Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)⁺/CD3⁺16^? T lymphocytes or TCR^?/CD3^?16^? natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1μg/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR^?/CD3^?16^? T lymphocytes. The proportion of TCR^?/CD3^?16^? NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR^?/CD3^?16^? NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR^?/CD3^?16^? NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1–0.3μg/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR⁺/CD3⁺8⁺ lymphocytes increased more rapidly relative to the TCR⁺/CD3⁺4⁺ lymphocytes resulting in an increased TCR⁺/CD3⁺8⁺ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR⁺/CD3⁺8⁺ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR⁺/CD3⁺4⁺, demonstrating a growth promoting effect of TCR⁺/CD3⁺4⁺ lymphocytes on TCR⁺/CD3⁺8⁺ lymphocytes in PBL bulk cultures.     

Helicobacter pylori vacuolating cytotoxin inhibits activation-induced proliferation of human T and B lymphocyte subsets

Helicobacter pylori are Gram-negative bacteria that persistently colonize the human gastric mucosa despite the recruitment of immune cells. The H. pylori vacuolating cytotoxin (VacA) recently has been shown to inhibit stimulation-induced proliferation of primary human CD4(+) T cells. In this study, we investigated effects of VacA on the proliferation of various other types of primary human immune cells. Intoxication of PBMC with VacA inhibited the stimulation-induced proliferation of CD4(+) T cells, CD8(+) T cells, and B cells. VacA also inhibited the proliferation of purified primary human CD4(+) T cells that were stimulated by dendritic cells. VacA inhibited both T cell-induced and PMA/anti-IgM-induced proliferation of purified B cells. Intoxication with VacA did not alter the magnitude of calcium flux that occurred upon stimulation of CD4(+) T cells or B cells, indicating that VacA does not alter early signaling events required for activation and proliferation. VacA reduced the mitochondrial membrane potential of CD4(+) T cells, but did not reduce the mitochondrial membrane potential of B cells. We propose that the immunomodulatory actions of VacA on T and B lymphocytes, the major effectors of the adaptive immune response, may contribute to the ability of H. pylori to establish a persistent infection in the human gastric mucosa.     

Interleukin-2 induces proliferation of T lymphocyte mutants lacking protein kinase C

We have identified a murine T lymphocyte clone that apparently lacks diacylglycerol- and phospholipid-activated protein kinase C (PKC): cell extracts do not display phosphatidylserine, Ca2+, or phorbol ester-dependent phosphotransferase activity; the enzyme was not detected in immunoblots with PKC-specific antibodies; phorbol ester binding sites are not detectable in intact cells; and activators of PKC do not stimulate proliferation or Na+/H+ exchange in intact cells. Only PKC beta mRNA was detected in normal murine T lymphocytes. The mutant T lymphocytes contained amounts of 4.4 kb PKC beta message similar to those in normal murine lymphocytes, but the 2.9 kb and 1.2 kb messages found in normal lymphocytes were barely detectable. No abnormalities were detected on Southern analysis, suggesting that the abnormality may be at the level of message splicing or stability. Since PKC-deficient cells proliferate in response to the T lymphocyte growth factor, interleukin-2, we conclude that activation of PKC is not essential for the growth-promoting action of interleukin-2.     

EGNA-specific LIF production of human lymphocyte subsets

Using the indirect leukocyte migration inhibition technique T cells have been identified as being responsible for Epstein-Barr virus nuclear antigen-induced specific leukocyte migration inhibitory factor production. The response was dependent on the presence of macrophages or their product, T-lymphocyte activating factor.     

Numerical alterations of ageing B lymphocyte subsets

The global population is ageing. Elderly people suffer from more severe infections than younger persons. The major reason for the increased susceptibility to infections in the elderly is the deregulated functions of the immune system. Immunosenescence affects both innate and adaptive immune reactions. Among these, quantitative alterations of B lymphocyte subsets determine outcome of infections and vaccination. The overall number of B cells seems to be stable or the decrease is moderate. Reduced input of naive B lymphocytes is compensated by anergic, exhausted memory cells. Concerning B lymphocyte subsets, experimental data obtained in the mouse model and in vivo studies conducted in old-age humans are frequently controversial. Further analysis of human B lymphocyte subpopulations is required that could be regarded as an important biomarker of human life span.     

Impact of cadmium in T lymphocyte subsets and cytokine expression: differential regulation by oxidative stress and apoptosis

Cadmium (Cd), a possible human carcinogen is a potent immunotoxicant. In rodents it causes thymic atrophy and splenomegaly, in addition to immuno-suppression and modulation of humoral and/or cellular immune response. Oxidative stress and apoptosis appear to be underlying mechanism of Cd induced thymic injury. To understand the involvement of reactive oxygen species (ROS), intracellular glutathione (GSH) and apoptosis in modulation of T-cell repertoire, we studied the effect of Cd (10, 25 and 50 μM) on primary T lymphocytes of BALB/c mice at different time intervals (6, 12 and 18 h). We observed a dose and time dependent decline in CD4⁺/CD8⁺ ratio (a bio-indicator of immunotoxicity) as a result of significant suppression of CD4⁺ subsets (helper T-cells) and enhancement in CD8⁺ cells (cytotoxic T-cells) At the same time, the CD4⁺CD8⁺ (DP) cell population was lowered while the CD4⁻CD8⁻ (DN) cells were increased. The oxidative stress and apoptotic data revealed almost similar ROS generation in both CD4⁺ and CD8⁺ cells, but relatively more marked GSH depletion and apoptosis in CD4⁺ than in CD8⁺ population. On further analysis of CD4⁺ T-subsets, cytokine release (IL-2 and IFNγ) by Th 1 cells and IL-4 by Th 2 cells were shown to be significantly suppressed in a dose responsive manner. The highest inhibition was observed in IFNγ, then IL-2 followed by IL-4. In conclusion, our data demonstrates that T-cell apoptosis by Cd, more in CD4⁺ than in CD8⁺ cells appear related to higher depletion of intracellular glutathione. Th 1 cells of CD4⁺ sub-population are more responsive to Cd than Th 2, leading to higher suppression of IL-2 and IFNγ than IL-4 and hence, the study unravels to some extend, the underlying events involved in Cd immunotoxicity.     

Endotoxin-induced T lymphocyte proliferation

The lymphocyte response to endotoxin (LPS) has been attributed largely to the action of this agent as a polyclonal activator of B lymphocytes. In this study we found that a cloned murine interleukin 2-dependent cytotoxic T cell line, CT 6, proliferates in response to LPS, thus providing the first evidence that T cells can be stimulated directly by LPS. The response was dose and time dependent and was blocked by polymyxin B, an inhibitor of LPS-induced mitogenesis. The fact that this is a cloned T cell line, free of other potentially contaminating lymphoid cell types, precludes the possibility that this proliferation is due to contaminating B lymphocytes or is mediated by macrophage-derived products such as interleukin 1. Moreover, highly purified splenic T lymphocyte populations (purified by negative/positive selection or by a rigorous column purification procedure) contain a small subpopulation (approximately 3%) of T cells that proliferate in response to LPS. This population is missing in the endotoxin-hyporesponsive C3H/HeJ mouse. As was observed in the CT 6 line, proliferation of splenic T cells in response to LPS was inhibited by polymyxin B. Furthermore, treatment of LPS-stimulated T cells with anti-T cell antibodies plus complement blocks the uptake of 3H-thymidine by these cultures. Exogenous interleukin 1 failed to stimulate the T cell cultures comparably to LPS and therefore cannot account for the degree of stimulation observed. These findings support and extend previous findings that suggested a role for an endotoxin-sensitive T cell population in the induction of certain responses, such as LPS-induced adjuvanticity of the lymphocyte-dependent LPS induction of macrophage procoagulant activity.     

Alternol inhibits proliferation and induces apoptosis in mouse lymphocyte leukemia (L1210) cells

Germline mutations of the serine/threonine kinase LKB1 (also known as STK11) lead to Peutz–Jeghers syndrome (PJS) that is associated with increased incidence of malignant cancers. However, the tumor suppressor function of LKB1 has not been fully elucidated. We applied yeast two-hybrid screening and identified that a novel WD-repeat protein WDR6 was able to interact with LKB1. Immunofluorescence staining revealed that WDR6 was localized in cytoplasm, similar to the localization of LKB1. Expression of LKB1 was able to inhibit colony formation of Hela cells. Interestingly, coexpression of WDR6 with LKB1 enhanced the inhibitory effect of LKB1 on Hela cell proliferation. Consistently, WDR6 was able to synergize with LKB1 in cell cycle G1 arrest in Hela cells. Coexpression of WDR6 and LKB1 was able to induce a cyclin-dependent kinase (CDK) inhibitor p27^Kip1. Furthermore, the stimulatory effect of LKB1 on p27^Kip1 promoter activity was significantly elevated by coexpression with WDR6. Collectively, these results provided initial evidence that WDR6 is implicated in the cell growth inhibitory pathway of LKB1 via regulation of p27^Kip1.     

Differential effects of cadmium on blood lymphocyte subsets

This work was designed to analyze the possible dose dependent effects of cadmium on the blood lymphocyte subset distribution and if these effects are related to circulating cadmium concentration. For that purpose, adult male rats were exposed for one month to 0, 5, 10, 25, 50 or 100 ppm of cadmium chloride (CdCl2) in the drinking water. B lymphocytes decreased in peripheral blood with the doses of 5 and 10 ppm of CdCl2. From the dose of 25 ppm on, B cells increased. T lymphocytes were increased with the doses of 25, 50 and 100 ppm of CdCl2. The lower doses of the metal induced opposite effects. CD4+ and CD8+ cells decreased with the doses of 5 and 10 ppm whereas they were increased with the dose of 25 ppm of CdCl2 on. From the dose of 10 ppm on, cadmium concentration was increased. The results on the distribution of blood lymphocyte subsets suggest that cadmium inhibits the humoral and cellular immune response with the lower doses of the metal used, and opposite effects were detected with the higher doses, the effect not being dependent on the circulating cadmium.     

Differential function of intestinal intraepithelial lymphocyte subsets.

It has been proposed that intestinal intraepithelial lymphocytes (I-IEL) perform immune surveillance of the epithelial layer (1) and regulate mucosal humoral responses to exogenous Ag (2). To better understand the functional potential of this unique population, purified murine I-IEL were analyzed phenotypically and functionally. Initial studies determined that I-IEL could be distinguished based on several phenotypic characteristics including: TCR (TCR-alpha beta vs TCR-gamma delta); Thy-1, CD45R/B220, CD5, and CD8 (CD8 alpha alpha vs CD8 alpha beta) expression. Using anti-TCR mAb, individual I-IEL subsets were activated and examined functionally. Both TCR-alpha beta and TCR-gamma delta I-IEL were found to synthesize an array of lymphokines that included IL-2, IL-3, and IL-6 but not IL-4 or IL-5. Additionally, a number of lymphokines were detected that directly influence epithelial function (IFN-gamma, TNF-alpha, and TGF-beta 1). However, the majority of the I-IEL function was localized within the Thy-1+, CD45R/B220- I-IEL subset. In addition those TCR-alpha beta I-IEL expressing the CD8 alpha beta heterodimer were more easily activated. Thus, a subset of I-IEL have the capacity to respond to TCR-mediated stimuli. The functional activities of these cells may influence both local immune cell populations as well as epithelial differentiation.     

肠道菌群对肠黏膜T淋巴细胞亚群的调节作用

肠道菌群在肠道免疫稳态中起到了至关重要的作用。大量研究表明肠道菌群通过调节T淋巴细胞亚群的增殖、分化和T细胞亚群分泌不同的细胞因子,可以改变肠道免疫系统的状态。本研究综述了肠道微生物对主要T淋巴细胞亚群的调节作用。     

Exploitation of differential homeostatic proliferation of T-cell subsets following chemotherapy to enhance the efficacy of vaccine-mediated antitumor responses

The 5-year survival rate for stage IB-III non-small-cell lung cancer (NSCLC) remains 15%. Surgical resection followed by adjuvant chemotherapy with cisplatin and vinorelbine is one standard-of-care. We sought to determine in a preclinical model whether (a) the combination of cisplatin and vinorelbine could positively modulate components of the immune system independent of antitumor activity, and (b) there were synergistic effects of this drug combination and vaccine immunotherapy. We examined the effect of cisplatin/vinorelbine on gene expression, cell-surface phenotype, and CTL-mediated cytolysis of murine lung carcinoma cells in vitro; we also assessed the effects of cisplatin/vinorelbine on immune subsets and function of Tregs in vivo. Finally, we evaluated the potential synergy between chemotherapy and a recombinant yeast-CEA vaccine in a murine model transgenic for CEA with mice bearing lung tumors. These studies demonstrate that exposure of lung tumor cells to the platinum doublet cisplatin/vinorelbine modulates tumor cell phenotype and increases sensitivity to CTL-mediated cytolysis. These studies also demonstrate that cisplatin/vinorelbine (a) induces sub-myeloablative leucopenia that differentially modulates reconstitution of Treg versus effector T-cell subsets and (b) can be employed synergistically with vaccine, exploiting homeostatic peripheral expansion of T cells. Antitumor studies show for the first time that cisplatin/vinorelbine combined with vaccine increases the survival of mice with established NSCLC. These findings provide the rationale for the potential clinical benefit of the combined use of vaccine with cisplatin/vinorelbine chemotherapy regimens.     

T lymphocyte subsets in patients with malignant melanoma

Summary T lymphocyte subset profiles were determined by monoclonal antibodies on cryopreserved peripheral blood lymphocytes from 57 patients with malignant melanoma and 19 healthy controls. Quantitation of percentages of total T cells (OKT3.PAN), helper (OKT4.IND) or suppressor (OKT8.SUP) cells, and the ratio of helper/suppressor subsets revealed no correlation of these markers with stage of disease or clinical outcome. A sequential study of these markers on peripheral blood lymphocytes from three stage I melanoma patients with subsequent recurrent disease showed no fluctuations that could be correlated to tumor progression. This study indicates that there is no systemic imbalance in T cell subsets in malignant melanoma and that quantitation of these subsets cannot predict the clinical course of this disease.     

Activation of human blood lymphocyte subsets for cytotoxic potential

Blood lymphocytes of individuals differ in the spontaneous cytotoxic potential exerted in vitro against certain cell lines (natural killing, NK). In the low NK donors, the activity can be enhanced by short-term IFN pretreatment of the effectors (interferon activated killing, IAK) and by addition of PHA to the short-term assay (lectin-dependent cellular cytotoxicity, LDCC). Lymphocyte subpopulations fractionated on the basis of nylon adherence, SRBC, and EA rosette formation differ in their response to these measures. The results obtained with IFN-treated lymphocytes of low NK donors were similar in strength to the spontaneous activity of the high NK donors. Therefore, the distinction between NK and IAK is only operational. The nylon passed E receptor-negative and low-avidity E receptor-positive cells had the strongest NK activity. These subsets can be triggered for enhanced activity by IFN. In the majority of the cases the high-activity E-receptor-positive subset which did not sediment with EA indicators had low NK effect and was not triggered by IFN. Addition of PHA to the lytic assay, however, induced activity in the subset. Realization of DNA synthesis was not necessary for the lytic performance. The PHA-imposed triggering event was not dependent on IFN production nor on induction of the competence for IFN response. The results showed that all non-B lymphocyte subsets separated on the basis of nylon wool adherence, SRBC, and EA rosetting contain cells with lytic potential if the appropriate stimulus is used. The relative activities of the subsets against K562 and Daudi differed. Cells which rosetted readily with EA indicators had weak effect against Daudi.     

Sequential monitoring of peripheral blood lymphocyte subsets in rats

The methodology of enumeration of T cell subsets in humans is well established and has widespread clinical application. Sequential monitoring of lymphocyte subsets in small animals requires a consistent and reliable methodology for staining and enumerating lymphocyte populations. The optimal conditions for enumerating subclasses of rat peripheral blood lymphocytes with the Spectrum III flow cytometer are described. The sources of biological variation and technical error that were considered in optimizing the techniques and the limitations in applying them are discussed.     

Mechanisms of serotonin-induced lymphocyte proliferation inhibition

When human peripheral blood lymphocytes were stimulated with phytohemagglutinin in the presence of serotonin, inhibition of [3H]thymidine incorporation occurred, the most marked inhibition occurring at high (10(-3)M) serotonin concentrations. This effect could not be reversed by the addition of Interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analysis showed that virtually all of the cells remained in the G0 phase (unactivated) at 24 hr while some of the cells entered the G1a and G1b phases of the cell cycle by 42 hr. The cellular production of IL-2 was not affected by serotonin, as supernatants of treated cultures contained essentially the same IL-2 titers as did control cultures. Serotonin seemed to primarily affect cell activation and had little or no effect on proliferating cells. This was further confirmed by the lack of effects of serotonin on a variety of established proliferating lymphocyte, macrophage, and fibroblast cell lines. By contrast, dose-dependent inhibition of IL-2-dependent CTLL cells occurred. Serotonin was not toxic even at 10(-3) M concentrations. A marked decrease in IL-2 receptors and a change in their distribution on responder cells was seen when treated cultures were examined with the anti-Tac monoclonal antibody. At 24 hr this effect was contrastingly not seen for the OKT-8 marker, although a slight decrease in OKT-4-positive cells was seen. Serotonin thus produced an inhibition of lectin-stimulated lymphocyte proliferation via a mechanism independent of IL-2 production, and caused a decrease in the expression and distribution of IL-2 receptors on the surface of responder cells.