BAT mAb induces lymphopoiesis in nude mice

The athymic nude mouse provides a powerful tool in the study of human tumors, as it enables growth of human tumors due to deficiencies in T cell functions. However, deficiencies in T cell functions might limit research on efficacy of immune modulators in cancer immunotherapy. BAT mAb mediates its anti-cancer activity through modulation of the immune system that involves both NK and T cells. We analyzed lymphocyte populations in blood 5 and 14 days following the injection of BAT antibody alone or following engraftment of human colon carcinoma cells. Our results demonstrate that BAT injection induced lymphopoiesis in the nude mouse. Percentage of CD3 cells increased up to 24%, CD4 cells up to 20% but no increase was found in CD8 T cells in BAT-injected nude mice. Injection of BAT 12 days post-tumor engraftment propagated CD3, CD4 and CD8 cells seen in the blood 5 days later but not seen in the blood 14 days post-BAT injection. It is possible that this decrease is associated with migration of the lymphocytes from the blood to the tumor sites in the livers. The percentage of CD56-positive NK cells increased (up to 18%) by BAT administration alone or post-tumor injection. The presence of tumors alone did not induce lymphopoiesis in the nude mice. Propagation and lymphopoiesis by BAT mAb might have future clinical implications.     

Differential regulation of NK cell proliferation by type I and type II IFN

IFN, produced during viral infections by accessory (type I IFN) or NK cells (type II IFN), play a primary role in the regulation of immune and anti-viral NK cell effector functions. Because IFN have anti-proliferative effects on several cell types, including hematopoietic cells, we asked whether they modulate proliferation of human NK cells, and whether IFN-alpha and IFN-gamma mediate distinct effects on NK cells at different developmental stages. Analysis of proliferation at the single-cell level in human NK cells indicated that both IFN types inhibit IL-4-induced accumulation of immature CD56(-) IL-13(+) NK cells in freshly separated peripheral blood lymphocytes and in cells derived from them after short-term cultures. However, IFN-gamma inhibited specifically the IL-4-dependent proliferation of these cells without affecting the IL-2-dependent one or that of the IL-13(-) cells, whereas IFN-alpha attenuated proliferation of NK cells at any developmental stage (both immature CD56(-)IL-13(+) and mature CD56(+)IL-13(-) IFN-gamma(+) NK cells) and contributed to their monokine-induced differentiation to IFN-gamma-producing cells. Adding to our previous report that IL-13 inhibits accumulation of mature IFN-gamma(+) NK cells, the present data unravel a mechanism by which peripheral immature IL-13(+) and mature IFN-gamma(+) NK cells can negatively regulate each other's accumulation.     

MAC technique (morphology antibody chromosomes) in phenotypic identification of proliferating NK and T cells in interleukin-2-stimulated lymphocyte cultures.

Human peripheral blood lymphocytes were activated with recombinant interleukin 2 (rIL-2) and cultured in fetal calf serum (FCS)- or human-AB-serum-supplemented media. Proliferative cells were identified by the MAC technique (morphology antibody chromosomes) which enables the immunoenzymatic identification of both mitotic and non-proliferating cells in unfractionated lymphocyte populations. The results indicate that the phenotype of more than 90% of proliferative lymphocytes can be characterized by using antibodies against T cells and NK cells. Substantial mitotic activity of CD4-positive (CD4+) T cells was observed only in FCS-supplemented cultures, whereas in serum-free or human-AB-serum-supplemented cultures mostly CD8+ T cells and NK cells proliferated. The proportion of NK cells among all mitotic cells varied between 14 and 32%. Interestingly, in unfractionated cultures approximately 13% NK cells entered mitoses in the presence of rIL-2, suggesting that the poor proliferative capacity of purified NK cells demonstrated previously may be due to the lack of accessory stimulatory signals. The proliferation of B cells was minimal in all experiments. The MAC technique is a useful addition to the techniques by which lymphocyte growth regulation is monitored.     

Activation of lymphocytes by BAT and anti CTLA-4: comparison of binding to T and B cells.

In this study we compare the binding and immune stimulatory properties of BAT and anti CTLA-4 monoclonal antibodies (mAbs). Both antibodies were previously shown to manifest effective immune responses against tumor cells. We have described that BAT antibody, produced against Daudi, a B lymphoblastoid cell line, binds and activates T cells. In this paper we demonstrate that anti CTLA-4, produced against the T-cell activation determinant CTLA-4, binds also to B lymphoblastoid cell lines like Daudi and Raji. Both antibodies do not bind resting B cells. BAT binds resting T lymphocytes as well as activated T lymphocytes, whereas anti CTLA-4 binds only activated T cells. Competitive binding experiments indicate that the binding sites of BAT and anti CTLA-4 on activated T cells are distinct. We have studied the in vitro stimulatory effect of BAT and anti CTLA-4 on lymphocytes cultured with or without tumor cells. In contrast to BAT that increased the proliferation of lymphocytes that have been cultured with tumor cells, anti CTLA-4 did not synergize with tumor cells to enhance lymphocyte proliferation.     

鳖血提取物对小鼠免疫功能正向调节作用的研究

目的 :研究鳖血提取物对小鼠的免疫调节功能。方法 :采用环磷酰胺为免疫抑制剂 ,获得免疫功能低下的小鼠动物模型。比较鳖血提取物作用前后 ,小鼠T细胞亚群的数量、NK细胞的杀伤功能 ,淋巴细胞的增殖功能以及细胞因子水平的变化。结果 :鳖血提取物对免疫功能低下小鼠的CD4 T细胞在外周血中的比例、NK细胞的杀伤活性、淋巴细胞的增殖功能 ,均具有正向调节作用 (P <0 .0 5 ) ,并呈剂量依赖性 ;能提高淋巴细胞分泌IFN γ的能力。结论 :鳖血提取物具有正向调节小鼠免疫功能的能力     

Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirus

The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3 K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCRgammadelta(+) and TCRalphabeta(+)CD8(+) T lymphocytes, but it has been also detected in CD4(+) T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4(+) T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4(+)NKG2D(+) T lymphocytes that coexpressed perforin. NKG2D was detected in CD28(-) and CD28(dull )subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4(+) T cells, triggering proliferation and cytokine production (i.e. IFN-gamma and TNF-alpha). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4(+) T lymphocytes and thus may have a role in the response against infected HLA class II(+) cells displaying NKG2D ligands.     

Immunomodulating activity of seaweed extract on human lymphocytes in vitro.

Effect of eight kinds of seaweed extract (SWE) on human lymphocytes was studied in vitro. The extracts of Hizikiafusiformis and Meristotheca papulosa (green) markedly stimulated human lymphocytes to proliferate, whereas Eucheuma muricatum and Meristotheca papulosa (red) weakly stimulated proliferation. The responder cells are T cells, because T cells purified by sheep red blood cell (SRBC) rosette-formation were significantly stimulated with SWE, but B cells were not. These extracts enhanced the induction of cytotoxic T lymphocyte (CTL) activity, but failed to enhance natural killer (NK) cell activity. These extracts had a stimulatory effect on immunoglobulin (Ig) production by B cells and tumor necrosis factor (TNF) production by monocytes. The activity of Hizikia fusiformis associated with polysaccharides which were extracted with ethanol and purified by ion-exchange and gel filtration chromatography, whose molecular weight was about 100 kDa. These results suggest that SWE has an immunomodulating activity on human lymphocytes and this ability might be useful for clinical application to treat several diseases such as tumors.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

Immunobiological effects of glucosamine in vitro

Glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) were assayed in vitro for their effects on proliferation, cytotoxicity and cytokine secretion in primary and secondary mixed lymphocyte cultures (MLCs). In addition, we studied the effect of GlcN and GlcNAc on the proliferation of purified CD4+ T cells exposed to immobilized anti-CD3 antibody. The present data show that GlcN, but not GlcNAc, inhibits CD4+ T-cell proliferation, the generation of alloreactive cytotoxic T lymphocytes (CTLs) and the secretion of interferon-gamma (IFN-gamma) and interleukin-5 (IL-5) in primary MLC. In secondary T helper-2 (Th2)-polarized MLC, GlcN, but not GlcNAc, inhibits IL-4 and IL-5 secretion, whereas no effect was found on IFN-gamma secretion in Th1-polarized MLC. Dendritic cells treated with GlcN showed a 75-80% decreased capacity for antigen cross-presentation and allostimulation. In cellular bioassays, GlcN was shown to inhibit the stimulatory activity of IL-4 and IL-2, as well as the cytotoxic activity of tumour necrosis factor-alpha (TNF-alpha). In conclusion, GlcN suppresses unprimed T-cell responses by interfering with antigen-presenting cell functions and by a direct inhibitory effect on T-cell proliferation. In addition, GlcN inhibits the secretion of cytokines in antigen-stimulated unprimed T cells and primed Th2-polarized cells.     

Interferon-gamma production by specific lung lymphocyte phenotypes in silicosis in mice

We recently described overproduction of interferon (IFN)-gamma by lung lymphocytes in mice with silicosis (11% of cells in air-control versus 19% of cells from silica-exposed mice; Davis and colleagues, Am. J. Respir. Cell Mol. Biol. 1999;20:813-824). We hypothesized that the increased IFN-gamma production might be due to selective enrichment of one lymphocyte phenotype. To test this hypothesis, small mononuclear cells from lung digest preparations of mice exposed 4 mo previously to cristobalite silica (70 mg/m(3), 12 d, 5 h/d) or to sham-air were stained for intracellular cytokines and surface antigen phenotypes, and examined by flow cytometry. Air-sham mouse lung digests included CD4(+) (16%) and CD8(+) (6%) T cells, gammadelta T-cell antigen receptor (TCR)(+) CD4(-)CD8(-) T cells (3%), natural killer (NK) cells (15%), B cells (6%), and macrophages (12%). The total number of lung lymphocytes was increased 1.7-fold in silicosis, but the phenotype frequencies did not change significantly. In the control lungs IFN-gamma was produced by three major phenotypes of lymphocytes: 5% of CD4(+) T cells, 5% of gammadelta-TCR(+) CD4(-)CD8(-) T cells, and 2% of NK cells. The percentage of each type producing IFN-gamma was increased 2- to 3-fold in silicosis. When multiplied by cell number, the increased percentages yielded a 3- to 5-fold increase in the total number of each IFN- gamma-producing phenotype in the lung. Our results demonstrate no selective phenotype enrichment but upregulated IFN-gamma production by at least three lymphocyte phenotypes. IFN-gamma may be an important signal driving lymphocyte differentiation and macrophage activation in silicosis.     

Activation of human peripheral blood mononuclear cells by nonpathogenic bacteria in vitro: evidence of NK cells as primary targets

The interaction of commensal bacteria with immunocompetent cells may occur in definite compartments of the mucosal immune system, as limited translocation through the epithelial barrier cannot be excluded. In this study the stimulation of human peripheral blood mononuclear cells and purified lymphocyte subsets by nonpathogenic gram-positive lactobacilli (Lactobacillus johnsonii and Lactobacillus sakei) and gram-negative Escherichia coli was investigated. The various bacterial strains induced a differential cytokine pattern. Whereas L. johnsonii and L. sakei strongly induced gamma interferon (IFN-gamma) and interleukin-12 (IL-12), E. coli and lipopolysaccharide (LPS) preferentially induced IL-10 after 16 h of stimulation. Expression of activation antigens CD69 and CD25 was observed on (CD3(-) CD56(+)) natural killer (NK) cells after stimulation of total human peripheral blood mononuclear cells. All bacteria mediated the proliferation of human peripheral blood mononuclear cells, and the strongest proliferative response was observed with L. johnsonii. Purified CD4(+), CD8(+), and CD19(+) lymphocyte subsets were not activated upon bacterial stimulation but showed normal response to a mitogenic stimulus. In contrast, purified NK cells upregulated the IL-2Ralpha chain (CD25) and underwent proliferation when stimulated by L. johnsonii. E. coli and LPS were less effective in inducing proliferation. Expression of CD25 or secretion of IFN-gamma from purified NK cells was significantly increased in the presence of bacterially primed macrophages, indicating that full activation required both bacterium- and cell contact-based signals derived from accessory cells.     

Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) inhibit the proliferation of HLA-unrelated T lymphocytes to allogeneic stimulation, but the mechanisms responsible for this activity are not fully understood. We show here that MSCs suppress the proliferation of both CD4+ and CD8+ T lymphocytes, as well as of natural killer (NK) cells, whereas they do not have an effect on the proliferation of B lymphocytes. The antiproliferative effect of MSCs was not associated with any effect on the expression of cell-activation markers, induction of cell apoptosis, or mimicry/enhancement of T regulatory cell activity. The suppressive activity of MSCs was not contact-dependent and required the presence of interferon (IFN)-gamma produced by activated T cells and NK cells. Accordingly, even activated B cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added IFN-gamma. The suppressive effect of IFN-gamma was related to its ability to stimulate the production by MSCs of indoleamine 2,3-dioxygenase activity, which in turn inhibited the proliferation of activated T or NK cells. These findings suggest that the beneficial effect on graft-versus-host disease induced by in vivo coinfusion with the graft of MSCs may be due to the activation of the immunomodulatory properties of MSCs by T cell- derived IFN-gamma.     

Evaluation of T cell immune responses in multi-drug-resistant tuberculosis (MDR-TB) patients to Mycobacterium tuberculosis total lipid antigens

Mycobacterium tuberculosis lipid antigens produce significant T cell responses in healthy tuberculin reactor [purified protein derivative (PPD-positive] individuals. In the present study, proliferation and interferon (IFN)-gamma/interleukin (IL)-4 responses were analysed to M. tuberculosis total lipid antigens in T lymphocytes from 25 patients with multi-drug-resistant tuberculosis (MDR-TB). The obtained results were compared with those of 30 asymptomatic healthy PPD-positive and 30 healthy tuberculin skin test negative (PPD-negative) subjects. Peripheral blood mononuclear cells (PBMCs) and T cells (CD4(+) and CD8(+)) were stimulated using autologous immature dendritic cells. Proliferation responses were assessed using 3-{4,5-dimethylthiazol-2-yl}-2,5 diphenyl tetrazolium bromide (MTT). IFN-gamma/IL-4 concentrations in the supernatant of the CD4(+) and CD8(+)T cells were measured by enzyme-linked immunosorbent assay. Proliferation assay showed that the peripheral blood mononuclear cells and CD4(+) T cells from the MDR-TB patients responded significantly less to the M. tuberculosis total lipid antigens than to the CD4(+) T cells in the PPD-positive subjects. Total lipid antigen-specific proliferative responses in the CD8(+) T cells from the MDR-TB patients were minimally detected and the responses were similar to those of the PPD-positive subjects. IFN-gamma production by the CD4(+) T cells stimulated by total lipid antigens from the MDR-TB patients was decreased significantly compared with the PPD-positive individuals, whereas IL-4 production in the patients was elevated. IFN-gamma and IL-4 production in the CD8(+) T cells of the MDR-TB patients was similar to those of the PPD-positive subjects. In conclusion, it is suggested that stimulated CD4(+) T cells by M. tuberculosis total lipid antigens may be shifted to T helper 2 responses in MDR-TB patients.     

Suppressive properties of human CD4+CD25+ regulatory T cells are dependent on CTLA-4 expression

It has been demonstrated that T cells with regulatory properties are present within the peripheral blood CD4(+)CD25(+) T cell compartment. Here, we describe an original method to purify human CD4(+)CD25(+)CD152(+) T lymphocytes as living cells by forcing the exportation of CTLA-4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4(+)CD25(+) T cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T cells is associated with an increased expression of Foxp3 and that CD4(+)CD25(+)CD152(+) T lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen-specific T cells than CD4(+)CD25(+)CD152(-) T lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA-4, we demonstrate that indeed CTLA-4 is involved in CD4(+)CD25(+)CD152(+) T cell regulatory activity, while suppressive cytokines are not.     

Novel human CD4+ T lymphocyte subpopulations defined by CD300a/c molecule expression

The CD300c (CMRF-35A) and CD300a (CMRF-35H) molecules are leukocyte surface proteins that are part of a larger family of immunoregulatory molecules encoded by a gene complex on human chromosome 17. The CMRF-35 monoclonal antibody binds to an epitope common to both molecules, expressed on most human leukocyte populations, apart from B lymphocytes and a subpopulation of CD4(+) and CD8(+) T lymphocytes. We describe the CMRF-35(pos) and CMRF-35(-) fractions of CD4(+) T lymphocytes. The CMRF-35(pos) fraction can further be divided into CMRF-35(++) and CMRF-35(+)CD4(+) T lymphocyte subpopulations. Resting peripheral CD4(+) T lymphocytes express CD300a mRNA and very low amounts of CD300c. Activation results in an initial decrease in CD300a gene expression before an increase in both CD300a and CD300c gene expression. The up-regulated expression of these genes was associated with increased CMRF-35 binding to activated T lymphocytes. The CMRF-35(-) fraction of CD4(+) T lymphocytes proliferated to a greater extent than the CMRF-35(pos) fraction, in response to mitogens or allogeneic antigen. The poor proliferation of the CMRF-35(pos) CD4(+) in response to mitogens was explained by increased apoptosis within this subpopulation. The recall antigen, tetanus toxoid, stimulated the CMRF-35(++)CD4(+)CD45RO(+) but not the CMRF-35(-)CD4(+)CD45RO(+) subpopulation. Resting CMRF-35(++) CD4(+) lymphocytes express low levels of IFN-gamma mRNA. Within 18 h following in vitro activation, CMRF-35(++) CD4(+) lymphocytes express more IFN-gamma mRNA and protein compared with the CMRF-35(-)CD4(+) lymphocytes, however, after 24 h, both the CMRF-35(+) and CMRF-35(-)CD4(+) T lymphocytes were able to produce IFN-gamma. The CMRF-35(++)CD4(+) T lymphocyte population contains the Th(1) memory effector cells.     

Combination treatment with IL-2 and anti-IL-2 mAbs reduces tumor metastasis via NK cell activation

Combination treatment consisting of IL-2 together with anti-IL-2 mAbs results in markedly larger increases in the numbers of CD8(+) T cells, dendritic cells (DCs) and NK cells in vivo compared with the results observed with injections of IL-2 or the antibodies alone. We previously showed that this combination treatment overcomes the problems associated with the short half-life of IL-2 in vivo. Importantly, the combination treatment but not IL-2 or the anti-IL-2 mAbs alone protected the mice against tumor metastases in the lungs. Here we have investigated which cell types are responsible for this protective immunity against tumors. We analyzed tumor metastases in mice that were depleted of DCs, CD8(+) T cells or NK cells. DC-deficient, diphtheria toxin receptor-expressing mice injected with diphtheria toxin as well as B cell- and T cell-deficient RAG-2-knockout mice were protected against tumors after they were administered the combination treatment. On the other hand, mice that were depleted of NK cells using anti-asialo-GM1 antibodies did not exhibit the anti-tumor activity after treatment with IL-2 combined with anti-IL-2 mAbs. Thus, these data demonstrate that NK cells, but not DCs, or CD8(+) T cells mediate the anti-tumor effect induced by this combination treatment. Therefore, combining neutralizing anti-IL-2 mAbs with IL-2 may be clinically useful to effectively enhance IL-2-mediated NK cell activities.     

IL-15 enhances the function and inhibits CD95/Fas-induced apoptosis of human CD4+ and CD8+ effector-memory T cells

Although the effect of IL-15 has been described on murine cells in vitro and in vivo, its effect on human memory CD8(+) T cells is not well characterized. We show here that IL-15 preferentially enhances the activation and effector function of human effector-memory CD45RA(-)CD62L(-) and CD45RA(+)CD62L(-) CD4(+) and CD8(+) T cells in both healthy and HIV-infected individuals. We find that IL-15 increases 2- to 5-fold both the activation and secretion of the effector cytokines IFN-gamma and tumor necrosis factor (TNF)-alpha by anti-CD3-stimulated purified CD4(+) and CD8(+) T cells and peripheral blood mononuclear cells from healthy and HIV-infected individuals. Furthermore, IL-15 potently inhibits CD95/Fas-induced apoptosis of the effector-memory CD4(+) and CD8(+) T cells from HIV-infected individuals. These findings suggest that in addition to being a growth and survival factor for memory CD8(+) T cells, IL-15 is also a potent activator of human effector-memory CD8(+) T cells both in healthy and in HIV-infected individuals.     

Cellular sources and targets of IFN-gamma-mediated protection against viral demyelination and neurological deficits

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.