Studies on immunoregulatory mechanisms in acute and chronic hepatitis B

Patients with acute hepatitis B and HBV-induced chronic hepatitis as well as normal control persons participated in the study. Hepatitis patients of both groups have decreased OKT4+/OKT8+T cell ratios due to an percental increase of OKT8+T cells in peripheral blood compared to the data of controls. Lymphocyte cultures of chronic hepatitis patients show reduced DNA synthesis after stimulation by allogeneic non-T cells, PHA, Con A and PWM. PWM-induced immunoglobulin secretion by B cells, determined by means of a reverse haemolytic plaque assay (RHPA) and a solid phase ELISA, showed comparable results in hepatitis B patients and controls. The AMLR, which is thought to reflect an autologous immunoregulatory phenomenon, is slightly impaired in cultures of hepatitis B patients in comparison to controls. Con A-induced suppressor cell activity on T cell reactions is decreased in hepatitis, whereas suppressor cell activity on B cell activation is within the same range as in cultures of controls. It is concluded from these data, that suppressor cell activity on T cell function is impaired in hepatitis B, whereas B cell functions and suppressor cell activity on B cell function are in the normal range. The results with the functional assays and the finding of increased proportions of OKT8+T cells in hepatitis B are considered to reflect properties of different T cell subpopulations, responsible for different immunoregulatory functions.     

Circulating thymic hormone activity in young cancer patients.

The natural killer (NK) cell cytotoxic activity (against K562 targets) of peripheral blood mononuclear leucocytes (MNL) from patients splenectomized for trauma was examined. The data were fitted to a mathematical model from which the values for maximal cytotoxicity, and the number of MNL required to achieve 25% cytotoxicity (LD25-, were computed. The results showed that the NK cell cytotoxicity of MNL from splenectomized patients was significantly elevated at all effector/target cell ratios tested (1:1 to 100:1), compared to normal subjects. In the splenectomized group there was a significant increase in the maximal cytotoxicity and a significant reduction in the LD25. Flow cytofluorometry studies revealed an increase in percentage and absolute numbers of cells bearing Leu-7 or the Leu-11 antigens in MNL of splenectomized patients. We found that there was a significant correlation between the percentage of Leu-11+ cells and the NK cytotoxicity (both maximal and LD25) but not between Leu-7+ and these parameters, for both the controls and splenectomized subjects. This suggests that the increased NK cytotoxicity by the MNL of splenectomized patients may be due wholly or partly to an increase in the proportion of cells expressing the Leu-11+ antigens.     

Abnormal T-cell activation in chronic hepatitis B viral infection: a consequence of monocyte dysfunction?

The process of T-cell activation in chronic hepatitis B virus (HBV) carriers has been investigated by measurement of membrane expression of lymphocyte-activation markers in response to a variety of mitogenic stimuli in order to delineate further the abnormality of T-cell-mediated immunity present in such patients. A substantial proportion of unstimulated T cells from the peripheral blood of patients but not controls expressed HLA-DR; in contrast the IL-2 and transferrin receptors were rarely expressed spontaneously in either group and there was no difference in spontaneous lymphocyte transformation. After stimulation with monocyte-dependent T-cell mitogens, phytohaemagglutinin (PHA) or anti-T3, patients had significantly reduced expression of the IL-2 and transferrin receptors and of HLA-DR in association with impaired lymphocyte transformations compared to controls. In contrast, lymphocyte activation was normal in response to the monocyte-independent T-cell mitogen phorbol-myristate-acetate (PMA). These data confirm that the process of T-cell activation is abnormal in chronic HBV carriers but suggest that the T cell is intrinsically normal. In allogeneic co-cultures, monocytes from patients inhibited the transformation of normal and patients' lymphocytes in response to PHA, suggesting that defects of T-cell-mediated immunity in chronic HBV carriers may be a consequence of monocyte dysfunction.     

Circulating Ki67 positive lymphocytes in multiple myeloma and benign monoclonal gammopathy.

AIMS--To estimate the proportion and nature of the proliferating (Ki67+) circulating lymphocytes in a series of patients with multiple myeloma and monoclonal gammopathy of unknown significance (MGUS) and to correlate this with other clinical and laboratory parameters, using blood from healthy adults as a control. To investigate the extent to which the B and T lymphoid components are involved in progression and/or control of disease. METHODS--Blood lymphocytes from 15 patients with multiple myeloma, 10 patients with MGUS and 10 healthy adults were analysed using a sequential double immunoenzymatic staining technique. Antibodies directed against Ki67 were used to detect cells in cycle, CD3, CD4, and CD8 to identify T cells, HLA-Dr as a marker for B cells and activated T cells, and CD11b as a marker for natural killer cells. Polyclonal antibodies directed against the kappa and lambda immunoglobulin light chains were also used to detect B cells. RESULTS--The proportion of proliferating (Ki67+) lymphocytes was significantly higher in patients with multiple myeloma (6.8 +/- 2.6) and MGUS (3.5 +/- 1.1) compared with the normal controls (1.69 +/- 0.3); this was also true when multiple myeloma and MGUS cases were compared. In multiple myeloma and MGUS over 50% of the Ki67+ cells were activated T lymphocytes (CD3+/HLA-Dr+); a minority (11%) were non-clonal B lymphocytes. In contrast to controls (6.7 +/- 1.9), in patients with multiple myeloma and MGUS the proportion of proliferating T cells expressing CD8 (23.6 +/- 12.5 and 15.3 +/- 7.7, respectively) and CD11b (13 +/- 8.7 and 11.6 +/- 3.9, respectively) was higher. In multiple myeloma there was a positive correlation between the proportion of Ki67+ lymphocytes, beta-2-microglobulin concentrations and disease stage. CONCLUSIONS--Although the number of patients investigated is small, this study suggests that Ki67 expression in blood lymphocytes from patients with multiple myeloma may be a good prognostic indicator for aggressive disease and may help to distinguish multiple myeloma from MGUS. The activated proliferating T cells in these diseases may represent an immunological reaction against the tumour.     

Involvement of the spleen in the control of the immunogenic and phagocytic function of thioglycollate-induced macrophages

The immunogenic capacity of thioglycollate-induced peritoneal macrophages of adult splenectomized animals was compared to that of macrophages of sham-operated controls. Macrophages from splenectomized animals were found to be impaired in their function as antigen-presenting cells, both in the education of virgin initiator T lymphocytes and in the stimulation of antigen-specific T memory cells. Macrophages from splenectomized animals were also severely impaired in their phagocytic capacity, as assessed in an opsonin-dependent bacterial phagocytosis assay. However, they were not impaired in their ability to pinocytose soluble keyhole limpet hemocyanin. These results indicate that the spleen may play a decisive role in controlling the differentiation of peritoneal macrophages.     

Increased cytolytic T lymphocyte activity and decreased B7 responsiveness are associated with CD28 down-regulation on CD8+ T cells from HIV-infected subjects.

The CD28 receptor on CD4+ and CD8+ T cells interacts with B7 molecules on antigen-presenting cells (APC) to generate essential costimulatory signals. The cytolytic potential of CD8+ T cells could be linked to CD28 expression. Since HIV induces dysfunction of both CD4+ and CD8+ T cells, we evaluated CD28 expression and function in both subsets during HIV infection. CD28 expression on CD8+ T cells from HIV+ subjects was strongly reduced in a disease stage-related fashion. CD28- CD8+ T cells preferentially expressed CD57 and CD11b, but lacked CD26 and IL-2R alpha. The CD8+ T cells from the patients showed a significantly reduced proliferative response to co-stimulation with cell-bound anti-CD3 and B7. Nevertheless, when stimulated with plate-fixed anti-CD3, CD8+ T cells from HIV-infected subjects proliferated normally, and normal levels of IL-2R alpha and transferrin-receptor could be induced on CD28- CD8+ T cells from the patients. In addition, stimulation with plate-fixed anti-CD3 induced proliferative responses in highly purified CD28- CD8+ T cells from both HIV- and HIV+ persons. Furthermore, the increased cytotoxic activity of peripheral blood mononuclear cells (PBMC) from HIV+ subjects, measured in an anti-CD3 redirected assay, was predominantly exerted by CD28- CD57+ T cells. CD4+ T cells from the patients showed a slight but significant CD28 down-regulation and were slightly hyporesponsive to B7 co-stimulation. Decrease of CD28 on CD8+ T cells from HIV+ subjects is associated with an impaired response to co-stimulation via B7. CD28- CD8+ T cells from seropositives, however, are not completely inert, since they contain in vivo activated CTL and they can be additionally activated through a B7-independent stimulation.     

Increased numbers but functional defects of CD56+CD3+ cells in lung cancer

CD56+ T cells were studied in samples of peripheral blood from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) patients compared with healthy controls. Relative numbers of CD56+CD3+ cells were increased in NSCLC (P = 0.001) and SCLC (P = 0.002) compared with normal subjects but their ability to respond to activation by up-regulating CD25 or producing IFN-γ were both significantly impaired. Expression of the killer-immunoglobulin-like receptor CD158a was significantly lower on CD56+CD3+ cells in SCLC than controls and also in early stage compared with late stage NSCLC patients. Mean levels of CD158e were higher in NSCLC patients than controls. CD158e levels on CD56+CD3+ cells were increased in the presence of its ligand HLA-Bw4 compared with controls. Although the precise role of CD56+CD3+ cells is not clear, they appear to be functionally impaired in lung cancer, which may have implications for a reduction of direct or indirect anti-tumour responses.     

T cell activation, apoptosis and cytokine dysregulation in the (co)pathogenesis of HIV and pulmonary tuberculosis (TB)

Immune parameters were compared in four groups of Ugandan subjects: HIV-and HIV+ adult patients with active pulmonary TB (HIV- PTB n = 38; HIV+ PTB n = 28), patients with HIV infection only (n = 26) and PPD+ healthy controls (n = 25). Compared with healthy controls, CD4 and CD8 T cells from patients with HIV and/or PTB expressed more activation markers (HLA-DR, CD38); their CD8 T cells expressed more CD95 (pre-apoptosis) and less CD28 (co-stimulatory receptor). Peripheral blood mononuclear cells (PBMC) of patients with either HIV or PTB were impaired in interferon-gamma (IFN-gamma) production upon antigenic stimulation. PTB (with or without HIV) was characterized by monocytosis, granulocytosis, increased transforming growth factor-beta 1 production and PPD-induced apoptosis. In vivo CD4 T cell depletion, in vitro increased spontaneous CD4 T cell apoptosis and defects in IFN-gamma responses upon mitogenic stimulation were restricted to HIV+ subjects (with or without PTB). Overlapping and distinctive immune alterations, associated with PTB and HIV, might explain mutual unfavourable influences of both diseases.     

Resistance/susceptibility to Echinococcus multilocularis infection and cytokine profile in humans. II. Influence of the HLA B8, DR3, DQ2 haplotype

Differences have been shown between HLA characteristics of patients with different courses of alveolar echinococcosis (AE). Notably the HLA B8, DR3, DQ2 haplotype was associated with more severe forms of this granulomatous parasitic disease. We compared IL-10, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) secretion by peripheral blood mononuclear cells (PBMC) isolated from eight HLA-DR3+, DQ2+, B8+ AE patients and from 10 HLA-DR3-, DQ2-, B8- patients after non-specific mitogenic and specific Echinococcus multilocularis antigenic in vitro stimulation. PBMC from seven HLA-DR3+, DQ2+, B8+ healthy subjects and nine HLA-DR3-, DQ2-, B8- subjects were also studied as controls. PBMC from AE patients with HLA DR3+, DQ2+ haplotype secreted higher levels of IL-10 without any stimulation and after specific antigenic stimulation than did patients without this haplotype. Higher levels of IL-5 and IFN-gamma were also produced by these patients' PBMC after stimulation with non-purified parasitic antigenic preparations; however, the specific alkaline phosphatase antigen extracted from E. multilocularis induced only Th2-type cytokine secretion. A spontaneous secretion of TNF by HLA DR3+, DQ2+ B8+ AE patients was also found. These results suggest that HLA characteristics of the host can influence immune-mediated mechanisms, and thus the course of AE in humans; specific antigenic components of E. multilocularis could contribute to the preferential Th2-type cytokine production favoured by the genetic background of the host.     

T and B lymphocytes in peripheral blood of splenectomized subjects]

The report is a part of more extensive studies on the role of the spleen in immune processes. Assessing the ability of lymphocytes T to non-immunological binding of sheep erythrocytes and the ability of B cells to bind immunological complexes through the receptor for C3 component of complement, and the presence of immunoglobulins on the surface of lymphocytes B the number of these cells in peripheral blood was determined in 14 healthy individuals who had been splenectomized. In all these subjects the degree of blastic transformation of lymphocytes in PHA-stimulated 3 day-old white blood cell cultures was determined. It was found that the number of T and B lymphocytes in the peripheral blood of splenectomized and non-splenectomized patients was similiar. Lymphocytes obtained from splenectomized patients had however, an impairment of transformation ability after PHA stimulation. It is suggested that, apart from determination of T and B cell pool in the peripheral blood, an evaluation of their transformation ability after PHA stimulation is necessary for assessment of the immunological state in vitro investigations.     

Peripheral blood leucocyte subpopulations in patients splenectomized for trauma.

Monoclonal antibodies were used to type leucocyte subpopulations in peripheral blood mononuclear leucocytes (MNL) from patients who had had spleens removed following trauma. The proportion of OKT3+ (T) cell and OKT4+ ('T helper/inducer') cell was significantly decreased in splenectomized subjects. While a decrease was also observed for the OKT8+ ('T suppressor/cytotoxic') cells, this was not significant. The ratio of OKT4+/OKT8+ cells was significantly decreased in the splenectomized group. Interestingly, the absolute numbers of OKT3+, OKT4+ and OKT8+ cells were increased. The % of B lymphocytes (identified as Ig+ and FMC1+ cells) was significantly increased in patients. The proportion of MHC Class II+ cells (FMC4+) was also increased although not significantly. A marked increase in % of monocytes (FMC33+) was observed in patients. The changes in proportion of regulatory T cells and monocytes may in part explain the depressed mitogen responses of MNL from splenectomized subjects.     

Systemic T-cell unresponsiveness during rush bee-venom immunotherapy

By rush bee-venom immunotherapy, subjects reacting allergically to the venom can be effectively anergized, although the mechanism of action is not known. Here we analyzed the systemic effects of rush desensitization on the T cells of allergic patients. In most patients, we found reduced frequencies of T cells recalled to express CD69 and the cytokines interleukin (IL)-4 and interferon-gamma (IFN-γ) after stimulation of peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as compared with normal donors. These frequencies are progressively reduced during immunotherapy. The frequency of cells expressing IL-2 does not change. A few patients show a different response to immunotherapy: frequencies of cells expressing CD69, IL-4, or IFN-γ do not change, and remain similar to those of normal donors. However, the frequency of cells able to express IL-2 is increased. The analysis of cytokine expression in CD45RO* vs CD45RO' T-cell populations revealed differences between normal and allergic donors. In allergic patients, higher frequencies of IL-4- and IFN-γ-expressing cells among the CD45RO subpopulation were found than in normal donors. This situation is not modified by immunotherapy. The results reveal a certain degree of heterogeneity in the response of allergic patients to bee-venom rush immunotherapy; however, all are clearly differentiated from normal controls as judged by cytokine expression of CD45RO T cells. In most allergic patients, a considerable percentage of TTi cells become unresponsive to mitogenic stimulation, and may be responsible for the desensitization itself     

T lymphocytes expressing CCR3 are increased in allergic rhinitis compared with non-allergic controls and following allergen immunotherapy

BACKGROUND: In T cell-associated allergic inflammation, homing of T-helper 2 (Th2) effector cells to mucosal sites may be influenced by chemokine receptor expression. Previous studies have identified CCR3 and CCR4 as putative markers of Th2 cells and CCR5 and CXCR3 as markers of Th1 cells. The aim of this study was to assess differential chemokine receptor expression from symptomatic atopic grass pollen-sensitive subjects, compared with patients on high-dose allergen injection immunotherapy (IT) and healthy controls. METHODS: We examined chemokine receptor expression (CCR1-7 and CXCR1-4) by flow cytometry of peripheral blood CD4+ and CD8+ T cells. We also depleted peripheral blood mononuclear cell (PBMC) populations of CCR3+ CD4+ cells by magnetic bead separation and cells were stimulated with grass pollen allergen for 6 days. Cytokine production was measured by enzyme-linked immunosorbent assay. RESULTS: On freshly isolated PBMC, atopic individuals exhibited increased numbers of CCR3+ CD4+ cells compared with normal controls (P < 0.01). CCR3 expression in IT patients was reduced compared with matched atopic rhinitic controls (P < 0.05) and comparable with that observed in normal subjects. Depletion of CCR3+ CD4+ cells from allergen-stimulated PBMC cultures resulted in decreased interleukin (IL)-5 production compared with whole CD4+ populations (P < 0.05). Freshly isolated CCR3+ CD4+ cells have significantly higher intracellular IL-4 and lower IFN-gamma levels than CCR3- CD4+ cells. CD4+ T cells cultured from both peripheral cells and nasal biopsies demonstrated increased expression of CCR3 in the presence of IL-4 (P < 0.05). CONCLUSION: CCR3+ CD4+ T cells are increased in allergic rhinitis, are reduced by allergen IT, have a Th2 phenotype and contribute to allergen-specific responses. Strategies against CCR3+ T cells may be effective in human allergic diseases.     

结核性胸膜炎合并慢性阻塞性肺疾病患者免疫功能的变化及意义

目的 通过观察T淋巴细胞亚群、B细胞、NK细胞活性来探讨结核性胸膜炎合并慢性阻塞性肺疾病(COPD)患者免疫功能变化.方法 采用流式细胞术分别测定45例结核性胸膜炎合并COPD患者(研究组)、45例COPD患者(COPD对照组)和45例健康体检者(健康对照组)外周血T淋巴细胞亚群、B细胞和NK细胞水平.结果 研究组CD3+、CD4+、CD4+/CD8+、B细胞和NK细胞表达率降低,与健康对照组差异有统计学意义(P ＜0.05);COPD对照组CD3+、CD4+、CD4+/CD8+、B细胞和NK细胞表达率与健康对照组差异有统计学意义(P＜0.05);研究组与COPD对照组的差异无统计学意义.结论 结核性胸膜炎合并COPD患者免疫功能明显低于正常健康人群,增强免疫治疗非常必要.     

Role of the spleen in peripheral memory B-cell homeostasis in patients with autoimmune thrombocytopenia purpura

The effect of splenectomy on circulating memory B cells in autoimmune thrombocytopenia purpura (AITP) patients has not yet been addressed. We therefore analyzed the distribution and phenotypic characteristics of B-cell subsets in non-splenectomized and splenectomized AITP patients and controls, as well as CD95 expression after B cell activation. Decreased frequencies of memory B cells in splenectomized individuals were observed, with a rapid decline of CD27+IgD+ and a slower decrease of CD27+IgD- and CD27-/IgD- cells. Similar results were noted following splenectomy in healthy donors (HD). CD95+ B cells were substantially increased in all subsets in patients with active AITP, indicating their enhanced activation status. After splenectomy, the percentage of CD95+ B cells were further increased in the CD27+IgD- post-switch memory population in AITP, but not in HD. CD95+CD27+ memory B cells largely reside in the region in the human spleen analogous to the murine marginal zone. Thus, the spleen plays a fundamental role in controlling peripheral memory B cell homeostasis in both AITP and HD and regulates activated CD95+ B cells in patients with AITP.     

Soluble and cellular markers of immune activation in patients with systemic sclerosis

The peripheral blood lymphocyte pattern, the lymphocyte responses in vitro, as well as the soluble markers of immune activation were studied in 24 patients with systemic sclerosis (SSc patients). The proportions of total T cells (CD3), their CD4 subset, as well as B lymphocytes were within the normal range. The relative proportion of CD8 lymphocytes, however, was significantly reduced. Patients with SSc had a slightly lower percentage of CD4/4B4+ cells, whereas their proportion of CD4/2H4+ cells was elevated as compared to healthy controls. The proportion of lymphocytes expressing the interleukin-2 receptor (IL-2R) was significantly higher in SSc patients. The proliferative responses of peripheral blood mononuclear cells to PHA stimulation were reduced in the patient group, while expression of IL-2R on lymphocytes after such in vitro stimulation was comparable to that of controls. Expression of IL-2R on patient but not control lymphocytes was increased after in vitro exposure to laminin. Such exposure failed to induce IL-2 production or cell proliferative responses. Soluble plasma IL-2R level (sIL-2R) and soluble CD8 (sCD8) molecule levels in SSc patients were significantly elevated. These results indicate the presence of an ongoing lymphocyte activation in this disease process.     

Polymorphonuclear neutrophils function in splenectomized patients.

Some essential functions of polymorphonuclear neutrophils (PMN) were evaluated in 30 patients splenectomized because of rupture of the spleen. These cells revealed normal random migration, adherence, unstimulated O2- and H2O2 production. Phagocytosis of viable staphylococci was higher than in controls, whereas chemotaxis, bactericidal capacity, aggregation and stimulated O2- and H2O2 production were significantly impaired. PMN from splenectomized patients manifested also the decreased intracellular myeloperoxidase activity. The percentage of cells with receptor for Fc IgG in peripheral blood was markedly decreased. Plasma of these patients induced increased adherence of autologous as well as control neutrophils. The possible mechanisms leading to the observed events are discussed.     

T cell proliferation and apoptosis in HIV-1-infected lymphoid tissue: impact of highly active antiretroviral therapy.

T cell turnover was studied in situ in tonsillar lymphoid tissue (LT) from HIV-1-infected individuals during 48 weeks of highly active antiretroviral therapy (HAART) and compared to that of HIV-1-negative controls. Prior to therapy, CD4 cell proliferation (%CD4+ Ki67+) and apoptosis (%CD4+ TUNEL+) were increased in HIV-1-infected LT and both parameters correlated with tonsillar viral load. CD8 cell proliferation (%CD8+ Ki67+) was increased 4- to 10-fold, mainly in the germinal centers. Apoptotic CD8+ T cell levels (%CD8+ TUNEL+) were raised preferentially in the tonsillar T cell zone. The frequency of CD8+ Ki67+ and CD8+ TUNEL+ T cells correlated with tonsillar viral load and with the fraction of CD8(+) T cells expressing activation markers. During HAART, CD4 cell turnover normalized while CD8 cell turnover was dramatically reduced. However, low level viral replication concomitant with slightly elevated levels of CD8 cell turnover indicated a persistent cellular immune response in LT. In conclusion, enhanced T cell turnover may reflect effector cells related to HIV-1 infection.