Effect of dendritic cells on flow cytometric quantification of cytomegalovirus-specific, interferon-gamma-producing T cells

Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen-specific T-cell response. As the frequency of antigen-specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen-pulsed dendritic cells (DCs) to increase the number of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV-seropositive and -seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV-seropositive donors was 23-86% lower compared to the corresponding fresh blood samples. Antigen-pulsed DCs could not improve the sensitivity of the intracellular cytokine-detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate-pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN-gamma-producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.     

T-Lymphocyte Subsets During Pregnancy and the Menstrual Cycle

ABSTRACT: Many observations suggest that cellular immunity is depressed during gestation. To define alterations in subsets of circulating lymphocytes during pregnancy, we compared the percentages of total T cells, T helper cells, and T suppressor cells in the peripheral blood of 31 pregnant women at various stages of gestation with those in that of nonpregnant controls. Control studies consisted of five women ingesting oral contraceptives and five women menstruating spontaneously. Isolated peripheral blood mononuclear cells were reacted with fluoresceinated monoclonal antibodies that were specific for Leu-2 cells, Leu-3 cells, and Leu-4 cells and that were subsequently analyzed on a fluorescence-activated cell sorter. No variation in the percentage of Leu-2, Leu-3, or Leu-4 cells was observed during the menstrual cycle or oral contraceptive cycle. No differences were seen when T-cell subsets at various stages of gestation were compared with the corresponding T-cell subset in nonpregnant controls. The data suggest that pregnancy does not alter the percentage of T-cell subsets in the peripheral blood.     

Cultured T cells from patients with T cell chronic lymphocytic leukemia demonstrate a normal phenotype

Peripheral blood mononuclear cells from eight patients with the rare T-cell form of chronic lymphocytic leukemia were isolated and cultured with Interleukin-2 (IL-2). In all but one case, cultured T-cells (CTC) were established. Various culture conditions were tested for their effectiveness; feeder layers proved valuable for expanding the cultures to large volumes. The CTC remained IL-2 dependent. Analysis of surface determinants on these CTC showed a polyclonal proliferation of T-cells. The distribution of subset markers in the patients' CTC population had completely changed in comparison to the "fresh" peripheral blood cell population but was similar to CTC initiated from healthy donors. Our data suggest that the patients' few contaminating normal T-lymphocytes expanded in culture, while the malignant cells were unresponsive to IL-2. This conclusion is supported by growth characteristics and morphology of the CTC.     

Variation in peripheral blood T cell subsets in serial assays

No significant differences were found in the T cell subsets of fresh and frozen peripheral blood mononuclear cells (PBM) from six healthy donors analysed with the Ortho series of monoclonal antibodies and a fluorescence-activated cell sorter. Analysis of replicates of cryopreserved PBM showed that considerably higher variation in T cell subsets occurred when samples were assayed in serial assays than when the samples were analysed together under the same conditions. These results indicate that errors introduced into a longitudinal study by serial analysis of samples may be reduced if samples are cryopreserved and subsequently thawed and analysed together at the end of the study.     

FcRgamma chain does not replace CD3zeta chain in CD3zeta-deficient T lymphocytes of patients with gastric adenocarcinoma

Defective CD3zeta chain expression has been reported in T lymphocytes of patients with inflammatory diseases, such as systemic lupus erythematosus or osteoarthritis, and with cancer. In lupus, the absent CD3zeta chain is replaced by the FcRgamma chain, rendering the T cells hyper responsive. However, there are no data on T lymphocytes from patients with cancer. In this study, the presence of the FcRgamma chain and its associated kinase, Syk, was analysed in patients with gastric adenocarcinoma and healthy subjects. Western blot and immunoprecipitation experiments were carried out with total cell or lipid raft extracts from fresh peripheral blood mononuclear cells or T lymphocytes, and Herpesvirus saimiri-derived T-cell lines (of blood or tissue origin). Our results revealed that the absent CD3zeta chain in cancer T lymphocytes was not replaced by FcRgamma either in fresh T cells or T-cell lines, in contrast to lupus T cells. This altered expression of signalling molecules in T lymphocytes of cancer patients, would explain their low proliferative capacity. Our T-cell lines represent tools to unveil the signalling abnormalities of cancer T lymphocytes.     

Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2

In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell‐based vaccine and concomitant low‐dose IFN‐α and IL‐2. The regulatory subset of CD4 T cells, characterized by CD25^high, was prospectively analysed in fresh blood, and treatment‐associated quantitative and qualitative changes were analysed. By the 4th vaccine, patients showed a marked increase in CD4⁺CD25^high T cell subset from 6% to 22% (P < 0.001). At the 6th vaccine, a general decline was observed and a significantly (P = 0.01) lower level of CD4⁺CD25^high Treg cells was reached in the group of patients who attained disease stabilization (9.5%) compared to patients with continued progressive disease (14.5%). However, when FoxP3 was employed for retrospective analysis of Tregs on frozen blood, this difference did not reach significance (P = 0.09). The vast majority of the Treg produced IL‐10 and, to a varying extent, TGF‐β. In addition, sorted CD4⁺CD25^highCD127^? Tregs were able to suppress proliferation of peripheral blood mononuclear cells in a dose‐dependent manner, thus suggesting a regulatory functionality. These findings emphasize the need for strategies to effectively eliminate Treg cells to optimize the clinical effectiveness of cancer immunotherapy.     

Effect of Theophylline on the Proportion and Function of T-Suppressor Cells in Asthmatic Children

T-cell subpopulations and their function were studied in asthmatic children with or without theophylline therapy. A decreased proportion and function of theophylline-sensitive T-suppressor cells was found in fresh peripheral blood of asthmatic children. Following peripheral blood mononuclear cells (PBMC) incubation in theophylline-free medium, the proportion of theophylline-sensitive T-cells in asthmatic children treated with theophylline was elevated to normal values. However, their suppressive effect on control lymphocyte proliferation was unchanged.     

Suppression of lymphocyte proliferation by Pseudomonas aeruginosa: mediation by Pseudomonas-activated suppressor monocytes.

Pseudomonas aeruginosa has been shown to suppress cell-mediated immunity in experimental animals, but recent reports have also demonstrated that there is a strong T-cell response to this bacteria. Our studies of human peripheral blood mononuclear cells showed a great variation in the in vitro proliferative response to killed P. aeruginosa, so we examined the interaction of the different mononuclear cells in cultures with this bacteria. P. aeruginosa stimulated the proliferation of T lymphocytes, specifically the surface-immunoglobulin-negative, T8- subset, which are felt to be T helper cells. P. aeruginosa added in coculture experiments to peripheral blood mononuclear cells stimulated with Staphylococcus aureus, Streptococcus pyogenes, or tetanus toxoid suppressed the proliferation to these latter antigens. This proliferation was not affected by the depletion of adherent monocytes from peripheral blood mononuclear cells, and the suppression was restored when monocytes were added back to these cultures. Moreover, monocytes pulsed with P. aeruginosa but not with S. aureus suppressed the antigen-induced proliferation of peripheral blood mononuclear cells. This monocyte suppression was not inhibited by indomethacin and was unlikely to be the result of prostaglandin synthesis by these cells. Thus, P. aeruginosa can induce monocytes to suppress antigen-stimulated T-lymphocyte proliferation in vitro, and these suppressor cells may facilitate the growth of this organism in disorders such as cystic fibrosis.     

The maintenance of B-cell and T-cell function in frozen and stored human lymphocytes

The objective of this study was to identify and test a convenient means for long-term storage of lymphocytes taken from clinically characterized patients without losing B- or T-cell function. Accordingly, peripheral blood lymphocytes were frozen and stored, and portions of each sample were subsequently assayed for T-cell blastogenic response and B-cell Jerne plaquing at various time intervals after freezing. A comparison of the cell counts of fresh and frozen cultures indicated that all cells were recovered after freezing. Furthermore, these cells showed no significant differences in (i) cell viability; (ii) blastogenic response to antigens ofActinomyces naeslundii, Bacteroides melaninogenicus, Fusobacterium nucleatum, and tetanus toxoid; (iii) blastogenic response to phytohemagglutinin and pokeweed mitogen; and (iv) polyclonal B-cell response to pokeweed mitogen as measured by the direct Jerne plaque assay. The retained blastogenic and plaquing responses seen in frozen cultures indicated the maintenance of both T-cell and B-cell function, respectively. This is the first reported demonstration of Jerne plaquing of normal human lymphocytes after freezing. It appears that freezing techniques provide a means for repeating and extending both T- and B-cell assays using frozen stored portions of the same cell sample.     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

T-cell subsets in human lymphocytes maintained in IL-2 medium after PHA or mixed lymphocyte reaction activation

The culture of human T lymphocytes in interleukin-2 (IL-2) containing growth factor medium results in a significant shift in the T-lymphocytes subsets isolated from such cultures at weekly intervals. If normal peripheral blood mononuclear cells are stimulated with phytohemagglutinin (PHA) or in a mixed lymphocyte reaction (MLR), the resulting T lymphoblasts can be propagated in growth factor medium. Staining of the cultured cells with monoclonal antibodies was evaluated by indirect immunofluorescence on a laser-activated flow cytometer (Ortho Spectrum III). The antibodies used were: OKT3 (mature T lymphocytes), OKT4 (helper/inducer T lymphocytes), OKT8 (cytotoxic/suppressor T lymphocytes, OKT10 (immature and "activated" lymphocytes), OKT11a (cells which rosette with sheep erythrocytes), and OKIa-I (HLA-DR constant region). Both PHA and MLR activation resulted in initial preservation of the OKT4+ subset predominance over OKT8+ T lymphocytes noted on normal circulating blood lymphocytes. However, during culture in T-cell growth factor medium, there was a progressive increase in the percentage of OKT8+ cells, and a concomitant decrease in OKT4+ lymphoblasts. The increase in OKT8+ cells in the MLR-stimulated cultures was paralleled by an increase in specific cell-mediated cytotoxicity against the stimulating lymphocyte population. In addition to the shift in T-lymphocyte subset, there was virtual 100% staining with OKT3 and OKT11a, indicating the T-cell nature of the proliferating cells. OKT10 which was present on a small subset of fresh blood lymphocytes appeared rapidly in stimulated cultures, and was retained on virtually all lymphoblasts of either OKT4+ or OKT8+ subset. OKIa-1 cells increased slowly in PHA-stimulated cultures. HLA-DR+ T cells were detected earlier in MLR cultures. The activation of T lymphocytes results in a significant increase in the number of molecules of OKT11a bound per cell, in concert with the increased avidity of T lymphoblasts for sheep erythrocytes. The significant change in the phenotype and function of lymphoblasts isolated from long-term cultures demonstrates the importance of monitoring cultures, and the potential hazards in equating a cultured cell population with a freshly isolated one.     

冷冻保存对肝癌患者粘附性LAK细胞生物学特性的影响

为了观察冷冻保存对肝癌患者粘附性LAK (A LAK )细胞生物学特性的影响。我们采用苯丙氨酸甲酯 (PME )处理肝癌患者外周血单个核细胞 (PBMC )以制备A LAK细胞 ,并观察冷冻保存后A LAK细胞增殖能力、杀伤活性及细胞表型的变化。实验结果显示 ,A LAK细胞在冷冻复苏后的短时间内 ,增殖能力、杀伤活性均受到损伤 ,但随着IL 2的刺激而迅速恢复。至复苏后第 5天 ,冻存的A LAK细胞的杀伤活性明显高于未经冻存的新鲜A LAK细胞。而A LAK细胞的表面标志则在冷冻前后无显著性改变。因此 ,冷冻保存可促进肝癌患者A LAK细胞的免疫活性     

Identification of B-cell and T-helper-cell defects, and of suppressor cell hyperactivity, in humoral immunodeficiency

Methods are described for distinguishing between intrinsic B-cell defects, T-helper-cell defects, and suppressor cell hyperactivity in patients who fail to secrete immunoglobulin when peripheral blood mononuclear cells are stimulated with pokeweed mitogen. Control cells which respond to pokeweed mitogen are made unresponsive by depleting B cells or OKT4+ cells, and the missing subset, purified from the patient's peripheral blood mononuclear cells, is added back to examine its functional activity. Alternatively, hyperactivity of OKT8+ putative suppressor T cells or suppressor monocytes is evaluated by depleting these populations from the patient's peripheral blood mononuclear cells. Four patients who produced few plaque-forming cells in response to pokeweed mitogen were investigated: two had intrinsic B-cell deficiencies, one had T-helper-cell deficiency, and one had T-suppressor-cell hyperactivity.     

γδ T-Cell Subset Distribution in Human Semen From Fertile Donors

PROBLEM : γδ T-cell subset distribution has not been fully investigated in normal human semen. METHODS : We therefore carried out experiments by using a direct immunofluorescence staining technique followed by two-color cytofluorimetric analysis on mononuclear cell (MC) suspensions from ejaculates of ten healthy, fertile volunteers. Autologous peripheral blood MC were simultaneously analyzed and the results used for statistical comparison. RESULTS : The proportion of normal human semen lymphocytes bearing the γδ T-cell receptor for antigen was greatly increased compared with autologous circulating counterparts. Interestingly, the rise was mainly due to an overexpansion of cells expressing Vδl gene-encoded determinants on their surface. This contrasts with the normal blood picture, where most γδ T cells express Vδ2 conformational epitopes. CONCLUSIONS : The numerical and phenotypical differences in semen γδ T lymphocytes provide further evidence of a defined migrating lymphocyte subset balance in anatomically and physiologically distinct areas of the body. Their functional role, in terms of both helper and suppressor-cytotoxic activities in the nonsterile proximal portions of the male genital tract, now needs to be explored in detail.     

Further evidence that antibody-dependent and spontaneous cell-mediated cytotoxicity are mediated by different processes or cell types.

We have observed that canine peripheral blood mononuclear cells are ineffective as mediators of spontaneous cell-mediated cytotoxicity (SCMC) despite being excellent mediators of antibody-dependent cellular cytotoxicity (ADCC). Canine lymphocytes were unable to kill seven cell lines in SCMC assays including Chang, K-562, dog kidney, and foetal intestine. One the other hand, they were able to kill Chang and K-562 cell line cells as well as chicken red blood cells in ADCC assay systems. Canine mononuclear cells were 40% E-rosette forming, 30% surface immunoglobulin bearing, 14% Fc receptor bearing, and 13% esterase staining. K-562 cell line cells inhibited the capability of human peripheral blood cells but not canine peripheral blood cells to kill CRBCs in an ADCC assay. Fc-receptor bearing human lymphocytes of both T-cell and null-cell subclasses mediated both SCMC and ADCC, while Fc-receptor bearing canine lymphocytes mediated ADCC but not SCMC. These observations add to the evidence of a dichotomy between SCMC and ADDC with regards to either cell type or cell processes.     

Coculture of Th cells with interleukin (IL)-7 in the absence of antigenic stimuli induced T-cell anergy reversed by IL-15

Interleukin-7 (IL-7) is an important survival factor for T cells. We report here for the first time that it has another important role, facilitating T-cell clonal unresponsiveness, or anergy. The anergy was induced by a 20-day coculture of activated-human CD4(+) T-cell clones with IL-7 and irradiated peripheral blood mononuclear cells without antigenic stimuli. T-cell survival, but not T-cell anergy induction, was dependent on direct cell contacts between T cells and irradiated peripheral blood mononuclear cells. The anergic T cells exhibited no or very low expression of IL-7 receptor alpha chain (IL-7Ralpha), IL-2 receptor alpha chain (IL-2Ralpha), and common gamma chain (gammac), and did not express cytotoxic T-lymphocyte-associated protein 4, but expressed IL-15Ralpha. Coculture for 3 to 9 days of anergic T cells with a T-cell-activating cytokine IL-15, but not IL-2, restored the responsiveness of IL-7-induced anergic T cells together with reexpressions of IL-7Ralpha, IL-2Ralpha, and gammac. The anergy induction by IL-7 and restoration of responsiveness by IL-15 suggest novel mechanisms for regulation of helper T-cell responses, induction of peripheral tolerance, and breakdown of T-cell self-tolerance.     

Priming with dendritic cells can generate strong cytotoxic T cell responses to chronic myelogenous leukemia cells in vitro

Dendritic cells (DC) are antigen-presenting cells that can elicit potent antigen-specific responses. Since the development of techniques to cultivate these cells from peripheral blood, there has been a great deal of interest in their use in immunotherapeutic strategies. Here we show that morphologically, phenotypically, and functionally characteristic DC can be generated in vitro from peripheral blood mononuclear cells (PBMC) isolated from frozen apheresis product (AP) of cancer patients. These DC, when pulsed with whole-tumor lysate, protein, or RNA from a chronic myelogenous leukemia (CML) cell line, can induce anti-CML specific cytotoxicity in vitro by autologous cytotoxic T lymphocytes (CTL). RNA and protein-pulsed DC were more effective than lysate-pulsed DC at inducing cytotoxicity at low effector:target (E:T) ratios. These results were comparable to those obtained when fresh healthy peripheral blood was used as the source of PBMC, indicating that neither the malignant state of the patient nor the storage period detrimentally affected the generation or functionality of DC. CML cells were found to increase their level of MHC class I expression after exposure to CTL and pulsed DC thereby becoming better targets. These investigations lend support for the utilization of DC to generate anti-tumor responses in CML.     

Alterations of T-cell subsets in primary biliary cirrhosis.

To determine whether abnormalities of immunoregulatory T cells are associated with primary biliary cirrhosis, we characterized peripheral blood mononuclear cells in 16 patients with primary biliary cirrhosis and compared them with 30 normal controls. For this analysis we used monoclonal antibodies to the surface antigens on helper/inducer (T4+) and suppressor (T8+) T cell subsets and to a common T cell antigen (T3+). In contrast to normal persons, patients with primary biliary cirrhosis had reduced percentages of T3+ cells. More importantly, there was a relative decrease in helper/inducer (T4+) cells in 9/16 patients and a decrease in suppressor (T8+) cells in 5/16 patients. Furthermore, clinical studies indicated that patients with a decreased suppressor cell population (increased T4+ : T8+ ratio) had more advanced disease, as reflected by serum bilirubin levels (P less than 0.05) and histological changes in the liver (P less than 0.001), than those patients with a reduced helper T cell population (decreased T4+ : T8+ ratio). These data suggest that abnormalities of immune responsiveness in primary biliary cirrhosis may have a more complex origin than a uniform alteration in one immunoregulatory T-cell subset and that these immunoregulatory cell changes vary according to the severity of the disease.     

Microbe-induced lymphocyte blastogenesis enhancement after preculture.

The in vitro blastogenic response of human peripheral blood mononuclear cells to Fusobacterium nucleatum and other oral microorganisms was enhanced if the peripheral blood mononuclear cells were cultured for 24 h at 37 degrees C prior to the addition of stimulant. The enhancement which occurred at optimal and supraoptimal concentrations of F. nucleatum (10 to 100 micrograms/ml) was detected after a preculture period of as little as 2 h. The blastogenic response was a result of T-cell proliferation, and enhancement occurred independently of monocytes. Suppressor activity was induced by culturing fresh lymphocytes for 24 h in the presence of supraoptimal concentrations of F. nucleatum. The enhancement phenomenon occurred independently of the prostaglandin effects on lymphocyte blastogenesis and was not abrogated by treatment with indomethacin.     

Increased Numbers of Suppressor-Cytotoxic Cells in a Patient with Carbamazepine Hypersensitivity

A patient is presented with a clinical syndrome of erythroderma, fever, liver function abnormalities, eosinophilia and atypical lymphocytosis due to carbamazepine hypersensitivity. Immunological analysis of peripheral blood mononuclear cells was performed using mouse monoclonal antibodies against T-cell and Ia antigens. A 12-fold increase in the absolute numbers of suppressor-cytotoxic T-cells was found, resulting in a reversed helper/suppressor ratio. Also the number of Ia-positive cells was greatly increased. Carbamazepine may induce a reversible proliferation and activation of the suppressor-cytotoxic subset of T-cells. Implications and pathogenetic possibilities are briefly discussed.