Cytokines ; metabolism ; Humans ; Leukocytes, Mononuclear ; immunology ; secretion ; Ubiquitin ; blood

OBJECTIVETo study the surface antigen of the dendritic cells (DC) and their Toll-like receptor 4 (TLR4) expression in patients with idiopathic thrombocytopenic purpura (ITP), and to explore their role in ITP pathogenesis.

METHODSThe peripheral blood mononuclear cells isolated from complete remission patients (CR), non-complete remission patients (n-CR) and normal controls were stimulated by rhGM-CSF and rhIL-4. The surface antigen of the DC was analyzed by flow cytometry. The level of IL-12p70 in the supernatant was detected by enzyme linked immunosorbent assay. The expression of TLR4 mRNA of DC was detected by real time PCR.

RESULTSIn the 21 CR ITP patients, the expression of both CD80 and CD86 in DC was significantly increased compared with that in normal controls \[(51.60 ± 13.47)% vs (36.03 ± 15.43)%, (61.50 ± 15.93)% vs (40.28 ± 11.49)%, respectively\] (P < 0.01). The expression of CD80 and CD86 in n-CR group was also significantly increased \[(53.29 ± 19.49)% and (62.91 ± 18.43)%, respectively\] (P < 0.01). After HD-DXM treatment, both CD80 and CD86 in CR patients were decreased (P < 0.01). There was no difference between the DXM treatment patients and the normal controls. In n-CR group, there was no difference in CD80 and CD86 expression before and after DXM therapy \[(52.30 ± 20.98% and (49.79 ± 20.28)%, respectively\] (P > 0.05). CD80 was still higher than normal (P < 0.05), while CD86 was not changed. The level of IL-12p70 in CR ITP patients before treatment was significantly higher \[(67.52 ± 14.43) pg/ml\] than that of the controls \[(39.78 ± 10.03) pg/ml\](P < 0.01), and after treatment, was significantly decreased to (43.90 ± 8.49) pg/ml, being no difference from that in control. In n-CR group, IL-12p70 was lower after treatment \[(48.45 ± 9.68) pg/ml\] than that before treatment \[(65.35 ± 12.52) pg/ml\] (P < 0.01), but still higher than that in control (P < 0.05). The TLR4 mRNA level in DCs of CR ITP patients before treatment were significantly higher 0.69 ± 0.17 than that of controls (0.31 ± 0.09) (P < 0.01) and after treatment, was reduced to 0.35 ± 0.11, being no difference from that in control. In n-CR group, TLR4 mRNA was decreased from 0.65 ± 0.09 to 0.52 ± 0.21 after treatment (P < 0.01), but still higher than normal (P < 0.01).

CONCLUSIONDC may play an important role in ITP by their Toll-like receptor and cytokine secretion.

Dendritic Cells ; immunology ; Humans ; Interleukin-12 ; metabolism ; Leukocytes, Mononuclear ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; Toll-Like Receptor 4

BACKGROUNDPrevious studies indicate that CD43 plays a role in regulating the adhesion of lymphocytes, cell mutation and activation, however, little is known about its effect on systemic lupus erythematosus (SLE). This study was designed to explore the clinical significance of CD43 in SLE patients.

METHODSWe used microarray and real-time PCR to detect the mRNA and protein expression of magnetic bead sorted T cells and B cells from peripheral blood mononuclear cells (PBMCs) of SLE patients, and analyzed the relationship between CD43 and the clinical indexes.

RESULTSBoth microarray and real-time PCR results showed that CD43 mRNA was significantly decreased in PBMCs of SLE patients compared with healthy controls (P < 0.001). There were no significant differences between lupus nephritis and non-lupus nephritis patients, and neuropsychiatric and non-neuropsychiatric patients. CD43 mRNA expression was significantly reduced in T cells but not in B-cells in SLE patients compared to healthy controls (P < 0.01). Compared with healthy controls, the percentage of CD43(+) cells in the PBMCs of SLE was significantly decreased (P = 0.004), and the CD43 fluorescence intensity in CD3(+)/CD43(+) cells and CD19(+)/CD43(+) cells was also significantly weaker than in healthy controls (P = 0.039 and 0.003). There was no significant difference in the percentage of CD3(+)/CD43(+) cells, CD19(+)/CD43(+) cells between the two groups. The CD43 fluorescence intensity in CD3(+)/CD43(+) cells was inversely correlated with the levels of IgG and IgM (r = -0.8 and -0.6).

CONCLUSIONSCompared to healthy controls, both CD43 mRNA and protein expressions were reduced in T cells from patients with SLE, and were inversely correlated with IgG.

B-Lymphocytes ; immunology ; metabolism ; Humans ; Leukocytes, Mononuclear ; Leukosialin ; genetics ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; metabolism ; Oligonucleotide Array Sequence Analysis ; Real-Time Polymerase Chain Reaction ; T-Lymphocytes ; immunology ; metabolism

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

The major house-dust-mite allergen, Der p I, stimulates the phospholipase D (PLD) in peripheral blood mononuclear cells (PBMC) from allergic patients with maximal responses after 30 min exposure. At 30 min, Der p I stimulated PLD activity by 1.4-fold in mild, 1.6-fold in moderate and 2-fold in severe allergic patients over control values (p < 0.05). When the cells were pretreated for 24 h with phorbol myristate acetate to down-regulate protein kinase C (PKC), PLD stimulation by Der p I was largely abolished. These results indicate that in PBMC from allergic patients, Der p I can stimulate PLD activity, and that PKC activation is involved in this stimulation.
Adult ; Allergens/metabolism* ; Allergens/immunology ; Animal ; Down-Regulation (Physiology) ; Glycoproteins/metabolism* ; Glycoproteins/immunology ; Human ; Hypersensitivity/metabolism ; Hypersensitivity/immunology ; Hypersensitivity/blood ; IgE/blood ; In Vitro ; Leukocytes, Mononuclear/metabolism ; Leukocytes, Mononuclear/immunology ; Mites/metabolism ; Mites/immunology ; Phospholipase D/metabolism* ; Phospholipase D/immunology ; Protein Kinase C/metabolism* ; Skin Tests ; Tetradecanoylphorbol Acetate/pharmacology

BackgroundDevelopment of innovative immunotherapy is imperative to improve the poor survival of the nasopharyngeal carcinoma (NPC) patients. In this study, we evaluated the T cell response to melanoma-associated antigen (MAGE)-A1, MAGE-A3, or synovial sarcoma X-2 (SSX-2) in the peripheral blood of treatment-naive NPC patients. The relationship of responses among the three proteins and the human leukocyte antigen (HLA)-A types were analyzed to provide evidence of designing novel therapy.

MethodsSixty-one NPC patients admitted into the Tumor Hospital affiliated to the Xinjiang Medical University between March 2015 and July 2016 were enrolled. Mononuclear cells were isolated from the peripheral blood before any treatment. HLA-A alleles were typed with Sanger sequence-based typing technique. The T cell response to the MAGE-A1, MAGE-A3, or SSX-2 was evaluated with the Enzyme-Linked ImmunoSpot assay. Mann-Whitney U-test was used to compare the T cell responses from different groups. Spearman's rank correlation was used to analyze the relationship of T cell responses.

ResultsHLA-A*02:01, A*02:07, and A*24:02 were the three most frequent alleles (18.9%, 12.3%, and 11.5%, respectively) among the 22 detected alleles. 31.1%, 19.7%, and 16.4% of the patients displayed MAGE-A1, MAGE-A3, or SSX-2-specific T cell response, respectively. The magnitudes of response to the three proteins were 32.5, 38.0, and 28.7 SFC/10 peripheral blood mononuclear cells, respectively. The T cell response against the three proteins correlated with each other to different extent. The percentage of A*02:01 and A*24:02 carriers were significantly higher in patients responding to any of the three proteins compared to the nonresponders.

ConclusionMAGE-A1, MAGE-A3, or SSX-2-specific T cell responses were detectable in a subgroup of NPC patients, the frequency and magnitude of which were correlated.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Alleles ; Antigens, Neoplasm ; immunology ; metabolism ; Carcinoma ; immunology ; metabolism ; Female ; HLA-A Antigens ; metabolism ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; Neoplasm Proteins ; metabolism ; Sarcoma, Synovial ; immunology ; metabolism ; Young Adult

The latent membrane protein 2 (LMP2) is a kind of protein expressed by EBV-infected cells. This study was aimed to investigate whether the stimulation of peripheral blood mononuclear cells with peptides induces EBV-specific cytotoxic T lymphocytes (CTL). The peptides were mixture of LMP2 protein and available for people with different HLA types. Peripheral blood sample was collected from a patient with EBV-associated hemophagocytic syndrome. The mononuclear cells were isolated and cultured to obtain dendritic cells (DCs). Immature DCs were pulsed with MIX-LMP2 and added with different maturation-promoting factors. The auto-T lymphocytes were stimulated weekly with the harvested mature DCs loaded with MIX-LMP2, and totally for two times. Part of isolated lymphocytes was cultured without any stimulation as control. T-cell receptor (TCR) beta spectratyping was used to analyze the distribution of different T cell subgroups before and after culture. The phenotype of T lymphocytes was determined by flow cytometry. The IFN-gamma assay was used to estimate specific cytoxic activity of the cultured T cells. The results showed that the distribution of TCRbeta was changed according to analysis of TCR spectratypes. From the distribution of gene families of TCRbeta, the T lymphocytes were oligoclonal before culture, but shifted to a polyclonal after culture in vitro like the normalization of TCR diversity, suggesting the subgroups of lymphocyte could return to normal. The percentage of CD3+, CD3+CD8+ CD3+ CD45RA- CD45 RO+ on T lymphocytes from freshly isolated mononuclear cells were 70.73%, 42.99%, 27.56% respectively. After being stimulated twice with DC loaded with MIX-LMP2, they further increased to 95.17%, 52.54% and 81.41%. The percentages of CD3-CD56+ NK cells and CD4+CD35+ FOXP3+ regulation T cells seldom changed, from 2.12%, 0.03% to 2.35%, 0.02% respectively. The increase of CD3+CD45RA-CD45RO+ cells obviously indicated that most naive T cells could be activated. ELISA for IFN-gamma showed that when DCs loaded with LMP2 peptide were used as target cells, IFN-gamma level secreted by the T cells stimulated with LMP2 peptide-pulsed DCs was 805+/-16 pg/ml and 1729+/-49 pg/ml, the IFN-gamma level secreted by T cells stimulated twice with LMP2 peptide-pulsed DCs was 956+/-23 pg/ml and 2325+/-58 pg/ml respectively at effector-target ratios of 10:1 and 10:2. They were both significantly higher than that secreted by T cells without any stimulation (441+/-27 pg/m and 557+/-19 pg/ml) (p<0.05). But DCs unpulsed with LMP2 peptide were used as target cells, there were no significant differences between the T cells stimulated with LMP2 peptide-pulsed DCs and the T cells without stimulation (p>0.05). It is concluded that the antigen specific T cells recognizing EBV epitopes can be obtained by using DCs pulsed with MIX-LMP2 peptide in vitro, meanwhile the distribution of T cell subgroups can be changed and normalized.
Antigens, Viral ; immunology ; Cells, Cultured ; Coculture Techniques ; Cysteine Endopeptidases ; immunology ; metabolism ; Dendritic Cells ; immunology ; metabolism ; Epitopes, T-Lymphocyte ; immunology ; Epstein-Barr Virus Infections ; immunology ; Herpesvirus 4, Human ; immunology ; Humans ; Leukocytes, Mononuclear ; cytology ; T-Lymphocytes ; cytology ; immunology

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

OBJECTIVETo analyze the drift of the complementarity-determining region 3 (CDR3) of T cell receptor beta (TCRbeta) chain variable region in T cells of healthy volunteers cultured with interleukin-2 (IL-2).

METHODST cells were isolated from the peripheral blood and cultured in vitro in the presence of IL-2. The non-specific killing effect of the cells was analyzed by LDH releasing assay, and the distribution of TCRbeta chain CDR3 in healthy volunteers by immunoscope spectratyping method to evaluate the clonality of the T cells.

RESULTSThe results showed Gaussian distribution of TCR Vbeta gene CDR3 in healthy volunteers. The T cell cultured with IL-2, however, displayed some anomalous and oligoclonal expansion in different TCR Vbeta families without killing effect against nasophargngal carcinoma cell line CNE2.

CONCLUSIONIL-2 may affect TCRbeta chain CDR3 distribution in T cells cultured in vitro.

Cells, Cultured ; Complementarity Determining Regions ; genetics ; Genetic Drift ; Humans ; Interleukin-2 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; T-Lymphocytes ; immunology ; metabolism

OBJECTIVETo explore the role of indirect antigen presentation pathway on the immunogenecity of epidermal cells.

METHODSHuman epidermal cells (HEC), allogeneic human peripheral blood lymphocytes (PBL) and mononuclear cells (PBM, including monocytes) were isolated and cultured in vitro. HECs were transfected by human-originated CTLA4Ig-adenovirus vector. The CTLA4Ig expression was observed. Allogeneic PBLs or PBMs were added to the transfected and non-transfected HECs with simple cultured PBLs and PBMs as the control. The proliferation of PBL and PBM was determined by (3)H-TdR incooperation.

RESULTSHECs could be successfully transfected by CTLA4Ig-adenovirus vector and expressed corresponding proteins. The non-transfected HECs could stimulate slight proliferation of allogeneic PBLs (P < 0.05) and stimulate remarkable proliferation of PBMs (including monocytes) (P < 0.05). The proliferation reaction of PBLs and PBMs decreased significantly (P < 0.05) after being stimulated by HEC which was modulated by CTLA4Ig genes.

CONCLUSIONIndirect antigen presentation pathway might play important roles in the HEC immunogenicity which could be evidently inhibited by CTLA4Ig.

Adenoviridae ; genetics ; Antigen Presentation ; immunology ; physiology ; Antigens, CD ; Antigens, Differentiation ; genetics ; immunology ; CTLA-4 Antigen ; Cell Division ; immunology ; Cells, Cultured ; Coculture Techniques ; Epidermis ; cytology ; immunology ; metabolism ; Genetic Vectors ; genetics ; Humans ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocytes ; cytology ; immunology ; Signal Transduction ; Transfection