Clinical, Laboratory, and Bone Marrow Findings of 31 Patients With Waldenström Macroglobulinemia

BACKGROUND: Waldenström macroglobulinemia (WM) is a subset of lymphoplasmacytic lymphoma (LPL) with bone marrow (BM) involvement and an IgM monoclonal gammopathy of any level. We aimed to identify the clinical, laboratory, and BM findings of patients with WM and to evaluate the usefulness of CD154 for the diagnosis and prognosis of WM.METHODS: We reviewed the medical records and BM studies and/or flow cytometric immunotyping of 31 patients with untreated WM. Semiquantitative immunohistochemistry (CD20, CD138, tryptase, and CD154) of BM was performed.RESULTS: Only six patients presented with symptoms of hyperviscosity syndrome. Eleven patients had solid cancer and/or another hematologic malignancy. Mast cells (MC) increased in all samples, with some in close contact with tumor cells. Tryptase-positive MC (17.1/ high-power fields [HPF], 1.2–72.0/HPF) and CD154-positive MC (8.6/HPF, 0.1–31.1/HPF) were observed. The high CD154-positive MC (≥8.6/HPF) group showed a lower overall five-year survival rate than the low CD154-positive MC (<8.6/HPF) group (71.9% vs. 100.0%; P=0.012). Flow cytometric immunophenotyping of BM aspirates showed increased B lymphocytes and plasma cells with a normal phenotype (CD138⁺/CD38⁺/CD19⁺/CD45⁺/CD56⁻).CONCLUSIONS: Approximately one third of WM patients showed other malignancies and all patients had increased MC. Immunohistochemistry and flow cytometric immunophenotyping are useful for diagnosing WM, and increased CD154-positive MC can indicate poor prognosis.

Effects of chimeric B7 antibodies on mononuclear cells in ankylosing spondlitis / 中华微生物学和免疫学杂志

Objective To study the effects of chimeric B7 antibodies on mononuclear cells from patients with ankylosing spondylitis (AS).Methods Chimeric antibodies of ch4E5 and chlD1 against B7 with a final concentration of 5 μg/ml were respectively added into culture media of mononuclear cells from patients with AS.Two other groups including huIgG-treated and untreated culture medium of mononuclear cells from patients with AS were set up as the controls.The culture medium of mononuclear cells from healthy subjects were prepared as normal control group.The disparities of mononuclear cell death in culture among each group were analyzed by using micro-lymphocyte cytotoxicity test (MLCT).The expression of membrane molecules were analyzed by FCM.The concentrations of cytokines in culture media were measured by ELISA.Results Both groups treated with the chimeric antibody showed lower scores for cell death than untreated group as indicated by MLCT (P＜0.01).FCM analysis demonstrated that both CD4/CD8 ratio (P ＜0.05) and membrane expression of CD154 (P＜0.01) decreased in chimeric antibody treated groups,but the number of CD4+CD25high Treg cells (P＜0.01) increased as compared with those in untreated group.The concentrations of IL-2 and TNF-α were down-regulated (P＜0.05) in chimeric antibody treated groups,while IL-4 and TGF-β were up-regulated (P＜0.02) as compared with those in untreated group.Conclusion Chimeric B7 antibodies might affect the expression of T cell membrane molecules and the cytokine production by mononuclear cells through B7/CD28 and CD4O/CD154 signaling pathways in patients with AS.

Detection of Foreign Antigen-specific CD4+Foxp3+ Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining

The unrestricted population of CD4+Foxp3+ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4+Foxp3+ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4+Foxp3+ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4+ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4+ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4+Foxp3+ Tregs using Foxp3GFP knock-in mouse, and found that LCMV GP66-77-specific CD4+Foxp3+ Tregs and polyclonal CD4+Foxp3+ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4+Foxp3+ Treg in various models.

Changes of T lymphocyte subsets and expression of costimulatory molecule CD154 on T-cells in peripheral blood from patients with ankylosing spondylitis / 中华风湿病学杂志

Objective To study T lymphocyte subsets and expression of costimulatory molecule CD154 on T-cells in peripheral blood from patients with ankylosing spondylitis and their changes after treated with Enbrel. Methods Sixty-six patients with AS(39 active and 27 inactive, 35 axial and peripheral joint involvement and 31 axial involvement only), 30 patients with rheumatoid arthritis(RA), 30 healthy volunteers were analyzed. The expression of CD154 on CD3+ T cell as well as T-cells subsets were evaluated using flow cytometry respectively. The changes of the expression of costimulatory molecule CD154 in 39 active AS patients(Enbrel treatment or placebo treatment) were observed in a randomized, double-blind, placebothan that of healthy volunteers, and CD154 expression on CD3+ T cells in peripheral blood in AS patients was on CD3+ T cells in the peripheral blood of active AS or AS patients with peripheral joint involvement were significantly higher than those in inactive or axial involvement only AS patients(P＜0.05), and CD154 on CD3+ placebo, in AS patients group, there was significant reduction in CD154 expression on CD3+ T cells in Enbrel group at week 6(P＜0.05), there was no significant difference between Enbrel group and healthy volunteers at week 6(P＞0.05). Conclusion T lymphocyte subsets are significantly abnormal and CD154 is overexpressed on T-cells in peripheral blood of patients with AS. A six-week course of treatment with Enbrel in active AS can induce a down-regulation of the expression of CD154 on T cells.

Platelets Induce Proliferation of Human Umbilical Vein Endothelial Cells via CD154-CD40 Pathway Independently of VEGF

BACKGROUND: Platelets take part in repairing the lesions of endothelial damage. To understand the molecular mechanism of this process, we tested the hypothesis that CD154 expressed on activated platelets stimulates proliferation of human endothelial cells. METHODS: The expression levels of CD154 and CD40 on platelets and endothelial cells, respectively, were measured by flow cytometry and confocal microscopy. Function-blocking monoclonal antibody against CD154 was developed after immunization with CD154- transfected L cells. RESULTS: An anti-CD40 agonist antibody and soluble CD154 both induced significant proliferation of endothelial cells. In addition, a function-blocking anti-CD154 antibody inhibited the platelet-induced proliferation of endothelial cells, indicating that the CD154-CD40 pathway is involved in these cellular interactions. An anti-VEGF antibody failed to inhibit the proliferation. This, in addition to the fact that very small amounts of VEGF are released from platelets or endothelial cells, suggests that VEGF does not play an important role in the platelet-stimulated proliferation of endothelial cells. CONCLUSION: Our results indicate that platelets induce proliferation of endothelial cells by CD154-CD40 interactions independently of VEGF.

CD40 and CD154 Expression in Hepatocellular Carcinomas

PURPOSE: CD40 expression has been reported in a variety of cells, including antigen presenting cells (dendritic cells), B lymphocytes, monocytes, and many epithelial malignancies (breast, lung, ovary, bladder, and melanoma). The interaction of CD40 and its natural ligand (CD154) is well known in the adaptive immune response. The CD40-CD154 pathway appears to have diverse effects in malignant disease. In this study, we investigated the expression of CD40 and CD154 and evaluated their clinical implication in hepatocellular carcinomas (HCC). METHOD: Immunohistochemical staining was performed for CD40 and CD154 in 96 surgically resected HCCs using the tissue microarray method. The clinicopathological data and outcomes were reviewed and compared to the expression of CD40 and CD154. RESULTS: Positive expression of CD40 and CD154 was observed in 64.2% and 37.5% of cases, respectively. Overexpression, defined by the stain intensity and area of CD40 staining had a positive correlation with tumor cell proliferation activity as measured by the Ki-67 labeling index (p=0.013) and CD154 had a similar tendency (p=0.094). CD40 overexpression was observed frequently in small sized HCCs (p=0.046). There were no other clinicopathological factors that were significantly correlated with CD40 and CD154 expression. CD40 overexpression was correlated with a poor overall survival according to the univariate analysis (p=0.054) and multivariate analysis (p=0.035). CD154 overexpression was not correlated with the overall survival rate. CONCLUSION: The results of this study showed that CD40 expression correlated with a more aggressive tumor potential in patients with HCC. CD 40 overexpression might provide a novel prognostic marker for survival in patients with HCC.

IL-2 Pathway Blocking in Combination with Anti-CD154 Synergistically Establishes Mixed Macrochimerism with Limited Dose of Bone Marrow Cells and Prolongs Skin Graft Survival in Mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.

IL-2 Pathway Blocking in Combination with Anti-CD154 Synergistically Establishes Mixed Macrochimerism with Limited Dose of Bone Marrow Cells and Prolongs Skin Graft Survival in Mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.

Role of NF-?B activation on spontaneous formation of germinal centers in spleen in BXSB mice / 中华风湿病学杂志

Objective To explore the role of NF-kB activation on spontaneous formation of germinal centers in spleen in BXSB mice and it's mechanisms.Methods Eighteen BXSB mice were divided to control group and pyrrolidine dithiocarbonate(PDTC)group randomly.PDTC group was given PDTC 120 mg/kg?BW ip every other day and control group was given the same dose of dissolving solution.NF-kB activity was deter- mined by electrophoretic mobility shift assay.Two color flow cytometry were used to detect CD154 expression on splenic B cells and germinal center B cells apoptosis.Germinal centers were stained for histochemical analysis.Results PDTC could inhibit the NF-kB activity in spleen tissue in BXSB mice.It decreased the NF-kB activity by 62.82%.Spontaneous germinal center formation was detected in spleen in BXSB mice.In- hibiting NF-KB activation could down-regulate CD154 expression on splenic B cell,retard spontaneous germi- nal center formation and increase germinal center B cell apoptosis.Conclusion NF-kB activation may induce spontaneous germinal center formation in spleen in BXSB mice by upregulating CD154 expression on splenic B cell and decreasing germinal center B cell apoptosis.The autoreactive B cells generated during spontaneous germinal center formation may escape apoptosis and then differentiate to autoantibody-producing plasm cells.It suggests that NF-kB can be a therapeutic target.

Abnormal expression of CD154 in the peripheral blood B lymphocytes from lupus patients and its implication in the pathogenesis / 中华风湿病学杂志

Objective To determine the expression level of CD154 in systemic lupus erythematosus(SLE) and its role in the pathogenesis. Methods CD154 were detected by flow cytometry in CD19+ peripheral blood lymphocytes from 16 active lupus patients and 14 healthy controls. Effects of anti-CD154, on the proliferation and IgG secretion by peripheral B lymphocytes were also observed. Results ① The positive rates and mean fluroscence intensity of CD154 in peripheral blood B lymphocytes from active lupus [(36?17)%,364?238] were significantly higher than from normal controls[(10?8)%, 124?97], (P

Effects of local gene transfection in CD154 extracellular domain on the survival of renal allografts / 第三军医大学学报

Objective To observe the efficacy of local gene transfection in CD154 extracellular domain on the survival of renal allografts. Methods The kidneys of Brown Norway (BN) rats were transfected with CD154 extracellular domain gene recombined adenovirus. The transfected kidneys were transplanted to Lewis rats (transfection group). BN→Lewis kidney transplantation with non transplanted kidneys served as the controls. The allograft survival time and the allograft function between the two groups were compared. Results The allograft survival time of the transfection group was longer than that of the controls significantly [(28?7.3)d vs (8.6?1.2) d, P

Cloning of hCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote / 中国病理生理杂志

AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 ?g/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.

Alloantigen-specific immunoregulatory function of anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways / 中国免疫学杂志

Objective:To investigate alloantigen-specific immunoregulatory function and phenotype of anergic cells induced by combined anti-CD154 and anti-CD80 monocolonal antibody(mAb) blocking.Methods:Anergic cells were generated in vitro by the addition of anti-CD154 and anti-CD80 mAbs to primary MLR (mixed lymphocyte reaction) consisting of BALB/C as responder and C3H as stimulator. Anergic or control cells were added to a newly formed MLR of naive BALB/C spleenocytes against the original (C3H) stimulator spleenocytes in assessing the regulatory capacity of anergic cells .Antigen specificity of the regulatory phenomenon was examined in MLR performed with third-party stimulator spleenocytes(C57BL/6J). To test the reversal condition of anergic cells,irradiated C3H spleenocytes,or recombinant mouse interleukin-2 (rmIL-2),or both C3H spleenocytes and rmIL-2 were added to the anergic cells. Anergic cells were phenotypically analyzed by double labeling procedure. Results:Anergic cells strongly suppressed the proliferation of naive BALB/C spleenocytes against the original (C3H) stimulator spleenocytes in a dose-dependent manner,but they failed to suppress the proliferation of naive BALB/C spleenocytes against the third-party stimulator spleenocytes(C57BL/6J).The anergic state was reversed by both original(C3H)stimulator and the addition of exogenous IL-2. There was an increased number of CD25 +CD4 +T cells observed in anergic cells,whereas there was no difference of CD45RB low CD4 + and CD28-CD8 +T cells between anergic and control cells.Conclusion:Anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways possess the alloantigen-specific immunoregulatory function and suppress the lymphocyte proliferation via infectious tolerance.

Effect of PRL on the expression of CD154 in Jurkat cells / 中华内分泌代谢杂志

Expression of PRL receptor mRNA in Jurkat D1.1 cell line was confirmed by RT PCR. CD154 expression of Jurkat cells was significantly increased when these cells were incubated with PHA and recombinant human PRL, suggesting that binding of PRL to its receptor promotes CD154 expression which plays a role in immune regulation.