PURPOSE: Forkhead box p3 (Foxp3) positive T regulatory cells (Tregs) have a functionally immunosuppressive property that prevents effector cells from acting against self in autoimmune diseases or a tumor. It is known that Tregs may be highly relevant in cancer progression. Dendritic cells (DCs) induce cutaneous immune response, however several studies have suggested that DCs are involved in immunosuppression. The aim of this study is to evaluate the prevalence of Tregs and DCs infiltration in cutaneous premalignant and malignant squamous lesions. MATERIALS AND METHODS: We evaluated Tregs and DCs in skin tissue samples obtained from 83 patients with actinic keratosis, Bowen's disease or squamous cell carcinoma by immunohistochemistry. RESULTS: The prevalence of Tregs and DCs was significantly higher in squamous cell carcinoma and Bowen's disease than in actinic keratosis. In addition, the number of DCs was closely correlated with the prevalence of Tregs, and DCs were also located in direct proximity to Tregs. CONCLUSION: Tregs is related to cutaneous squamous tumor progression.
Bowen's Disease/immunology/metabolism/pathology ; Carcinoma, Squamous Cell/immunology/metabolism/pathology ; Dendritic Cells/immunology/metabolism/pathology ; Forkhead Transcription Factors/immunology/*metabolism ; Humans ; Immune Tolerance ; Keratosis, Actinic/immunology/metabolism/pathology ; Skin Neoplasms/*immunology/metabolism/pathology ; T-Lymphocytes, Regulatory/*immunology/metabolism/pathology

M1-type macrophages are capable of inducing lysis in various types of cancer cells, but the mechanism of action is unclear. It has been noted that an "unknown protein" produced together with protease by activated macrophages is responsible for this action. Activated M1 macrophages have been recently reported to produce family 18 chitinases, all of which have been named chitotriosidase. Our experiments have demonstrated that family 18 chitinases work together with proteases and can damage various cancer cells both in vitro and in vivo. Thus, in this article, we suggest that the 50-kDa chitotriosidase is the reported "unknown protein". In addition, we discuss how to properly stimulate activated M1 macrophages to produce 50-kDa chitotriosidases and proteases for destroying cancer cells. Because family 19 chitinase has recently been reported to kill cancer cells, we also discuss the possibility of directly using human family 18 chitotriosidase and the humanized plant family 19 chitinase for cancer treatment.
Animals ; Antineoplastic Agents, Alkylating ; pharmacology ; Chitinases ; metabolism ; Cyclophosphamide ; pharmacology ; Hexosaminidases ; metabolism ; Humans ; Immunosuppressive Agents ; pharmacology ; Macrophage Activation ; immunology ; Macrophages ; classification ; enzymology ; immunology ; pathology ; Neoplasms ; immunology ; pathology ; Peptide Hydrolases ; metabolism ; T-Lymphocytes, Regulatory ; metabolism ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism

PURPOSE: Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8+T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8+CLA+T cells might be underlying mechanism in AD. MATERIALS AND METHODS: CD8+CLA+T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8+CLA+T cells were evaluated. The proliferative responses of CD8+CLA+T cells were assessed by flow cytometry, and the levels of transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8+CLA+T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8+CLA+T cells in AD. Meanwhile, the levels of TGF-beta1 produced by Tregs were significantly lower in AD, and anti-TGF-beta1 abolished such suppression. CONCLUSION: The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8+CLA+T cells, mediated by TGF-beta1, plays an important role in the pathogenesis of AD.
Adult ; Aged ; CD8-Positive T-Lymphocytes/drug effects/*immunology ; Case-Control Studies ; Cell Proliferation ; Cell Separation ; Dermatitis, Atopic/*immunology/pathology ; Female ; Granzymes/metabolism ; Humans ; Interleukin-10/metabolism ; Lymphocyte Count ; Male ; Perforin/metabolism ; Skin/*immunology/pathology ; T-Lymphocytes, Cytotoxic/drug effects/immunology ; T-Lymphocytes, Regulatory/drug effects/*immunology ; Transforming Growth Factor beta1/pharmacology

OBJECTIVESTo study the expression and distribution of CD4+CD25+ regulatory T cells (Treg) in liver tissues of patients with fibrosing cholestatic hepatitis (FCH) after liver and kidney transplantation and to investigate their roles in the pathogenesis of FCH.

METHODSLiver biopsy specimens from five patients with FCH were studied histopathologically. A specific marker for CD4+CD25+ regulatory T cells in those specimens was detected with anti-FOXP3 monoclonal antibody by immunohistochemistry. Apoptoses of hepatocytes were detected with in situ apoptosis detection TUNEL kit.

RESULTSFibrosis in portal and around portal areas, cholestasis in some of the hepatocytes and canaliculi, widespread ballooning and ground-glass appearance of liver cells, and positivity of HBsAg and HBcAg and Pre-S1 protein were seen in the livers of all cases. The positive signal of FOXP3 was located in the cytoplasm of lymphocytes and the positive cells were mainly aggregated in the portal areas as well as occasionally appearing in the hepatic sinusoids. There were many more apoptotic hepatocytes near the portal areas.

CONCLUSIONFibrosing cholestatic hepatitis has specific pathological characteristics which might be caused by high expressions of FOXP3 in liver tissues.

Adult ; Apoptosis ; Biopsy ; Cholestasis, Intrahepatic ; immunology ; metabolism ; pathology ; Forkhead Transcription Factors ; metabolism ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Kidney Transplantation ; Liver ; immunology ; metabolism ; pathology ; Liver Transplantation ; Male ; Middle Aged ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVE: To investigate the expression levels of Th9, Th17 and Treg cells in peripheral blood of patients with chronic rhinosinusitis with nasal polyps (CRSwNP), and explore the role of Th9, Th17 and Treg cells in the progression of CRSwNP. METHOD: Forty-six cases with CRSwNP served as an experimental group, while 22 cases with simple nasal bleeding or nasal septum deviation served as a control group. The peripheral blood of patients in both groups was collected and analyzed. (1) Using flow cytometry (FCM) to detect the expression rates of Th9, Th17 and Treg cells in peripheral blood. (2) Using qRT-PCR to detect the expression of relevant transcription factor of Th9, Th17 and Treg cells (IL-9mRNA, PU. 1, IRF-4, RoRc, and Foxp3). (3) Using SPSS16.0 to analyse the differentiations and the revelance among these three cells. RESULT: (1) The expression rates of Th9 and Th17 cells in patients with CRSwNP (1.29% ± 0.18%, 4.03% ± 0.69%) was higher than the control group (0.45% ± 0.14%, 1.35% ± 0.26%). But the expression rates of Treg cells in the experimental group (2.98% ± 0.13%) was significantly lower than the control group (5.44% ± 0.57%). The differences were statistically significant (P < 0.05). (2) The expression of revelant transcription factor (IL-9mRNA, PU.1, IRF-4, RoRc) in NP group was also higher than the control group. The expression of Foxp3 in the control group was higher than NP, the differences both were statistically significant (P < 0.05). (3) The difference between Th9 and Th17 in patients with NP was not significant (P > 0.05), and the negative correlation was found between Th17 and Treg (r = -0.549, P < 0.05). CONCLUSION The high expression level of Th9 and Th17 cells might promote the development of NP, whereas the low expression level of Treg cells might further aggravate the occurrence of NP. The main function of the imbalance of Th17/Treg cells may be immune regulation in the pathogenesis of nasal polys.
Case-Control Studies ; Cell Differentiation ; Disease Progression ; Epistaxis ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Humans ; Nasal Polyps ; immunology ; pathology ; Nasal Septum ; abnormalities ; Rhinitis ; immunology ; pathology ; Sinusitis ; immunology ; pathology ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology ; Transcription Factors ; metabolism

The aim of this study was to evaluate whether the Th17 and Treg cell infiltration into allograft tissue is associated with the severity of allograft dysfunction and tissue injury in acute T cell-mediated rejection (ATCMR). Seventy-one allograft tissues with biopsy-proven ATCMR were included. The biopsy specimens were immunostained for FOXP3 and IL-17. The allograft function was assessed at biopsy by measuring serum creatinine (Scr) concentration, and by applying the modified diet in renal disease (MDRD) formula, which provides the estimated glomerular filtration rate (eGFR). The severity of allograft tissue injury was assessed by calculating tissue injury scores using the Banff classification. The average numbers of infiltrating Treg and Th17 cells were 11.6 +/- 12.2 cells/mm2 and 5.6 +/- 8.0 cells/mm2, respectively. The average Treg/Th17 ratio was 5.6 +/- 8.2. The Treg/Th17 ratio was significantly associated with allograft function (Scr and MDRD eGFR) and with the severity of interstitial injury and tubular injury (P < 0.05, all parameters). In separate analyses of the number of infiltrating Treg and Th17 cells, Th17 cell infiltration was significantly associated with allograft function and the severity of tissue injury. By contrast, Treg cell infiltration was not significantly associated with allograft dysfunction or the severity of tissue injury. The results of this study show that higher infiltration of Th17 cell compared with Treg cell is significantly associated with the severity of allograft dysfunction and tissue injury.
Acute Disease ; Creatinine/metabolism ; Forkhead Transcription Factors/metabolism ; Graft Rejection/*etiology/pathology ; Humans ; Immunoenzyme Techniques ; Interleukin-17/*metabolism ; Kidney Transplantation/*adverse effects ; Retrospective Studies ; T-Lymphocytes, Regulatory/*immunology/pathology ; Th17 Cells/*immunology/pathology ; Transplantation, Homologous

OBJECTIVETo explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice.

METHODSB16 melanoma cells were treated with cisplatin at the final concentration of 20 µg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-β were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin (100 µg/mouse i.p., for sequential 3 days) and intratumoral injection of DC cells (3×10(6)/mouse, twice with a 7-day interval) in the tumor-bearing mouse models was evaluated. Expression of MHC II, ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination. Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation. The distribution of T-reg and CD8(+) T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8(+) T/T-reg was determined. The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay.

RESULTSCisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC II, ICAM-1 and CD86 on DC cells were (47.5 ± 8.8)%, (35.5 ± 8.3)% and (36.2 ± 9.2)%, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was (2.1 ± 0.6) g, that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g, showing a significant inhibition of tumor growth (P < 0.01). Furthermore, the CD8(+) T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine. When the effector-to-target ratio was 20:1, 10:1 and 5:1, the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)%, (22.0 ± 6.0)% and (14.0 ± 4.0)%, respectively, significantly higher than (8.2 ± 3.6)%, (6.7 ± 1.8)% and (3.6 ± 1.9)%, respectively, in the control group (all P < 0.01).

CONCLUSIONCisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; B7-2 Antigen ; metabolism ; CD8-Positive T-Lymphocytes ; pathology ; Cancer Vaccines ; pharmacology ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Dendritic Cells ; immunology ; metabolism ; Female ; Genes, MHC Class II ; HMGB1 Protein ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; T-Lymphocytes, Cytotoxic ; immunology ; T-Lymphocytes, Regulatory ; pathology ; Tumor Burden ; drug effects

As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and economic value. We developed a low protein serum-free medium for large-scale GS-NS0 myeloma cell culture and anti-CD25 monoclonal antibody production. Further study focused on the characteristics of GS-NSO cell growth, glucose and amino acid metabolism, and antibody production. In the serum-free medium, the maximal viable cell density and antibody concentration reached above 3x10(6) cells/mL and 300 mg/L in batch culture. Compared with the commercial serum-free medium (Excell 620 + 0.2% Primatone), the maximal viable cell density doubled and the maximal antibody concentration increased 46%. Results also showed the specific growth rate decreased when the glucose concentration was lower than 6 mmol/L. And the production of lactate increased when glucose concentration was excessively high (> 30 mmol/L). These results were important to provide technique and theory basis for developing optimized GS-NS0 cell culture and anti-CD25 monoclonal antibody production processes.
Antibodies, Monoclonal ; biosynthesis ; immunology ; Cell Line, Tumor ; Culture Media, Serum-Free ; pharmacology ; Culture Techniques ; methods ; Humans ; Interleukin-2 Receptor alpha Subunit ; immunology ; Multiple Myeloma ; metabolism ; pathology ; Recombinant Proteins ; biosynthesis ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo observe the pathology of AIDS-related lymphadenopathy and its relationship to the expression and distribution of CD4 + CD25 + regulatory T cells in lymphoid node tissue.

METHODSTotally 22 biopsy and 13 autopsy lymphoid node tissues from HIV-positive patients were examined under microscopy and pathological staging was performed. Specific marker for CD4 + CD25 + regulatory T cells in lymphoid node tissue was detected with anti-Foxp3 monoclonal antibody by immunohistochemistry.

RESULTSAmong all the 35 specimens, 5, 4, 14, and 12 specimens were histopathologically staged from 1 to 4, respectively. FoxP3 were detected in all lymphoid node tissues. The distribution of FoxP3-positive lymphocytes were mainly in intermediate zone of follicle and cortical area in stages 1 and 2. The counts of FoxP3-positive lymphocytes remarkably decreased in stages 3 and 4, following depletion of lymphocytes.

CONCLUSIONSCD4 + CD25 + regulatory T cells exist in lymphoid node tissue of patients with HIV infection. Their amounts decrease or deplete along with the progression of AIDS-related lymphadenopathy.

Acquired Immunodeficiency Syndrome ; immunology ; pathology ; Adult ; CD4 Lymphocyte Count ; Female ; Forkhead Transcription Factors ; analysis ; Humans ; Immunohistochemistry ; Lymph Nodes ; immunology ; pathology ; Lymphatic Diseases ; immunology ; Male ; Middle Aged ; T-Lymphocytes, Regulatory ; metabolism

OBJECTIVETo study the relationship between the change of regulatory T cell number in CD4+ T subset and the growth of tumor in H22 hepatocellular carcinoma-bearing mice.

METHODSTumor-bearing mice were established by subcutaneous inoculation of H22 hepatocelluler carcinoma cells. Flow cytometry was used to detect the expression of CD4 and CD25 molecules of the T cells which came from the tumor-bearing mice. The Foxp3 gene expression was detected by RT-PCR and flow cytometry. CD4+ CD25+ T cells and CD4+ CD25- T cells were separated and purified by immuno-magnetic beads. The proliferation and suppressive function of the CD4+ CD25+ T cells coming from tumor-bearing mice was measured by [3H]-thymidines incorporation experiment in vitro, and then effect of CD4+ CD25+ T cells originated from hepatocellular carcinoma-bearing mice on tumor growth was observed in vivo.

RESULTS(1) Compared with mice of the control group, the percentage of CD4+ CD25+ T cells of CD4+ T cells in tumor-bearing mice is not only higher in draining lymph nodes (18.80% < or = 0.06%) vs. (9.50% +/- 0.03%), (P < 0.01), but also higher in non-draining lymph nodes (LN) and spleen (SP), LN: (16.28% +/- 0.02%) vs. (9.50% +/- 0.03%), P < 0.01; SP: (17.28% +/- 0.06%) vs. (11.08% +/- 0.04%), (P < 0.05). The expression of regulatory T cell specific marker Foxp3 gene was also increased. In the same tumor-bearing mice, the number of CD4+ CD25+ T cells in draining lymph node was relatively higher than the contralateral nondraining lymph node, but the difference was statistically not significant (18.8% +/- 0.06%) vs. (16.28% +/- 0.02%), (P > 0.05). (2) The CD4+ CD25+ T cells purified from tumor-bearing mice--like naturally occurring regulatory T cells--were anergic to anti-CD3 monoclonal antibody stimulation in vitro, but it could suppress CD4+ CD25- T cells proliferation. (3) The percentage of CD4+ CD25+ T cells was positively related to tumor size. It could also suppress the anti-tumor effect of CD4+ CD25- T cells in vivo. Conclusion The growth of hepatocellular carcinoma in mice can boost the amount of regulatory T cells. The amount of regulatory T cells is positively related to tumor size, indicating that attack on regulatory T cells could be used as one of modalities in cancer treatment in the future.

Animals ; CD4 Antigens ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Female ; Flow Cytometry ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Interleukin-2 Receptor alpha Subunit ; immunology ; Liver Neoplasms ; immunology ; metabolism ; pathology ; Liver Neoplasms, Experimental ; immunology ; metabolism ; pathology ; Lymph Nodes ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism