2 patients with VH1-69 had CDR3 sequences highly similar to those reported in literature.

<b>CONCLUSIONb>There is difference in IgVH gene segment usage and mutational status in different area CLL patients. Recurrent CDR3 sequences were found in specific IgVH gene segments, which highlights the importance of immunoglobulin mediated stimulation in the development of CLL.

Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; pathology ; Male ; Middle Aged ; Mutation

<b>OBJECTIVEb>To construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20 positive primary chronic lymphocytic leukemia (CLL) cells and provide a promising tool for tumor adoptive immunotherapy.

<b>METHODSb>The recombinant vectors were transduced into PA 317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection for one week. Then transduced T lymphocytes and primary CLL cells were co-cultured. The status of primary chronic lymphocytic leukemia cells were observed by microscope. The level of IL-2 and IFN-gamma in the culture medium were measured.

<b>RESULTSb>Primary T cells expressing anti-CD20scFv/IgGFc/CD80/CD28/zeta could be constructed successfully. These T cells were able to lyse CD20+ targets and secrete high levels of IL-2 (1301.00 pg/ml) and IFN-gamma (602.18 pg/ml) in vitro.

<b>CONCLUSIONb>(1) Recombinant gene modified T cells can be constructed successfully. (2) Recombinant gene modified T cells can specially kill CD20 positive primary CLL cells in vitro.

Antigens, CD20 ; genetics ; B7-1 Antigen ; genetics ; CD28 Antigens ; genetics ; Genetic Vectors ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Leukemia, Lymphocytic, Chronic, B-Cell ; pathology ; Retroviridae ; genetics ; T-Lymphocytes ; immunology ; secretion ; Transfection ; Tumor Cells, Cultured

<b>OBJECTIVEb>To investigate whether CpG-oligodeoxynucleotide (CpG-ODN) can improve the detection rate of the karyotypic abnormalities in chronic lymphocytic leukemia (CLL).

<b>METHODSb>The bone marrow (BM) or peripheral blood (PB) cells from 57 cases of CLL were collected and cultured with CpG-ODN DSP30+interleukin-2 (IL-2), phytohemagglutinin (PHA), pokeweed (PWM) or IL-2, respectively. Five days later cells were harvested for chromosome preparation. Karyotypic analysis was done using R banding technique. Panel fluorescence in situ hybridization (FISH) was carried out on 19 cases of CLL with normal karyotypes using the following probes: Cen12, D13S25, Rb1, ATM, p53, MYB and IgH. Genomic DNA from 21 cases of them was extracted from BM or PB leukocytes. The immunoglobulin variable heavy chain (IgVH) was amplified by polymerase chain reaction (PCR) and sequenced. CD38 and ZAP70 expressions in the leukemic cells were determined by flow cytometry (FCM).

<b>RESULTSb>The detection rate of karyotypic abnormalities in the CpG-ODN+IL-2 group (43.85%) was obviously higher than that in the PHA (15.09%), PWM (17.31%) and IL-2 (3.13%) groups (P<0.01). Fifty-two types of karyotypic abnormalities were found. Among them, trisomy12 (+12) or +12 with other abnormalities were the most common, while translocations were the most frequent structural abnormalities including 3 unbalanced and 11 balanced translocations, among them 7 had rearrangements involving 14q32. Thirteen cases showed one or more abnormalities on FISH including trisomy 12 and p53 deletion each in one case, IgH rearrangement and partial deletion each in one case, 13q14.3 deletion in 11 cases of which 5 cases also had Rb1 deletion, 1 case had Rb1 partial deletion. No case with ATM or MYB deletions was found. PCR detected IgVH mutations in 10/21 cases. FCM showed 10/45 cases were CD38 positive, but 35 /45 were CD38 negative, 11/27 cases expressed ZAP70, but 16/27 did not. Among the 26 cases examined for CD38 and ZAP70 expressions simultaneously, 5 cases were CD38+ZAP70+, 13 were CD38-ZAP70-, 6 were CD38-ZAP70+, and 2 were CD38+ZAP70-, respectively. Statistic analysis showed a correlation between complex karyotype and IgVH without mutation, but no association between karyotype and CD38 or ZAP70 expression was observed.

<b>CONCLUSIONb>CpG-ODN immunostimulation can obviously raise the detection rate of abnormal karyotypes, especially translocations in CLL. FISH is an important complement to conventional karyotypic analysis. The combination of both methods can provide more comprehensive genetic information for CLL.

Adjuvants, Immunologic ; genetics ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow Cells ; cytology ; immunology ; Cells, Cultured ; Chromosome Aberrations ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; In Situ Hybridization, Fluorescence ; Interleukin-2 ; genetics ; Karyotyping ; methods ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; genetics ; immunology ; Male ; Middle Aged ; Oligodeoxyribonucleotides ; genetics ; immunology ; Phytolacca americana ; genetics