Functional heterogeneities among concanavalin A-activated OKT4+ and OKT8+ cells by using autologous erythrocyte rosette technique.

Normal human peripheral blood T lymphocytes activated by concanavalin A (Con A) were fractionated into OKT4+ and OKT8+ populations by complement-dependent cell lysis using OKT8 and OKT4 antibodies, respectively. By using the preferential ability of some, but not all, Con A-activated T cells to form rosettes with autologous erythrocytes, each population was further divided into autorosetting cells and nonautorosetting cells, and thus Con A-activated OKT4+ autorosetting, OKT4+ nonautorosetting, OKT8+ autorosetting, and OKT8+ nonautorosetting cells were obtained. The immune regulatory function of these populations was then investigated using a pokeweed mitogen-driven B cell plaque-forming cell system. These studies demonstrated that (a) autorosetting cells can exert potent suppressor activity regardless of their phenotypes of OKT4+ and OKT8+ antigens, and fail to help B cell differentiation; suppressor function mediated by these cells is radiosensitive; moreover, receptors for autologous erythrocytes may constitute either the interleukin 2 (IL2) receptors themselves or a component of an IL2 receptor-effector complex involved in modulating the growth signal that IL2 transmits to T cells; (b) OKT4+ nonrosetting cells serve adequately as radioresistant helper cells, but are devoid of suppressor cells; and (c) OKT8+ nonrosetting cells are found to lack either suppressor or helper activity, suggesting that they may belong to a T lymphocyte subset distinct from the subsets related to immune regulation. The results lead us, therefore, to the conclusion that there may exist functional heterogeneities among both the OKT4+ and OKT8+ populations; these heterogeneities can be dissected by virtue of the autologous erythrocyte rosette technique.     

Antigen specific suppressor T cells from chronic hepatitis B virus (HBV) carriers inhibit the responsiveness to HBsAg of allogeneic high-responder lymphocytes

Immunoregulatory mechanisms in chronic HBsAg carriers have been investigated through the study of in vitro proliferative responses to HBsAg by allogeneic coculture experiments between T lymphocytes from HBsAg + chronic active hepatitis (CAH) patients (HBsAg no responder) and PBMC from subjects boosted with anti-hepatitis B vaccine (high responder). When high-responder PBMC have been challenged with the hepatitis B surface antigen (HBsAg) in the presence of HBsAg no-responder T lymphocytes, HBsAg no-responder T lymphocytes caused an antigen specific, dose-dependent, suppression of the responsiveness of high-responder PBMC. On the other hand, T cells from patients with autoimmune CAH did not exert any suppressor effect in our system. The suppressor T lymphocytes were mitomycin C resistant and were positive for OKT8, but were negative for OKT4. When T8 + cells were depleted from HBsAg no-responder PBMC, the in vitro immunoproliferative response to HBsAg in chronically HBV infected patients was markedly improved. Out data clearly demonstrate the existence of T8 + suppressor T lymphocytes that can control low responsiveness to HBsAg in chronic HBV patients.     

Hepatitis B core antigen specific CD4 response in peripheral blood

The proliferative response of peripheral blood CD4+ T cells to recombinant hepatitis B core antigen (rHBcAg) has been studied in patients with chronic active hepatitis (CAH) type B (CAH-B), CAH-nonA nonB, and normal volunteers. CD4+ T cells from patients with CAH-B indicated a significant proliferative response to rHBcAg in the presence of non-T antigen presenting cells. In contrast, there was no apparent T cell reaction to rHBcAg in patients with CAH-nonA nonB and healthy volunteers. We suggested the possibility of CD4-mediated HBcAg specific response even in the peripheral blood compartments of HBcAg-responsive CAH-B patients.     

Depressed effector activity of OKT4+ and OKT8+ T cell subsets in lectin-dependent cell-mediated cytotoxicity to HEP-2 cells in patients with systemic lupus erythematosus

The role of OKT4+ and OKT8+ T cell subsets was studied in depressed lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 target cells by peripheral blood mononuclear cells (PBMC) from patients with active systemic lupus erythematosus (SLE). LDCC activity was evaluated by detachment from the monolayer of 3H-TdR-prelabelled HEp-2 cells in a 24 hr assay at 50:1 effector-target cell ratio in the presence of 25 micrograms/ml concanavalin A (Con A). Decreased levels of LDCC were performed by all studied effector cell populations of SLE patients, including both OKT4+ and OKT8+ T cell fractions. LDCC by isolated OKT8+ T cells was superior to that by OKT4+ and unfractionated T lymphocytes from all healthy and SLE subjects. This suggests that the defect of LDCC activity in SLE did not affect the inherently higher LDCC effector activity of OKT8+ to OKT4+ cells. In parallel studies a reduced proliferation of PBMC in response to Con A and failure of OKT8+ T cells to suppress Con A-induced blastogenesis was observed in patients with SLE.     

Comparisons of peripheral blood and hepatic lymphocyte subpopulations and interferon production in chronic viral hepatitis.

Studies were undertaken to clarify the immunological mechanism in patients with chronic hepatitis C compared with chronic hepatitis B. Phenotypic study by flow cytometry showed that CD8+ T cells and HLA-DR+ cells isolated from liver biopsy specimen were significantly increased in their proportions as compared with those in peripheral blood, while intrahepatic CD4+ T cells were significantly lower than peripheral blood CD4+ T cells in both types of patients with chronic active hepatitis (CAH). Furthermore two-color analysis revealed that CD8+ CD11- and CD3+ HLA-DR+ cells were significantly elevated in liver tissue than in peripheral blood in both patients groups. In in vitro tests, mononuclear cells obtained from the liver of type B and type C CAH had a reduced capacity to produce IFN-gamma in response to pokeweed mitogen, while they produced equal amounts of IFN-alpha under stimulation with Newcastle disease virus as compared with control peripheral blood mononuclear cells. In contrast, spontaneous productions of both IFNs were greater in liver infiltrates of the two patients groups. These results indicate that functionally activated T cells are distributed in a similar manner in the liver of both chronic hepatitis B and C, suggesting that cytotoxic T cell plays a major role in the hepatocellular injury of both groups of patients.     

Ecto-5′-Nucleotidase Activity in Human T Cell Subsets. DECREASED NUMBERS OF ECTO-5′-NUCLEOTIDASE POSITIVE CELLS FROM BOTH OKT4+ AND OKT8+ CELLS IN PATIENTS WITH HYPOGAMMAGLOBULINEMIA

T lymphocytes from control subjects were separated into subsets using monoclonal antibodies of the OKT series and complement lysis and analyzed for ecto-5'-nucleotidase activity both by quantitative radiochemical assay and a histochemical stain. T cells from 15 control subjects contained 54+/-4% OKT4(+) (helper/inducer) cells and 32+/-3% OKT8(+) (cytotoxic/suppressor) cells. Total T cell ecto-5'-nucleotidase activity was 10.9+/-2.1 nmol/h per 10(6) cells with 25+/-7% positive by histochemical stain. Ecto-5'-nucleotidase activity in OKT4-enriched populations was 5.43+/-1.8 nmol/h per 10(6) cells with 14+/-2% positive by histochemical stain; that in OKT8-enriched populations was 17.1+/-5.9 nmol/h per 10(6) cells with 35+/-8% positive by histochemical stain.Two of four patients with congenital agammaglobulinemia and four of seven patients with common variable immunodeficiency had decreased proportions of OKT4(+) T cells with corresponding increases in the proportions of OKT8(+) T cells (OKT4/OKT8 = 0.60 to 1.0 as compared with 1.7+/-0.2 for control subjects). All four patients with congenital agammaglobulinemia, and three of seven patients with common variable immunodeficiency also had low T cell ecto-5'-nucleotidase activity (<5.5 nmol/h per 10(6) cells). Ecto-5'-nucleotidase activity in OKT4- enriched populations isolated from four patients with low total T cell activity was 2.85+/-0.90 nmol/h per 10(6) cells with 10+/-4% positive by histochemical stain; that in OKT8-enriched populations was 6.82+/-1.7 nmol/h per 10(6) cells with 7.5+/-3% positive by histochemical stain. Thus, the number of ecto-5'-nucleotidase positive cells is decreased, especially in the OKT8(+) subpopulation, and the low total T cell ecto-5'-nucleotidase activity seen in these patients is due to fewer positive cells rather than to substantially less activity per cell.Our data indicate that ecto-5'-nucleotidase activity defines two subpopulations of T lymphocytes (ecto-5'-nucleotidase positive and negative), the proportions of which are markedly altered in many patients with hypogammaglobulinemia. In preliminary studies with seven patients, increased numbers of ecto-5'-nucleotidase negative T cells appeared to correlate with increased suppressor T cell activity toward in vitro immunoglobulin synthesis. Therefore, ecto-5'-nucleotidase may be a useful cell surface marker in the study of imbalances of regulatory T cell subsets in patients with antibody synthesis disorders.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. IV. Induction of suppressor cells within the OKT4+ population

In this report, we explored the functional heterogeneity within the OKT4+ subset of human T cells. Evidence was obtained that although in vitro pokeweed mitogen-activated OKT4+ cells can function as radioresistant helper cells, these activated OKT4+ cells could also exert potent feedback suppression. Despite the induction of suppressor cells after pokeweed mitogen activation, the OKT4+ population maintains its original OKT3+, OKT4+, nd OKT8- surface phenotype. The suppressor cells contained within the activated OKT4+ population were found to be radiosensitive. Importantly, the suppression mediated by activated OKT4+ cells required the presence of radiosensitive cells contained within the resting OKT4+ population. Taken together, these results suggest that the OKT4+ subset of human T cells contains cells that can be activated to differentiate into suppressor cells independent of OKT8+ cells.     

Control of polyclonal immunoglobulin production from human lymphocytes by leukotrienes; leukotriene B4 induces an OKT8(+), radiosensitive suppressor cell from resting, human OKT8(-) T cells.

We report that leukotriene B4 (LTB4), a 5-lipoxygenase metabolite of arachidonic acid, is a potent suppressor of polyclonal Ig production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood lymphocytes, while LTC4 and LTD4 have little activity in this system. Preincubation of T cells with LTB4 in nanomolar to picomolar concentrations rendered these cells suppressive of Ig production in subsequent PWM-stimulated cultures of fresh, autologous B + T cells. This LTB4-induced suppressor cell was radiosensitive, and its generation could be blocked by cyclohexamide but not by mitomycin C. The LTB4-induced suppressor cell was OKT8(+), while the precursor for the cell could be OKT8(-). The incubation of OKT8(-) T cells with LTB4 for 18 h resulted in the appearance of the OKT8(+) on 10-20% of the cells, and this could be blocked by cyclohexamide but not by mitomycin C. Thus, LTB4 in very low concentrations induces a radiosensitive OKT8(+) suppressor cell from OKT8(-) cells. In this regard, LTB4 is three to six orders of magnitude more potent than any endogenous hormonal inducer of suppressor cells previously described. Glucocorticosteroids, which block suppressor cell induction in many systems, may act by inhibiting endogenous production of LTB4.     

不同亚型乙型病毒性肝炎患者T细胞亚群调查研究

目的研究不同亚型乙型肝炎患者外周血T淋巴细胞亚群变化及其临床意义。方法选择40例慢性活动性乙型肝炎、30例乙型肝炎病毒携带者、30例乙型肝炎肝硬化、20例急性乙型肝炎患者和30例健康对照者,采用流式细胞仪检测各组外周血T淋巴细胞亚群的表型和频率。结果与正常对照组比较,慢性活动性肝炎、肝炎肝硬变患者外周血CD4 、CD4 /CD8 比值均有显著性下降(P<0.01),以肝炎肝硬变下降最显著,慢性病毒携带者肝炎、急性乙型肝炎外周血T细胞亚群数量和比值变化不明显(P>0.05)。HBV DNA病毒载量与患者外周血CD4 T细胞数量无相关性(P>0.05)。结论慢性活动性乙型肝炎和肝硬化患者的细胞免疫功能降低,其下降程度与病毒复制水平无相关性。     

慢性乙型病毒性肝炎患者CD5^＋B细胞的变化

目的 研究CD5^＋B细胞在慢性乙型病毒性肝炎发病中的作用．方法 采用免疫荧光双标记技术和流式细胞仪对39例慢性乙型病毒性肝炎患者和18例健康时照者周围血中CD5^＋B细胞（CD5^＋CD19^＋）的百分率进行测定，同时检测慢性乙型病毒性肝炎患者肝功能的变化。结果 在慢性乙型病毒性肝炎患者周围血CD5^＋B细胞百分率显著高于对照组，而且在慢性乙型病毒性肝炎患者各分组（按肝功能损害程度分组）中CD5^＋B细胞均明显高于对照组；但在慢性乙型病毒性肝炎患者各分组之间CD5^＋B细胞百分率无显著性差异。结论 CD5^＋B细胞与慢性乙型病毒性肝炎发病有关，但与肝功能损害程度无显著相关。     

"Anti-Ia reactivity in sera from patients with chronic active hepatitis (CAH)"

Anti-Ia reactivity in sera from patients with chronic active hepatitis (CAH) were characterized by determining cross-reacting specificities with the antigen defined by anti-Ia monoclonal antibody (MoAb) and by studying the effect of CAH sera on the autologous mixed leukocyte reaction (MLR). Preincubation with autoimmune CAH sera lowered the percentage of Ia+ non-T cells stained by anti-Ia MoAb. HBsAg+ve/HBeAg+ve sera did not exert any blocking activity while 4 out of 11 HBsAg+ve/anti-HBe+ve sera exerted a significant blocking effect. Preincubation of cells with normal human serum (NHS) plus aggregated IgG did not block the binding of MoAb anti-Ia. Sera from patients with autoimmune or HBsAg+ve/anti-HBe+ve CAH, that blocked the binding of anti-Ia MoAb to Ia positive target cells by more than 20%, clearly inhibited the autologous mixed lymphocyte reaction (MLR). Both IgG and IgM fractions obtained by affinity chromatography from CAH sera inhibited the autologous MLR and blocked the binding of anti-Ia antibody to Ia positive target cells. A significant positive correlation (p less than 0.001) between serum anti-Ia reactivity and serum liver membrane antibodies (LMA) was observed. In 4 "autoimmune" CAH patients, steroid treatment induced a dramatic decrease in the anti-Ia reactivity.     

CD4^＋、CD25^＋调节性T细胞在重型乙型肝炎发病中的作用

目的探讨CD4＋、CD25＋调节性T细胞（CD4＋、CD25＋ Tr）在重型乙型肝炎病人发病机制中的作用。方法采用流式细胞术检测重型乙型肝炎病人及对照组外周血CD4＋、CD25＋ Tr的水平及应用PCR测定重型乙型肝炎病人及对照组外周血中HBVDNA滴度。结果重型乙型肝炎组外周血CD4＋、CD25＋ Tr的百分率为（2.63±0.83）%,较慢性乙型肝炎组的（4.15±1.17）%差异有显著性（P〈0.05）,较无症状HBV携带者组及健康对照组差异有非常显著性（P均〈0.01）;重型乙型肝炎组外周血HBVDNA滴度为1.2×10^4copies/ml,较慢性乙型肝炎组（2.3×10^6copies/ml）和无症状HBV携带者（7.8×105copies/ml）差异有非常显著性（P均〈0.01）;不同乙型肝炎病人外周血CD4^＋、CD25^＋ Tr的百分率与HBVDNA滴度正相关。结论CD4^＋、CD25＋^ Tr能抑制T细胞对HBV的免疫反应;重型乙型肝炎患者发病与CD4^＋、CD25^＋ Tr表达过低有关。     

OKT4+ and OKT8+ cell subset values and HLA-DR phenotypes

Blood levels of T cell subsets evaluated by use of monoclonal antibodies OKT4 and OKT8 were analyzed according to HLA-DR phenotypes. The results suggest that gene(s) associated with HLA-DR could be one of the factors which affects blood levels of T cell subsets, modulating the absolute values of T cell population.     

Distribution of marker-specific lymphocyte subsets in healthy human subjects

Cell membrane surface markers on peripheral blood lymphocytes of healthy human subjects were analysed by flow cytometry using monoclonal antibodies. Peripheral blood was obtained from 156 healthy individuals of both sexes, and ages ranging from 2-82 yr. The distribution of the lymphocytes with specific cell markers according to differences in sex and age was calculated. There was a significant difference (p less than 0.05) between sexes in the mean percentages of OKT4+ and OKT8+ cells and in the OKT4/8 ratio while the OKT3+, OKT10+ and OKIa1+ cells, and OKT4/8 ratio clearly correlated with age. A significant correlation among OKT3+, OKT4+, OKT8+, OKT11+ and OKIa1+ cells was obtained, and the results indicated that the percentage of a specific marker-positive cell varied under the influence of other marker-positive cells.     

The lymphocyte subpopulations involved in cytotoxicity generated by co-culture with autologous and allogeneic Epstein-Barr virus-transformed cell line

When peripheral blood lymphocytes from healthy adults are cultured with autologous (auto) or allogeneic (allo) Epstein-Barr virus-transformed cells (LCL), non-specific killer activity against NK-sensitive K562 and NK-resistant Raji, as well as specific killer activity against LCL is enhanced or generated. We analyzed the cell subsets possessing such cytotoxicity using monoclonal antibodies (MoAb). OKT3, a MoAb to T cell receptor-associated molecule, added in the effector phase suppressed the killer activity against LCL but not against Raji or K562. In contrast, OKT3 added in the induction phase abolished the generation of cytotoxicity against all targets. The addition of OKT8 in either the effector or induction phase inhibited anti-LCL killing induced by stimulation with alloLCL. This suggests that CD8 is required for recognition of alloLCL. The treatment of effector cells with MoAb and complement(C) revealed that killers against LCL were OKT8+ Leu11-, and those against K562 were OKT8- Leu11+. When auto-LCL were used as stimulator, removal of OKT4+ cells in the induction phase diminished the cytotoxicity against all targets, indicating that CD4+ T cells recognize autoLCL. Elimination of CD8+ cells from responder did not decrease the generation of killer activity. Further experiments suggested that this was caused by the coexistence of CD4+ killer cells or by the increase of residual CD8+ effector cells.     

活动性肺结核与潜在感染者CD8+、CD4+记忆T细胞表达水平的比较

[目的]探讨CD8+、CD4+记忆T细胞表达水平在活动性肺结核与潜伏感染者中的表达差异及其意义.[方法]本院确诊的25例活动性肺结核患者(A组)、25例肺结核潜伏感染者(B组)、30例健康人群作为健康对照组(C组),检测各组外周血CD4+记忆T细胞、CD8+记忆T细胞的表达水平,并探讨其与高分辨率CT评分(HRCT)的相关性.[结果]B组的CD4+ Tcm、CD8+ Tcm水平显著高于A组、C组(P＜0.05),A组的CD4+ Tcm、CD8+ Tcm水平显著的高于C组(P＜0.05).B组、A组的CD4+Tem、CD8+Tem水平显著的低于C组(P＜0.05),B组、A组的CD4+ Tem、CD8+ Tem水平相比差异无显著性(P＞0.05).A组患者CD4+ Tcm、CD8+ Tcm水平与HRCT评分呈显著的负相关关系(P＜0.05);A组患者CD4+Tem、CD8+ Tem水平与HRCT评分无显著的相关系(P＞0.05).[结论]CD4+、CD8+中心记忆性T细胞在活动性肺结核与潜伏感染者中水平差异显著,并且与肺结核患者病例损伤程度有关.     

Spinal fluid lymphocytes responsive to autologous and allogeneic cells in multiple sclerosis and control individuals.

Spinal fluid lymphocytes from multiple sclerosis (MS) patients and controls were stimulated with either autologous non-T cells or with allogeneic non-T cells followed by stimulation with autologous non-T lymphocytes. Cells responding to these stimuli were cloned and their proliferative responses to autologous and allogeneic MS and normal non-T cells were measured. Large numbers of clones with specific patterns of reaction to both autologous and allogeneic cells were obtained from lymphocytes in MS cerebrospinal fluid (CSF), but only occasionally from cells in control CSF. Patterns of responses among clones from a particular CSF were similar and often identical, which suggested that cells in MS CSF were relatively restricted in their specificities. Surface antigen phenotyping of the clones showed them to be predominantly OKT4+, with 13% OKT8+ and 11% OKT4+8+. Peripheral T cells that were stimulated and cultured in parallel with CSF cells were different in that they usually did not give rise to as many clones nor were their patterns of response similar. Many CSF clones were heteroclitic, that is they responded to particular allogeneic cells but not autologous cells. Lymphocytes in MS CSF thus appear to represent a selected population of cells with a high frequency of responsiveness to autologous and allogeneic antigens. Such responses may be evidence for immune regulation within the central nervous system or could represent responses to altered-self antigens.     

Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection

Few hepatitis C virus (HCV) infections resolve spontaneously but those that do appear to afford protective immunity. Second infections are usually shorter in duration and are less likely to persist but mechanisms of virus control in immune individuals have not been identified. In this study we investigated whether memory helper and/or cytotoxic T lymphocytes provide protection in chimpanzees serially reinfected with the virus. Clearance of the first infection took 3-4 mo and coincided with the delayed onset of CD4+ and CD8+ T cell responses. High frequencies of memory T cells targeting multiple HCV proteins were stable over 7 yr of follow-up. Animals were infected for a second time to assess the protective role of memory T cells. In contrast to the prolonged course of the first infection, viremia was terminated within 14 d. Control of this second infection was kinetically linked to rapid acquisition of virus-specific cytolytic activity by liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in blood. The importance of memory CD8+ T cells in control of HCV infection was confirmed by antibody-mediated depletion of this lymphocyte subset before a third infection. Virus replication was prolonged despite the presence of memory CD4+ T helper cells primed by the two prior infections and was not terminated until HCV-specific CD8+ T cells recovered in the liver. These experiments demonstrate an essential role for memory CD8+ T cells in long-term protection from chronic hepatitis C.     

Cell-mediated immunity in acute and chronic hepatitis.

Peripheral lymphocytes from patients with hepatitis-B surface antigen (HBsAg)-positive and -negative acute hepatitis (AH), chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), and normal controls were tested for in vitro cytotoxicity and blast transformation. Cytotoxicity was measured by chrominum (21Cr) release into the medium from 51Cr-labeled Chang liver cells after incubation for 6 h with peripheral lymphocytes at a lymphocyte target cell ratio of 200:1. Concomitant 72-h incubation studies were performed to assess thymus cell-dependent (T) lymphocyte function as measured by conccanavalin A (Con A)- stimulated incorporation of tritiated thymidine (blast transformation) and by cytotoxicity. It was found that (a) lymphocytes from patients with AH are cytotoxic to Chang liver cells compared to controls (P less than 0.001); (b) lymphocytes from patients with acute and chronic hepatitis are less cytotoxic when incubated with autologous and homologous HB2Ag-positive and -negative AH, CAH, and CPH are as cytotoxic as normal controls when stimulated with a nonspecific mitogen such as Con A; and (d) lymphocytes from patients with CAH while on prednisone therapy showed marked depression of cytotoxicity when stimulated with Con A. Thus these studies show that patients with AH have circulating T lymphocytes which are capable of causing the destruction of Chang liver cells. There is no defect in T-cell function as measured by Con A-stimulated cytotoxicity. There is a serum factor (s) in patients with acute and chronic hepatitis which inhibits spontaneous and induced lymphocyte cytotoxicity and blast transformation. Finally, prednisone treatment appears to inhibit lymphocyte cytotoxicity in patients with CAH.