Thymic hormonal activity on human peripheral blood lymphocytes, in vitro. I. Reciprocal effect on T and B rosette formation.

One hour incubation with the thymic extract TP-1 induced reciprocal effect on B and T rosette formation in lymphocytes of human peripheral blood. The percentage of mouse erythrocyte rosette-forming cells among lymphocytes of chronic lymphatic leukaemia was decreased by TP-1 from 54.5% to 27.1% (P < 0.001). No such effect was observed in healthy adult or cord blood lymphocytes. On the other hand, the percentage of sheep erythrocyte rosette forming cells increased significantly after TP-1 treatment, but only under conditions of active rosette formation and not in the total rosette assay. This increase was highly significant in three conditions with relative deficiency of cell-mediated immunity: newborns (17.1 to 28.3%), cancer patients (24.5 to 31.7%) and patients with lepromatous leprosy (19.8 to 31.8%). Only a small increase was noticed in healthy adults. A similarly prepared spleen extract was not active in either B or T rosette assays.     

A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E. coli (ATCC 11303) rosette test

A new bacterial rosette technique for enumerating T lymphocytes is described. E. coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 μl) and after incubation at 4°C, slides are made, stained and counted. The nature of the lymphocytes forming E. coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E. coli and E, mouse E rosette and Fc receptor tests, and by mixed E. coli rosette tests and anti-Ig staining. E. coli and E rosette tests in controls and pediatric patients were also compared. The results show that T_μ and T_γ cells rosette with E. coli.     

Enumeration of T lymphocytes subsets in autoimmune disease using monoclonal antibodies.

The numbers of T lymphocytes, B lymphocytes, helper and suppressor T lymphocytes were measured in peripheral blood of patients with autoimmune disease (rheumatoid arthritis, erythema nodosum, Sjögren''s disease, Wegener''s disease, idiopathic thrombocytopenia, pernicious anaemia and Hashimoto''s disease). B lymphocytes were enumerated by direct immunofluorescence and T lymphocytes by E rosette tests and by indirect immunofluorescence with OKT3.PAN Helper and suppressor T lymphocytes were determined by indirect immunofluorescence with OKT4.IND and OKT8.SUP respectively. The numbers of T lymphocytes, B lymphocytes and helper T lymphocytes in patients with autoimmune disease were normal, but the numbers of suppressor T lymphocytes were significantly lower.     

重症肌无力患者外周血中CD4~+CD25~+调节性T细胞的变化及意义

研究CD4 + CD2 5 + 调节性T细胞在重症肌无力 (MG )发病中的作用。本文采用三色流式细胞术对 2 9例MG患者和 2 3例健康对照者外周血中CD4 + CD2 5 + T细胞 (CD3+ CD4 + CD2 5 + )的百分率进行测定。结果显示病情未能很好控制的MG患者外周血CD4 + CD2 5 + T细胞比率略低于健康对照组 (分别为 3 79%± 1 4 0 %、 4 5 3%± 0 96 % ,P =0 12 ) ,病情稳定或缓解的MG患者CD4 + CD2 5 + T细胞比率 (8 4 5 %± 1 96 % )显著高于健康对照组 (P =0 0 0 0 1) ;胸腺切除的MG患者CD4 + CD2 5 + T细胞比率 (8 4 4 %± 2 39% )显著高于非胸腺切除的MG患者 (5 88%± 2 89% ,P =0 0 38)和健康对照组 (4 5 3%± 0 96 % ,P =0 0 0 3)。提示MG患者外周血中存在异常比例的CD4 + CD2 5 + 调节性T细胞 ,可能参与疾病的发生与发展。     

REACTIVITY OF LYMPHOCYTE SUBPOPULATIONS IN HUMAN MIXED LYMPHOCYTE CULTURE

Loss of antigenicity in the mixed lymphocyte culture (MLC) reaction of lymphocytes precultured at 22°C for 7–10 days was accompanied by a decrease in bone-marrow-derived lymphocytes (B cells) from 22 ± 1% to 13 ± 1%, and an increase in thymus-derived lymphocytes (T cells) from 65 ± 2% to 83 ± 1% (P < 0.001). Depletion of B cells from a fresh lymphocyte suspension by either antihuman immunoglobulin-coated column fractionation or by sheep red blood cell (SRBC) rosette formation resulted in a significant reduction of the cell's ability to stimulate in MLC (P < 0.001). Coating of lymphocytes with rabbit antihuman brain serum abrogated their ability to respond but not the ability to stimulate in MLC.     

慢性乙肝病人外周血T细胞表面HLA-DR抗原的表达

本文采用单克隆抗体间接免疫荧光法,对15个慢性活动性肝炎病人以及1对照者外周血中T淋巴细胞膜表面的HLA-DR表达进行了测定。结果是慢性乙型活动性人外周血中的TDR~+细胞的百分率明显高于正常对照(P<0.001),并且增高的T细胞是以性细胞为主。     

T and B lymphocytes in myasthenia gravis.

Peripheral blood lymphocytes from seventeen non-thymectomized and nine thymectomized patients with myasthenia gravis (MG) and thirteen healthy controls were examined for the presence of surface markers characteristic of T and B lymphocytes by rosette formation with sheep red blood cells (SRBC). T cells were identified by their capacity to spontaneously form rosettes with SRBCs. The percentage of B lymphocytes was determined by the erythrocyte antibody complement (EAC) rosette-forming test. The EAC complex was prepared with either whole rabbit anti-SRBC serum or with the IgM fraction of rabbit anti-SRBC serum. The two kind of erythrocyte complement rosette-forming cells (EAC-RFC) are designated erythrocyte-haemolysin-complement RFC (EA(H)C-RFC), and erythrocyte-IgM-complement RFC (EA(M)C-RFC). The percentage of total lymphocytes and T cells was not altered in MG patients. The percentage of 'active' T cells, which have been considered to be more actively involved in cellular immunity, was also similar in MG patients and controls. A significant increase in EA(H)C-RFC occurred in both thymectomized and non-thymectomized MG patients, while in B cells detected by EA(M)C-RFC no alterations were found. The increase in EA(H)C-RFC in lymphocytes from MG patients may be due to an increase in the 19S antibody-forming B lymphocytes or to an increase in T cells which have Fc receptors on their surface.     

Enumeration of T cells, B cells and monocytes in the peripheral blood of normal and lymphocytotic cattle.

The rosette-forming capacity of bovine peripheral blood lymphocytes (PBL) was determined with dextran and 2-aminoethylisothiouronium bromide (AET)-treated sheep erythrocytes (SRBC). Both dextran and AET-enhanced rosette formation; however, AET-treated SRBC detected a larger percentage of rosette-forming cells and thus was used in this study. The specificity of rosette formation by bovine thymus-derived (T) lymphocytes was shown by (1) demonstration of rosettes and surface-membrane immunoglobulins sIg) on different cells in PBL and nylon-wool fractionated lymphocyte populations and (2) rosette formation by a large percentage (83--90%) of thymocytes from three bovine foetuses and two 14-month-old heifers. A procedure was also developed to identify bovine monocytes by latex phagocytosis and 10--30% latex-ingesting cells were detected in PBL preparations isolated by Ficoll-Hypaque flotation. The frequency of sIg-bearing latex-ingesting, and sIg-bearing latex non-ingesting cells in bovine peripheral blood was also determined. These procedures were utilized to determine the distribution of T and bone-marrow derived (B) lymphocytes in peripheral blood of normal and lymphocytotic cattle. PBL from twenty normal cattle contained approximately 63% T and 11% B (sIg+ latex non-ingesting) lymphocytes. In peripheral blood of three cattle with persistent lymphocytosis, a prodromal stage of bovine leukaemia, the percentage of B cells was elevated approximately to 59% whereas T lymphocytes decreased to 35%, thus providing additional evidence that persistent lymphocytosis is a B-cell disease.     

Analysis of B-lymphocyte differentiation in patients infected with hepatitis C virus

To clarify whether some of the functions of B lymphocytes could be affected during hepatitis C virus (HCV) infection, phenotypic characteristics of B lymphocytes from HCV-infected patients and their capacity to differentiate into immunoglobulins (Ig)-secreting cells were studied. B lymphocytes differentiation was investigated for patients untreated and non-responders (n = 9), treated and non-responders (n = 6), responders (n = 6), long-term responders (n = 9) to therapy and seronegative controls (n = 14) following in vitro stimulation with S. aureus strain Cowan I mitogen. HCV sequences in purified B lymphocytes were detected by RT-PCR. It was found that HCV-patients harbor a similar mean percentage of B cells and a normal level of naïve B cells (% IgM⁺/IgD⁺ cells = 79.7 ± 15.4 for untreated non-responders, 57.1 ± 22.9 for treated non-responders, 44.3 ± 29.1 for responders, 75.7 ± 16 for long-term responders) as compared with controls. It was also found that peripheral blood mononuclear cells (PBMCs) of patients or controls produced similar amounts of IgG, A, and M in vitro. A total of 57% of untreated non-responders versus 17% of treated non-responders were able to produce HCV-specific antibodies. Interestingly, B lymphocytes from PBMCs able to secrete anti-HCV antibodies contained HCV positive strand RNA, although no systematic detection of the negative strand was found. These data suggest that signaling through the B cell receptor (BCR) in B lymphocytes of HCV-infected patients appears normal whatever their response to therapy. The capacity to secrete HCV-specific IgG seemed to be linked to the presence of positive strand RNA rather than virus replication. J. Med. Virol. 72:566–574, 2004. © 2004 Wiley-Liss, Inc.     

Rosette formation with mouse erythrocytes by lymphocytes from normal donors and patients with various diseases.

Rosette formation of human lymphoid cells with mouse erythrocytes has recently been proposed as a marker for a subpopulation of B lymphocytes. In this work we studied the percentage of mouse rosette forming cells (MRFC) in normal and pathological conditions and compared them to the percentage of sheep rosette forming cells (SRFC) a marker for T lymphocytes. Peripheral blood lymphocytes (PBL) from normal donors contained 6.2 +/- 1.1% (mean +/- 1 S.D.) MRFC. High percentages of MRFC were found in CLL patients, and a slight increase was observed in patients with systemic lupus erythematosus. MRFC were absent in Bruton's type agammaglobulinaemia, but were normally present in patients with T cell defects. Cryopreservation of lymphocytes in 10% DMSO did not significantly affect the mean percentages of SRFC and MRFC, though a slight increase of the former and a small reduction of the latter was observed. Double binding experiments on peripheral blood lymphocytes showed a predominant association of MRFC with cells staining for surface IgM and/or IgD. In all samples tested, we also observed a small population of MRFC negative for sIgM or sIgD and a few sIgM or sIgD positive cells that did not rosette with mouse erythrocytes.     

Peripheral blood T and B lymphocytes in patients with thyrotoxicosis and Hashimoto''s thyroiditis and in normal subjects

Lymphocytes were separated from the peripheral blood of three groups of subjects (normal controls, untreated thyrotoxicosis and confirmed Hashimoto's thyroiditis) by two separation methods: dextran sedimentation and Ficoll–Triosil gradient centrifugation. It has been shown that there is a selective loss of T lymphocytes (as measured by sheep red cell rosettes) with relative enrichment of B lymphocytes (as measured by surface immunoglobulins) in the Ficoll–Triosil-separated suspensions. This distortion of the T/B ratio was seen to a similar extent in each of the three groups of subjects. Furthermore, the mean percentage T and B lymphocytes of both patient groups were not significantly different from those of the controls when separated by the same method. Optimal E-rosette formation occurred after prolonged incubation at 4°C and in the absence of serum. Direct counts using Toluidine Blue were superior to indirect counts with unstained rosette suspensions.     

Polyclonal B Cell Activation and Antigen-Driven Antibody Response as Mechanisms of Autoantibody Production Insle

We studied 16 patients affected by autoimmune hemolytic anaemia (AIHA), both idiopathic and associated with other diseases (B and T lymphoma, B hepatitis, gastric carcinoma, systemic lupus erythematosus) or a-methyldopa therapy, in order to value T- and B-cell activation. We determined the count of T- and B-cell subsets in peripheral blood, the proliferative response of peripheral blood lymphocytes (PBL) to phytohe-magglutinin (PHA) and to pokeweed mitogen (PWM), the percentage of CD25 ⁺ cells in culture and interleukin (1L)-lα, IL-2, IL-4, tumor necrosis factor (TNF)α and soluble IL-2 receptor (sIL-2R) levels in sera and in culture. Except for an increase in CD4 ⁺ and CD8 ⁺ T cell number in a case of AIHA associated with a T lymphoma and an increase in the percentage of CD5 ⁺ and PCAl⁺ B cells in two cases of AIHA associated with B lymphoma and with SLE, no further data showed a relationship with the disease possibly associated with AIHA, so both idiopathic and secondary AIHA cases were analyzed together. CD4 ⁺ T cells were reduced in number in 9 cases, while CD8 ⁺ T cells were reduced in 6 cases. The percentage of CD5 ⁺ B cells was increased in 5 cases. The percentage of PCAl⁺ cells was increased in all cases (mean ± sd: 18 ± 22 vs 0,2 ± 1 in controls). The average PBL proliferative response to PHA was reduced (S.I. 71 ± 55 vs 138 ± 45 in controls) as well as that to PWM (S.I. 27 ± 21 vs 75 ± 24 in controls), despite IL-2 high levels, in all cases, in both sera (meanfsd: 648 ± 351 pg/ml vs 16 ± 4 pg/ml in controls) and culture supernatants (meanksd: 1045 f 677 pg/ml vs 195 ± 51 pg/ml in controls). In PHA stimulated cultures the percentage of CD25⁺ cells was reduced (meanasd: 37 ± 18 vs 63 ± 14 in controls), sIL-2R levels were like controls in 7 cases. In sera sIL-2R levels were increased in all cases (meanfsd 1256 ± 465 U/ml vs 256 ± 114 U/ml in controls), IL-lα was increased in all cases too, while IL-4 levels were increased only in 7 cases. Linear regression analysis generally showed a low relationship between S.I. and IL-2, IL-4 and sIL-2R levels in supernatants of PHA stimulated culture as well as between S.I. and the percentage of CD25 ⁺ cells. Taken together these data suggest a state of B- and T-cell hyperactivation in AIHA. The low PBL proliferative response in vim, explained in previous studies as a temporary functional exhaustion, might be itself a sign of the complete lymphocyte activation occurring in vivo in AIHA.     

Serotonin and the immunologic reactivity of the body in taeniasis

Blood serotonin level, 5-HIAA in 24 h urine, serum immunoglobulins-IgG, IgM, IgA, percentage of lymphocytes T and B in rosette test were ascertained in 3 groups of patients with taeniosis, treated with Radeverm (32 patients), Praziquantel (22 patients) and Yomesan (10 patients). Primary hyperserotoninemia and higher level of 5-HIAA in post-treatment values were not uniformly differentiated. The same tendency was observed in post-treatment values of immunoglobulins and lymphocytes T and B as compared with values before treatment.     

Surface markers on lymphocytes of multiple sclerosis patients.

Peripheral blood lymphocytes of twenty-two multiple sclerosis (MS) patients and thirty-five healthy controls were examined for the presence of surface markers characteristic for B lymphocytes (surface immunoglobulin, receptor for C3 (EAC), reporter for Fc (EA) and the spontaneous rosette-forming capacity characteristic of T cells. The results obtained indicate that the number of B and T cells in MS is similar to controls, as evaluated by the presence of surface immunoglobulin and E rosette-forming capacity. However, a statistically significant reduction in the percentage of lymphocytes bearing C3 receptors has been found in MS patients. It might have resulted from a reduction in the lymphocyte population bearing C3 receptor but no surface immunoglobulin. The EA rosette test revealed the greatest difference between the groups. The difference indicated a reduction in the density of the receptor for 7S Fc on lymphocytes from MS patients. The results obtained are consistent with the hypothesis of an immune deficit in multiple sclerosis.     

Altered Proportion of Tμ and Tγ-Cell Subpopulations in Patients with Hodgkin''s Disease

The ability of peripheral blood lymphocytes from patients with Hodgkin's disease (HD) to form rosettes with ox red blood cells (ORBC) sensitized by anti-ORBC purified rabbit IgM and IgG was investigated. The mean percentage of cells capable of forming rosettes with ORBC coated with IgM (EAIgM-RFC) in the peripheral blood of either untreated or X-ray-treated patients with HD was significantly lower than that of normal individuals. In the same groups of patients with HD the mean percentage of T lymphocytes equipped with receptor for IgG (T gamma lymphocytes), evaluated by a mixed fluorescent rosette assay, was significantly higher than in normal controls. These data suggest that the altered proportion between T mu-and T gamma-cell subpopulations in patients with HD probably represents a disease-related phenomenon.     

Distribution of peripheral blood T and B lymphocyte markers in atopic children and changes during immunotherapy.

Peripheral blood lymphocytes from children undergoing evaluation for allergic disease were examined for T and B lymphocyte markers. Patients were evaluated at intervals to determine differences in these markers between atopic and nonatopic children and relative changes during immunotherapy. T lymphocytes were identified by the sheep RBC rosette technique. Surface immunoglobulin was detected by immunofluorescence following incubation with fluorescein-labeled antihuman IgG, IgA, IgM, and IgE. At initial examination, atopic patients differed from controls only in the increased percentage of lymphocytes bearing surface IgM. There were no differences between patient and control values in T lymphocyte distribution or in cells bearing surface IgG, IgA, or IgE at any point in the study. The increased percentage of IgM-bearing lymphocytes is reduced to the control level after four months of immunotherapy but remains elevated in the untreated atopic group. Serum IgM levels remained constant. This study shows that the distribution of lymphocyte markers may be altered in atopic children. Patients treated with immunotherapy demonstrated a return to control values of IgM-bearing lymphocytes. The elevated serum IgE seen in atopy was not reflected in an elevated percentage of IgE-bearing lymphocytes.     

Higher Percentage of CD3+CD154+ T Lymphocytes Predicts Efficacy of TNF-α Inhibitors in Active Axial SpA Patients

The objective of this study was to evaluate which subtypes of T lymphocytes (CD3⁺CD28⁺ and CD3⁺CD154⁺) could predict clinical efficacy after TNF-α inhibitor treatment in active axial SpA patients. Patients who fulfilled Assessment of SpondyloArthritis international Society (ASAS) criteria for axial SpA had a BASDAI of ≥40 mm. All patients received TNF-α inhibitor treatment for 12 weeks. ASAS20 was used to evaluate the effect of the treatment at week 12. We detected the percentage of CD3⁺CD28⁺ and CD3⁺CD154⁺ T lymphocytes on lymphocyte cells in the peripheral blood in patients and healthy controls. We evaluated whether the percentage of the above subtypes of T lymphocytes could predict clinical efficacy by ROC curve analysis. Fifty-eight healthy controls and 74 active axial SpA patients were included. Mean age was 26.28?±?9.08 and 26.95?±?8.13 years for healthy controls and patients, respectively (p?=?0.767). The percentage of CD3⁺CD154⁺ T lymphocytes was significantly higher in axial SpA patients than in healthy controls (1.62?±?1.89 % vs 0.79?±?0.52 %, p?+CD154⁺ T lymphocytes was significantly higher in HLA-B27(⁺) patients than HLA-B27(^?) ones (HLA-B27⁺ vs HLA-B27^?:1.77?±?1.95 % vs 0.41?±?0.27 %, p?=?0.005). Compared with baseline, the percentage of CD3⁺CD154⁺ T lymphocytes significantly decreased to 0.87?±?0.49 % at week 12 (p?+CD154⁺ T lymphocytes could predict clinical efficacy of SpA patients with TNF-α inhibitor treatment (AUC?=?0.733, p?=?0.014). High percentage of CD3⁺CD154⁺ is over-expressed on lymphocytes in peripheral blood of active SpA patients and can be down-regulated by TNF-α inhibitor therapy. High-percentage of CD3⁺CD154⁺ T lymphocytes may predict clinical efficacy of TNF-α inhibitor treatment in active axial SpA patients.     

The Status of Tumor Immunology and Cancer Immunotherapy

To explore if the increased percentages of Regulatory T (Treg) cells, as well as, overexpression of Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) are involved in laryngeal-squamous cell carcinoma (SCC), 45 patients with laryngeal-SCC and 27 healthy controls were enrolled. Flow cytometry was performed to investigate, in the peripheral blood, the prevalence of CD4+CD25+FoxP3+ Treg cells, as well as, surface and intracellular expression of CTLA-4 by the main lymphocyte subsets (CD4+, CD8+ and CD19+). The results indicated intracellular (In)CTLA4 with considerable higher expression in the CD8+ lymphocytes among patients with laryngeal-SCC compared with the control group (8.2 ± 8.7 versus 2.3 ± 3.5, P = 0.001). The mean percentage of InCTLA4+CD4+ and InCTLA4+CD19+ lymphocytes was also significantly higher in patients (8.7 ± 7.8 versus 4.4 ± 4.2, P = 0.018 and 0.6 ± 0.8 versus 0.2 ± 0.2, P = 0.024, respectively). With respect to surface (Sur)CTLA4, the difference between patients and controls was, however, significant only in the case of CD8+ lymphocytes (0.7 ± 0.6 versus 0.3 ± 0.3, P = 0.003, respectively). The percentage of Treg cells was observed to be significantly higher in patients (7.5 ± 6.3 and 3.2 ± 1.9, P < 0.0001). Furthermore, association analysis revealed the association of Treg cell increase with the higher tumor-size and lymphnode stage (P < 0.005). These data collectively suggest that patients with laryngeal-SCC may benefit from immunotherapy targeting CTLA4 and Treg cells.     

Levamisole and human lymphocyte surface markers.

The action of various concentrations of levamisole on normal human lymphocytes was investigated in vitro. The action of levamisole (10(-1) mg-10(-8) mg%) upon rosette formation was studied using two rosette assays with SRBC (the active and the total T-rosette tests) to quantify T cells and the EAC assay to quantify B cells. Levamisole at only 10(-3) mg% significantly increased the percentage of both active and total T cells. In sharp contrast, levamisole between 10(-2) and 10(-7) mg% significantly decreased the EAC percentage. It is concluded that levamisole promotes a better expressivity of T-cell receptor and decreases the expressivity of C3 receptor.     

传染病医院不同年龄职工细胞免疫状态的分析

目的 分析传染病医院健康职工不同年龄段T、B细胞亚群表达的差异,探讨其与年龄及工作环境的关系.方法 采用多色免疫荧光标记和流式细胞术对不同年龄体检职工(分为A、B、C、D四组)外周血T、B细胞水平进行检测,比较各组差异.结果 A、B、C、D各组CD3+T细胞占淋巴细胞的百分比分别为(65.48±5.22)％、(66.24±6.03)％、(66.15±7.11)％及(65.29±5.43)％,各组之间表达水平相近,无统计学差异;CD3+ CD4+T细胞占T淋巴细胞的百分比分别为(33±1.84)％、(31.57±2.11)％、(34.63±2.56)％及(41.53±3.41)％,A、B、C各组两两比较无统计学差异,但D组与其它三组比较均有统计学差异(P＜0.05);各组CD3+ CD28-T细胞占T细胞的百分比分别为(17.84±1.21)％、(21.81±1.43)％、(22.37±1.56)％及(14.22±1.36)％,D组分别与B、C两组比较差异具有统计学意义(P＜0.05);B细胞的百分比分别为(12.8±2.21)％、(11.79±1.79)％、(11.33±1.86)％及(8.17±2.36)％,差异具有统计学意义(P＜0.05).结论 不同年龄层次的职工机体的免疫功能存在差异.