First report of expansion of CD4+/CD28 null T-helper lymphocytes in adult patients with idiopathic autoimmune hemolytic anemia

ABSTRACT CD28 null T helper (Th) cells are rare in healthy individuals, but they are increased in various inflammatory and immune-mediated diseases. In this study, we determined the size of the CD4+/CD28 null T lymphocyte compartment in the peripheral blood of 40 autoimmune hemolytic anemia (AIHA) patients (idiopathic and secondary) and 20 healthy control subjects, using tri-color flow cytometry. The frequency and absolute count of CD4+/CD28 null T helper (Th) cells was significantly higher in idiopathic AIHA patients, compared to healthy controls (p = 0.001 and 0.001, respectively) and to patients with secondary AIHA (p = 0.04 and 0.01, respectively). The percentage of CD4+/CD28 null Th cells was also negatively correlated to the hemoglobin (Hb) level (p = 0.03). These findings demonstrate, for the first time, the expansion of this phenotypically-defined population of T lymphocytes in patients with idiopathic AIHA and indicate that it likely plays an etiological role in the development of this disease. However, establishing the use of this marker for diagnosis or monitoring treatment of such patients needs further studies.

CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Distribution of Telocytes in Vital Organs of ApoE Mice / 中国医学科学院学报

Objective To identify and verify the distribution of Telocytes derived from heterogeneous interstitial cells in the vital organs of ApoE mice.Methods Heart,kidney,and liver tissues were harvested from ApoE adult mice. Immunohistochemical assays were performed by using different immunobiological markers.Results Telocytes were found in these vital organs. The expressions of immunobiological markers differed among different organs. CD34,CD117,and CD28 were positively expressed in Telocytes in cardiac tissue;CD117 and plateled-derived growth factor-Α were negatively expressed in Telocytes in renal tissue;and CD117 and plateled-derived growth factor receptor-Α had negative expression in Telocytes in hepatic tissue. Furthermore,the distribution of Telocytes also differed in the same organ.Conclusions Telocytes exist in the vital organs of ApoE mice,as demonstrated by immunohistochemisty assay. The expressions of immunobiological markers differ among Telocytes in different organs.

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

Changes of CD8+CD28- T cell percentage in patients with multiple injuries and their clinical significance / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the correlation of the changes in CD8(+)CD28(-) T cell percentage with platelet (PLT) and D-dimer (D-D) levels in patients with multiple injuries (MI).</p><p><b>METHODS</b>Twenty-six patients with MI, 31 with a single injury (SI group) and 26 healthy individuals were examined for peripheral blood CD8(+)CD28(-) T cells and intracellular transformation growth factor-β1 (TGF-β1) and interleukin 10 (IL-10) contents using flow cytometry at 24, 48, and 72 h after the injuries. PLT and D-dimer levels were compared among the 3 groups.</p><p><b>RESULTS</b>CD8(+)CD28(-) T cells, TGF-β1 and IL-10 were significantly higher in MI group than in SI group and healthy control group (P<0.05) without significant differences between the latter 2 groups. The levels of PLT and D-D differed significantly among the 3 groups, the highest in MI group and the lowest in the control group. In MI group, CD8(+)CD28(-) T cells, TGF-β1 and IL-10 significantly increased at 48 h after the injury (P<0.05) but decreased significantly at 72 h (P<0.05) compared with the measurements at 24 h. The levels of PLT and D-D trended to decrease with time after the injuries and showed significant differences among the 3 groups at any of the 3 time points (P<0.05). CD8(+)CD28(-) T cells, TGF-β1 and IL-10 were all positively correlated with the levels of PLT and D-D in MI patients (r>0.70, P<0.05 for all comparisons).</p><p><b>CONCLUSION</b>In MI patients, CD8(+)CD28(-) T cell percentage and their cytokines tend to increase early after the injury but decrease significantly at 72 h in close relation with the changes of the coagulation function following the injuries.</p>

Influence of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice / 中国实验血液学杂志

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.

Effects of blocking co-stimulatory signals on immunotolerance rejection of sensitized recipient after hematopoietic stem cell transplantation / 中国实验血液学杂志

This research was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on engraftment of hematopoietic stem cell in the sensitized recipient so as to provide the experimental evidence for the treatment of sensitized recipient's immune rejection after clinical allogeneic hematopoietic stem cell transplantation (HSCT). The BALB/c mice were divided into 4 groups: (1)mice sensitized on 7 day before transplant; (2)mice were sensitized on 7 day before transplant, and injected CTLA4Ig+anti-CD154 applied; (3)normal mice injected by corresponding isotype control IgG of CTLA4Ig and anti-CD154; (4)normal blank control mice. Each group had 15 mice. On day 0, mice of each group were irradiated lethally 8 Gy by linear accelerator, and the bone marrow cells of C57BL/6 labeled by fluorescence staining were injected via the tail vein. The fluorescent cell level in peripheral blood and organ tissue at different time points were detected by flow cytometry (FCM) for homing assessment. Survival rates and hematopoietic reconstitution were also monitored and recorded. The results showed that application of CTLA4Ig anti-CD154 could promote implantation of allogeneic HSC in sensitized recipients, induce the immune tolerance, prolong their survival time and accelerate the hematopoietic reconstitution within 28 days, compared with the sensitized group. It is concluded that applying CTLA4Ig and anti-CD154 can enhance the engraftment of HSCT and induce immune tolerance in the sensitized recipient after allogeneic HSCT and accelerate the hematopoietic reconstitution.

Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses

OBJECTIVE@#To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8(+) CD28(+) cytotoxic T lymphocyte (CTL) responses.@*METHODS@#Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2K(b): Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8(+)CD28(+) CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs.@*RESULTS@#Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8(+)CD28(+)CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K(b): Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells.@*CONCLUSIONS@#The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8(+) CD28(+) CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

Expression of PD-1/PD-L1 in peripheral blood mononuclear cells in lung cancer patients and its biological significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To analyze the expression of co-stimulatory molecules PD-1/PD-L1 in peripheral blood mononuclear cells in lung cancer patients, and to explore its biological significance.</p><p><b>METHODS</b>One hundred and thirty-three lung cancer patients, 25 lung infection patients and 23 healthy donors were enrolled in this study. 100 µl of whole blood from these subjects were collected. Multi-color immunofluorescence staining and flow cytometry were used to detect PD-1/PD-L1 expression. The results were statistically analyzed.</p><p><b>RESULTS</b>The expression level of CD3⁺CD8⁺ T cells in the lung cancer patients was (38.83 ± 1.74)%, significantly lower than that in the control group [(43.25 ± 3.35)%, P < 0.05]. CD8⁺CD28⁺ T cell subset in the peripheral blood of lung cancer patients was (17.73 ± 1.21)% significantly lower than that of the healthy donors [(27.96 ± 2.72)%, P < 0.01]. The CD8⁺CD28⁻ T cell subset was (21.19 ± 1.92)% in the lung cancer patients, significantly higher than that of the healthy control group [(15.18 ± 2.93)%, P < 0.05]. The expression level of PD-1 on the surface of CD8⁺CD28⁺ T cells was (10.67 ± 1.12)% in the group of lung cancer patients, significantly higher than that of the control group [(5.32 ± 1.58)%, P < 0.01]. It was also found that the expression of PD-1 on CD8⁺CD28⁻ T cells was up-regulated in the group of lung cancer patients (7.46 ± 1.25)%, significantly higher than that of the healthy control group [(2.68+1.07)%, P < 0.01]. The expression level of PD-L1 on CD68⁺ cells in the lung cancer patients was (16.03 ± 2.06)%, significantly higher than that of the healthy control group [(9.32 ± 2.00)%, P < 0.05].</p><p><b>CONCLUSION</b>Up-regulation of PD-1/PD-L1 on peripheral blood cells in lung cancer patients negatively regulates the lymphocytes, inhibits the immune response for killing tumor cells, and promotes tumor development and immune escape.</p>

Anti-human 4-1BBL monoclonal antibody stimulates the nuclear translocation of NF-κB and the co-location of 4-1BBL/CD28 isoform in U937 cells / 中国实验血液学杂志

This study was purposed to investigate the molecular mechanism of 4-1BBL reverse signals in the human acute monocytic leukemia cell line of U937. The U937 cell line was used as target cells, and stimulated by the mouse anti-human 4-1BBL monoclonal antibody 1F1. The nuclear translocation of NF-κB and the co-location of 4-1BBL and CD28i molecules in U937 cells were observed with confocal laser scanning microscopy. The protein and m-RNA expression levels of 4-1BBL and CD28i were detected by flow cytometry and RT-PCR respectively. The results showed that the significant nuclear translocation of NF-κB and co-localization of 4-1BBL and CD28i on membrane of U937 cells appeared after being stimulated by mAb1F1. It is concluded that the 4-1BBL reverse signals transduction mediating the growth of U937 cells relates with the nuclear translocation of NF-κB. CD28i may be involved in intracellular 4-1BBL reverse signaling pathways.

The "time-window" effect of early allergen exposure on a rat asthma model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The hygiene hypothesis has been proposed to explain the pathogenesis of asthma. Allergen exposure was shown to inhibit asthma in an animal model. But the optimal timing of allergen exposure remains unclear. This study aims to explore the time effcct of allergen exposure and the possible mechanisms.</p><p><b>METHODS</b>Neonate Wistar rats were randomly divided into asthma group, control group and day 1, day 3, day 7, and day 14 groups. The day 1, day 3, day 7 and day 14 groups were injected with ovalbumin (OVA) subcutaneously on days 1, 3, 7 and 14 after birth, respectively. Six weeks later, all groups, except the control group, were sensitized and stimulated with OVA to make the asthma model. We observed the pulmonary pathologic changes, detected the regulatory T cells, and CD28 expression level in thymus and spleen by flow cytometry.</p><p><b>RESULTS</b>The asthmatic inflammation in the day 1, day 3 and day 7 groups, but not the day 14 group, was alleviated. The asthma group and day 14 group had lower proportions of regulatory T cells in the thymus compared with the control group, day 1, day 3, and day 7 groups. There was no significant difference in the CD28 expression levels on the regulatory and conventional T cells among groups. But the control group and the day 1, day 3, and day 7 groups had relatively higher proportions of CD28 positive regulatory T cells in the thymus than the day 14 group and the asthma group.</p><p><b>CONCLUSIONS</b>There is a "time-window" for early allergen exposure. The impairment of regulatory T cells may promote the development of asthma. Allergen exposure in the "time-window" can make the thymus produce normal quantity of regulatory cells. The CD28 signal on regulatory T cells may participate in the production of regulatory T cells.</p>

Repressor of GATA-3 can negatively regulate the expression of T cell cytokines through modulation on inducible costimulator / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The transcription factor, repressor of GATA-3 (ROG), can simultaneously suppress the expression of T helper cells (Th1 and Th2) cytokines. Since the suppression of Th2 cytokines by GATA-3 is well understood, it is postulated that there are other molecular targets of ROG that can suppress the expression of the Th1 cytokines. We hypothesized that ROG might suppress the stimulators of T lymphocyte cytokines such as CD3, CD28, and inducible costimulator (ICOS), or indirectly enhance the expression of cytokine suppressors such as T lymphocyte-associated antigen-4 (CTLA-4) and CD45. The objective of this study was to clarify the molecular targets of ROG involved in suppressing Th1 or Th2 cytokines.</p><p><b>METHODS</b>Real-time quantitative PCR (RT-PCR) and Western blotting were performed to evaluate the mRNA and protein levels of CD3, CD28, ICOS, CTLA-4, and CD45 in Th1 and Th2 cells during various levels of ROG expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of interferon-γ (IFN-γ) and interleukin (IL)-4 in culture media of Th1 and Th2 cells.</p><p><b>RESULTS</b>The results showed that the mRNA and protein levels of ROG were relatively low in Th1 and Th2 cells (P < 0.01). After ROG-pcDNA3.1 transfection, the mRNA and protein level of ROG was significantly elevated, while the expression of ICOS, IFN-γ, and IL-4 was markedly down-regulated (P < 0.01). Conversely, transfection of ROG-siRNA led to inhibition of ROG expression and up-regulation of ICOS, IFN-γ and IL-4 (P < 0.01). However, the expression levels of CD3, CD28, CTLA-4 and CD45 did not change in either ROG-pcDNA3.1 or ROG-siRNA-transfected Th1 and Th2 cells (P > 0.05).</p><p><b>CONCLUSION</b>It is concluded that ROG can inhibit the expression of Th1 and Th2 cytokines by down-regulating the expression of ICOS, which might be a potential molecular target for asthma treatment.</p>

Suppression of acute rejective response following orthotopic liver transplantation in experimental rats infected with Echinococcus multilocularis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hepatic alveolar echinococcosis (AE) is a parasitic disease in humans and caused by the Echinococcus multilocularis (Em). Orthotopic liver transplantation (OLT) may be the only effective treatment for end-stage hepatic AE. However, in some AE patients, extrahepatic Em can not be completely eliminated after OLT. We aimed to study whether the immunological changes caused by Em evasion may influence the rejective response.</p><p><b>METHODS</b>Rat modles of AE were established by injecting the Em suspension into abdomen of Brown Norway (BN) rats. Three months later, in the experimental group, the liver was transplanted from Lewis (LEW) rats to Em-infected BN rats. In the control group, transplantation was from LEW rats to healthy BN rats. Liver tissue and peripheral blood (PB) samples were collected on days 1, 3, 5, and 7 after OLT. Liver tissue was analyzed after hematoxylin and eosin (H&E) staining; numbers of CD4, CD8, and CD28 on peripheral blood cells were detected by flow cytometry; and expression of the chemokine fractalkine (Fkn) was detected by reverse transcription PCR (RT-PCR). Interleukin-10 (IL-10) was measured in the serum by enzyme-linked immunosorbent assay (ELISA). In every group, eight BN rats were retained for observing survival time.</p><p><b>RESULTS</b>The survival times of recipients in the experimental group were prolonged compared with those in the control group. The rejective response occurred later and was milder in the experimental group. percentage of CD4, CD8, CD28 T-cells and Fkn mRNA expression were lower in the experimental group. While the serum IL-10 levels were higher in the experimental group than those in the control group.</p><p><b>CONCLUSIONS</b>Acute rejective response after OLT was attenuated in the rats with Em infection, and the recipients` survival time was prolonged. Em may play a role in this process by elevating IL-10 secretion, decreasing the effector T cells, inhibiting the expression of Fkn, which lead to reduce the inflammatory cells infiltration into the liver.</p>

Anti-cD20scFv/CD80/CD28/zeta specific T lymphocytes eradicate primary chronic lymphocytic leukemia cells in vitro / 中国应用生理学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>(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.</p>

Expression of co-stimulators in ulcerative colitis and its pathologic significance / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression and localization of co-stimulators in the mucosa of patients with ulcerative colitis (UC), and to explore its role in the pathogenesis of UC.</p><p><b>METHODS</b>Expression of co-stimulators CD86 and inducible co-stimulator (ICOS) was studied by immunohistochemistry on paraffin-embedded mucosal tissue from patients with active UC (64 cases), inactive UC (51 cases) and normal controls (20 cases). Immunostaining for CD28 was also carried out on frozen fresh mucosal tissue sampled from patients with active UC (7 cases), inactive UC (2 cases) and normal controls (5 cases). In addition, expression of CD4, CD8 and CD20 were also examined.</p><p><b>RESULTS</b>In active UC, increased expression of CD86 was not only observed in lamina propria mononuclear cells but also in the intestinal epithelial cells, as compared with inactive UC and the normal controls (P < 0.01). Increased ICOS expression in lamina propria mononuclear cells was detected in active UC, as compared with inactive UC and the normal controls (P < 0.01). Increased ICOS expression in intestinal epithelial cells was also seen in active UC, as compared with that of inactive UC (P < 0.01). The expression of CD86 was higher in inactive UC than in the normal controls (P < 0.05 or P < 0.01). However, the expression of ICOS showed no statistically significant difference between inactive UC and normal controls. Increased expression of CD28 in active UC, compared with that in inactive UC and normal controls, was also noticed (P < 0.05 or P < 0.01). The number of CD4 or CD8-positive intraepithelial lymphocytes and lymphocytes infiltrating in the lamina propria and small vessel walls was much higher in active UC than in inactive UC and normal controls (P < 0.01). Moreover, the ratio of CD4/CD8 was highest in active UC (P < 0.01). The number of CD20-positive B lymphocytes in lamina propria was also higher in active UC than in inactive UC and normal controls (P < 0.01).</p><p><b>CONCLUSIONS</b>In active UC, CD86 and ICOS were over-expressed in the intestinal epithelial cells and lamina propria mononuclear cells. The phenomenon suggests that abnormal expression of co-stimulators may contribute to the deregulation of acquired immune responses in UC.</p>

Altered CD28 and CD95 mRNA expression in peripheral blood mononuclear cells from elderly patients with primary non-small cell lung cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The expression of the co-stimulatory molecule CD28 and death receptor CD95 on T cells, which change with age, are considered as important immunological parameters of immunosenescence. It is well established that CD28 and CD95 are associated with tumorgenesis and tumor progression, but the relationship between the age-related changes of these two immunological markers and cancer in the elderly is largely unknown.</p><p><b>METHODS</b>The levels of CD28 and CD95 mRNA in peripheral blood mononuclear cells (PBMCs) from sixty-three elderly patients (aged > or = 60 years) with primary non-small cell lung cancer (NSCLC) were analyzed by real-time fluorescence-based quantitative polymerase chain reaction (FQ-PCR). In addition, twenty young patients (aged < 60 years) with NSCLC, thirty elderly healthy donors and thirty young healthy donors were enrolled as controls.</p><p><b>RESULTS</b>CD28 mRNA levels were significantly lower and CD95 mRNA levels were significantly higher in elderly patients with NSCLC than in the other groups. Similar results were found in elderly healthy donors comparing with young healthy donors. By Logistic regression analysis an increased risk of NSCLC was markedly associated with aging, down-regulation of CD28 mRNA and up-regulation of CD95 mRNA, and CD28 mRNA had an obvious negative correlation with the CD95 mRNA. In addition, the mRNA levels of CD28 and CD95 in the peripheral blood of the elderly patients was closely associated with the tumor node metastasis (TNM) stages, grade of cell differentiation and lymph node metastasis status, but not related to pathological types.</p><p><b>CONCLUSIONS</b>The results suggest a close relationship between T cell senescence and NSCLC tumour progress in the elderly, and that up-regulation of CD28 mRNA or down-regulation of CD95 mRNA in peripheral blood T cells may play an important role in inhibiting oncogenesis and development of primary NSCLC in the elderly.</p>

Flow cytometric study of T-cell subsets and costimulatory molecules in clinical forms of human schistosoma mansoni chronic infection

Helminthic parasites cause widespread, persistent infections in humans. Schistosomiasis mansoni infected patients being in a chronic immune-activation state enabled us to investigate the effects of such immune activation on immune responses. We performed by flow cytometry aphenotypic analysis of peripheral blood T lymphocytes from 64 Schistosoma mansoni infected patients, in different clinical forms of the chronic disease. The main findings in the patient group in comparison with the non-infected controls were: [i] decreased CD3, CD4 and CD8 lymphocyte counts; [ii] elevated levels of activated T cells [CD4 expressing HLA-DR]; [iii] decreased numbers of CD28+ CD8+ lymphocytes. These findings support the notion that chronic helminthic infections cause persistent immune activation that result in hyporesponsiveness and anergy. Such impaired immune functions may diminish the capacity of these individuals to cope with infections and to generate cellular protective immunity after vaccination

Kv1.3 potassium channel expression changes after CD4(+) and subsets CD28(null)/CD28(+)T cells activation in peripheral blood of patients with acute coronary syndrome / 中华心血管病杂志

<p><b>OBJECTIVE</b>To study the Kv1.3 channel expression changes after CD4(+) and subsets CD28(null)/CD28(+)T cells activation in peripheral blood of patients with acute coronary syndrome (ACS).</p><p><b>METHODS</b>CD4(+)T cell in 27 ACS patients and CD4(+)CD28(null)/CD4(+)CD28(+)T cells in 12 out of these 27 ACS patients were isolated from peripheral blood with magnetic cell sorting. The whole-cell Kv1.3 currents for three T cells were recorded with patch-clamp technique before and 72 hours after activation by purified anti-human CD3 Interferon gamma, tumor necrosis factor alpha (TNF-alpha), granzyme B mRNA expression were determined by reverse transcription-PCR before and 72 hours after activation by purified anti-human CD3 in the presence or absence of recombinant Margatoxin (rMgTX, 0.1, 1, 10 nmol/L), a specific Kv1.3 channel blocker.</p><p><b>RESULTS</b>Peak Kv1.3 channel currents of CD4(+), CD4(+)CD28(null), CD4(+)CD28(+)T cells were significantly increased and the mean Kv1.3 channel numbers per cell of these cells were increased by about 90%, 60%, 80% (402 +/- 88 vs. 752 +/- 275, 553 +/- 328 vs. 874 +/- 400, 392 +/- 133 vs. 716 +/- 251, all P < 0.05) after activation compared to baseline values. Baseline CD4(+)CD28(null)T cell numbers were about 40% more than those of CD4(+)CD28(+)T cell (P < 0.05) and were similar after activation (P = 0.102). The mRNA expression of interferon gamma, TNF-alpha and granzyme B were dose-dependently down-regulated by rMgTX.</p><p><b>CONCLUSIONS</b>Kv1.3 channels of peripheral CD4(+)T cell and CD28(null)/CD28(+)T cells from ACS patients significantly increased after activation and Kv1.3-specific channel blocker rMgTX could effectively abolish this effect suggesting a potential role of Kv1.3 channel blocker on plaque stabilization in ACS patients.</p>

A study of costimulatory molecules in allergic allergic rhinitis patients / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the expression of costimulatory molecules CD28/B7 and CD40/CD40L in T and B lymphocytes as well as its relations with total IgE (TIgE), eosinophil cationic protein (ECP) in serum and nasal allergic symptoms in patients with allergic rhinitis (AR). The effect of specific immunotherapy (SIT) on them were also investigated.@*METHOD@#Thirty allergic allergic rhinitis patients were chosen as observation group, and 30 healthy patients as control group. Cytofluorometric analysis was used to compare the expression level of CD28/B7-1, B7-2 and CD40/CD40L on T cells and B cells in the two groups. The relationship between the CD28/ B7-1, B7-2 and CD40/CD40L expression level and serum Total IgE, ECP level were analyzed.@*RESULT@#The expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells in allergic rhinitis patients were significantly higher than in the healthy, and serum level of TIgE has a positive relationship with the expression level of CD40L on T cells. ECP has a positive relationship with the expression level of B7-2 on B cells. The expression level of B7-1 showed no significant difference between the two groups. After specific immunotherapy for 6 months, the expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells were decreased in allergic rhinitis patients but still higher than in healthy.@*CONCLUSIONS@#The upregulated level of costimulatory molecules CD28/B7-2 and CD40/ CD40L on T cells and B cells may play an important role in the pathogenesis of allergic rhinitis, specific immunotherapy can downregulate the expression level of CD28/B7-2 and CD40/CD40L, and decrease the serum level of TIgE, it may be a possible mechanism in the treatment of allergic rhinitis.

Mechanism of enhancement of the CTL activity in mice co-expressing CD80, CD86 and CD137L genes / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the mechanism of enhancement of the CTL activity in mice co-expressing of CD80, CD86 and CD137L genes.</p><p><b>METHODS</b>The mice were randomly divided into five groups, named A, B, C, D and E. The group A and B were control groups (CG). H22-BAL B/c HCC mouse model was established by subcutaneous injection with hepatocellular carcinoma cells of cell line H22-Wt (group A), H22-neo (group B), H22-CD80/CD86(+) (group C), H22-CD137L(+) (group D) and H22-CD80/CD86/CD137L(+) (group E), respectively. On the 14th, 35th, 56th and 84th day after the first inoculation of tumor cells, TUNEL staining and DNA ladder examination were used to detect apoptosis of splenic T lymphocytes in all groups at each post-inoculation time point. Electrophoretic mobility-shift assay (EMSA) method was used to detect the activity of nuclear factor kappaB (NF-kappaB) in splenic T lymphocytes in each group at each time point post-inoculation.</p><p><b>RESULTS</b>Apoptosis was found in a great number of T lymphocytes in CG on the 14th day, much more than that in group C and E. The number of apoptotic T cells in group C had a significant difference compared with that in the group E from 14th to 84th day (P = 0.003). DNA ladder analysis showed typical positive results in group C and E. The significant apoptosis fragments were found in group C on 21st, 35th and 84th days. NF-kappaB activity of T cells in groups C and E was remarkably higher than that of groups CG and D, with higher in group D than that of CG (P = 0.002), and with no significant difference between group C and E on 14th day. The activity in group E was stable and remarkably higher than that of group C on 56th and 84th days after the first inoculation.</p><p><b>CONCLUSION</b>H22-CD80/CD86/CD137L(+) induces higher NF-kappaB activity of the host T cells by synergistic action of CD28 and CD137, which may be one of the mechanisms of enhancement of the host CTL activity induced by co-expression of CD80, CD86 and CD137L genes.</p>