Autologous mixed lymphocyte reaction in man. I. Relationship with age and sex

Coculture of normal human T lymphocytes with autologous cells enriched in B lymphocytes results in increased DNA synthesis. In our system, the responder cells are mononuclear cells stimulated by irradiated autologous non-T cells in the presence of 20% inactivated autologous plasma. The results indicate that the proliferative response is age and sex related; significantly higher levels of thymidine incorporation are observed with female peripheral blood lymphocytes and, moreover, it appears that this strong stimulation is more pronounced in older female subjects. In contrast, the proliferative responses against allogeneic cells are not modified irrespective of the age and sex group considered. The involvement of autorosette-forming cells (A-RFC) in such autologous mixed lymphocyte reaction (A-MLR) is demonstrated by the marked impairment of the A-MLR after removal of A-RFC from responding cells. These data suggest that older healthy subjects, particularly females, exhibit a higher incidence of in vitro autologous reactions.     

Immunophenotype of human lymphocytes after interaction with mesenchymal stromal cells

Human multipotent mesenchymal stromal cells (MMSCs) were cocultured with allogenic blood-born mononuclear cells (MNCs). The MNCs consisted of cells that differed in their maturity or functional state, such as lymphocytes from adult peripheral blood vs. umbilical cord blood (cb) or nonstimulated vs. phytohemagglutinin (PHA)-activated lymphocytes from peripheral blood, respectively. The share of T, B, and natural killer (NK) cells or T cell subsets within the initial MNCs or cbMNCs were within physiological reference range for adult peripheral blood. After coculturing with the MMSCs, the populations of B cells decreased in both MNCs and cbMNCs, whereas the populations of the T and NK cells decreased among cbMNC only (p < 0.05). A decrease in the subset of T-NK cells was observed in the T cells of both MNCs and cbMNCs. In the coculture of MMSCs and PHA-MNCs, we found decrease in the number of CD8⁺ and HLA-DR⁺ cells and an increase in the number of CD25⁺ lymphocytes compared to monocultured PHA-MNCs. Our data show that the interaction with MMSCs did not substantially modify the composition of allogenic lymphocytes independent of their maturation (MNCs vs. cbMNCs) or activation (MNCs vs. PHA-MNCs), and the means were within the physiological limits. Moreover, exposure to the MMSCs did not reduce the viability of lymphocytes and even promoted the survival of cells in case of cbMNCs.     

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.     

Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression

Subclinical endometritis (SCE) is an important postpartum disease in dairy cows, but conventional cytobrush diagnosis often gives imprecise results. The aim of this study was to analyze disease-associated changes in peripheral blood as potential diagnostic parameters. Cellular subpopulations of blood leukocytes from cows with or without SCE (45–55 days postpartum) were flow-cytometrically quantified. Gene expression of whole blood leukocytes was assessed by PAXgene analysis. Subclinical endometritis cows showed significantly higher number of blood mononuclear cells and neutrophils. Among mononuclear cells, numbers of B-cells, NK-cells, and CD172a-positive monocytes were significantly elevated. Compared with non-SCE cows, blood leukocytes of SCE cows significantly expressed higher copy numbers of CXCL8, TNF, and IL12. To test whether circulating plasma factors are responsible for these changes, leukocytes, polymorphonuclear cells, and monocyte subpopulations (classical, intermediate, nonclassical) of healthy cows were stimulated with plasma of SCE and non-SCE cows. Although gene expression of whole leukocytes and polymorphonuclear cells remained unaltered, plasma from SCE animals significantly elevated expressed messenger RNA copy numbers of CXCL8, CXCL1, and IL1B in intermediate monocytes. In conclusion, elevated number of selected mononuclear subpopulations in peripheral blood and enhanced expression of distinct genes encoding for inflammatory mediators in blood leukocytes reflect the subclinical uterine inflammatory process in cows. Whether the observed changes in the periphery of SCE cows are the consequence of the uterine inflammatory process, or whether they affect the pathogenesis of the disease is currently unknown.     

Establishment of dose-response relationships between doses of Cs-137 γ-rays and frequencies of micronuclei in human peripheral blood lymphocytes

It has been suggested that the yield of micronuclei in human peripheral blood lymphocytes could be used as a biological dosimeter in cases of radiation exposure. In the present study micronuclei were induced in lymphocytes by exposing human blood samples in vitro to various doses of Cs-137 γ-rays. The blood samples were then cultivated using the cytokinesis block method. Coded programs were employed to establish the relationships between the frequencies of micronuclei and various doses of γ-rays. The best fit was obtained by the linear-quadratic model, Y = c + aD + bd², where Y is the yield of micronuclei, D is the dose in Gy and c, a, b, are constants. It seems there is a correlation between the yields of MN in mononuclear cells and the corresponding doses of radiation. Therefore an attempt was made to include these MN in the calculation of the dose-response relationship.     

Lymphocyte transformation in casein-induced murine amyloidosis.

Spleen lymphocytes from casein-induced amyloidotic mice demonstrate diminished transformation in vitro to PHA-P, concanavalin A, and pokeweed mitogen but a normal response to lipopolysaccharide. Thymic and peripheral blood lymphocytes respond normally to these mitogens. The amyloidogen casein acted as a mitogen in both normal and casein injected mice. The diminished PHA responsiveness of spleen lymphocytes in vitro could have resulted from an inhibitory effect of amyloid fibrils on lymphocyte proliferation and did not indicate a generalized diminished cellular immunologic responsiveness of amyloidotic mice.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Phenotypic and Functional Characterization of Lymphocytes from Different Age Groups of Bolivian Squirrel Monkeys (Saimiri boliviensis boliviensis)

Due to many physiological and genetic characteristic similarities to humans, squirrel monkeys provide an ideal animal model specifically for studying malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). While squirrel monkeys three years and older are generally considered adult subjects suitable for use in medical research studies, little is known about the functional properties of lymphocytes in relation to the age of these animals, which could significantly impact the quality and quantity of innate and adaptive immune responses. In this study, we investigated differences in the phenotype and function of lymphocytes subsets of young (3–4 years), adult (8–10 years) and aged (16–19 years) squirrel monkeys. In general, animals in all three age groups exhibited comparable numbers of different lymphocyte subsets except for CD20+ B cells that were significantly lower in aged relative to young animals and T cells subsets expressing both CD4 and CD8 (double positive) were significantly higher in aged relative to young animals. With increasing age, phenotypic differences in central and effector memory T cells subsets were observed, that were more pronounced for the CD8+ T cells. Despite equal proportions of CD3+ T cells among the three age groups, responses of peripheral blood mononuclear cells to T cell mitogens PHA and Con A showed lower IFN-γ producing cells in the aged group than that in the young group. Furthermore, aged animals showed significantly higher plasma levels of inflammatory cytokines IL-6, IFN-γ, TNF-α, IL-10 and IL-12. These findings suggest that while the squirrel monkeys in general share phenotypic and functional similarities of lymphocyte subsets with humans in relation to age, specific differences exist in immune function of lymphocytes between young and old animals that could potentially impact experimental outcomes for which the measurement of immunologic endpoints are critical.     

Tuberculin response of lymphocytes from human skin test nonreactors; evidence for in vitro primary sensitization of T lymphocytes.

Tuberculin-purified protein derivative (PPD) is a B-lymphocyte mitogen in a variety of experimental animals. Although peripheral blood mononuclear cells (PB MNC) from healthy human tuberculin responders consistently responded to PPD by increased incorporation of [³H]thymidine, cell fractionation studies showed this to be due to T-lymphocyte rather than B-cell blastogenesis. Moreover, utilizing thymidine suicide experiments, the T-lymphocyte response could be categorized as antigenic rather than nonspecific mitogenic reactivity. Kinetic studies revealed a delayed peak of PPD-induced thymidine incorporation in PB MNC from tuberculin skin test-negative as compared to skin test-positive donors. This suggested in vitro primary sensitization of T lymphocytes to PPD, which was corroborated in experiments demonstrating tuberculin reactivity of human umbilical-cord blood lymphocytes.     

Cell types in peripheral blood of the nurse shark: An approach to structure and function

Ultrastructural and functional studies were carried out on nurse shark (Ginglymostoma cirratum) peripheral blood cells in order to identify cells of definitive morphology and specific function. Along with erythrocytes and thrombocytes, four morphologically distinct leucocytes are recognized in peripheral blood: two types of granulocytes, the ‘eosiniphil’ and the ‘granulocyte’, and two mononuclear agranulocytic cells, one resembling mammalian macrophage and monocyte, the other resembling mammalian lymphocyte. Also present in peripheral circulation are blast-like cells and mitotic cells. In vitro phagocytosis was demonstrated by the monocyte-macrophage and the granulocyte while thrombocytes, eosinophils and lymphocytes showed no phagocytic activity in the system studied. It is stressed that care must be used in drawing functional analogies between blood cells of a mammal and an elasmobranch on the basis of morphological similarity alone.     

Immune modulation of connective tissue functions: studies on the production of collagen synthesis inhibitory factor by populations of human peripheral blood mononuclear cells

It has been shown previously that a soluble factor(s) from human peripheral blood mononuclear cells was capable of specifically suppressing collagen synthesis by normal human dermal fibroblasts (S. A. Jimenez, W. McArthur and J. Rosenbloom, J. Exp. Med.150, 1421, 1979). In this communication, the cell sources and the conditions for synthesis of this collagen synthesis inhibitory factor (CSIF) are identified. CSIF production by mononuclear cells was directly related to the number of cells in culture and was significantly enhanced by a variety of mitogens and by antigens. Homologous serum or bovine serum albumin was required for CSIF production and maximal levels were reached 48 hr after stimulation. Thymus-derived lymphocytes appeared to be the main cells responsible for CSIF synthesis but B lymphocytes also produced the factor in response to proper B-cell mitogens. Preparations of plastic-adherent mononuclear cells were also found to produce increased CSIF but it was not possible to exclude completely the presence of T lymphocytes in these preparations and therefore, the cell source of CSIF in these preparations was not clearly established. Through the use of metabolic inhibitors it was shown that CSIF production required de novo protein synthesis but not cell division. Indo-methacin had no effect on either the production of CSIF or on CSIF-mediated inhibition of collagen synthesis. The results indicate that CSIF has the classic characteristics of a lymphokine and suggest a mechanism by which the immune response could modulate connective tissue function.     

PNA-binding glycans are expressed at high levels on horse mature and immature T lymphocytes and a subpopulation of B lymphocytes

In mammals, the binding of peanut agglutinin (PNA) on the plasma membrane defines subpopulations among lymphocytes from peripheral blood and lymphoid organs. PNA binds Gal 1,3GalNAc residues provided that they are not sialylated. Here, we studied the expression of PNA-binding glycans on healthy horse peripheral blood, thymus, lymph node and spleen lymphocytes. We first demonstrated the binding specificity of PNA for galactose residues by competition experiments and the inhibitory role of sialic acids in PNA binding by sialidase digestion. Unlike human and murine lymphocytes, all equine lymphocytes were found positive by flow cytometry analysis. Double-staining analyses showed that lymphocytes expressing high levels of PNA-binding glycans (PNA^high lymphocytes) were made up of the great majority of CD5⁺, CD4⁺ and CD8⁺ cells, and of 30 and 50% of sIg-bearing lymphocytes in peripheral blood and in lymph nodes or spleen, respectively. Lectin histochemistry suggested that lymph node germinal centres contained PNA^high B cells. Contrary to what is found in humans and mice, PNA staining intensity on CD5⁺, CD4⁺ and CD8⁺ cells did not differentiate immature from mature T lymphocytes in the equine thymus. The functional consequences of these differences are discussed. Published in 2005.     

Induction of SerpinB2 and Th1/Th2 Modulation by SerpinB2 during Lentiviral Infections In Vivo

SerpinB2, also known as plasminogen activator inhibitor type 2, is a major product of activated monocytes/macrophages and is often strongly induced during infection and inflammation; however, its physiological function remains somewhat elusive. Herein we show that SerpinB2 is induced in peripheral blood mononuclear cells following infection of pigtail macaques with CCR5-utilizing (macrophage-tropic) SIV_mac239, but not the rapidly pathogenic CXCR4-utilizing (T cell-tropic) SHIV_mn229. To investigate the role of SerpinB2 in lentiviral infections, SerpinB2^−/− mice were infected with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV infected SerpinB2^−/− mice produced significantly lower anti-gag IgG1 antibody titres than infected SerpinB2^+/+ mice, and showed slightly delayed clearance of EcoHIV. Analyses of published microarray studies showed significantly higher levels of SerpinB2 mRNA in monocytes from HIV-1 infected patients when compared with uninfected controls, as well as a significant negative correlation between SerpinB2 and T-bet mRNA levels in peripheral blood mononuclear cells. These data illustrate that SerpinB2 can be induced by lentiviral infection in vivo and support the emerging notion that a physiological role of SerpinB2 is modulation of Th1/Th2 responses.     

The anti-tumor effect of human monocyte-derived dendritic cells loaded with HSV-TK/GCV induced dying cells

Herpes simplex virus thymidine kinase (HSV-TK) gene and dendritic cells (DC) have been used as the pioneering in cancer therapy. HSV-TK gene can induce apoptosis and necrosis in tumor cells in the presence of the non-toxic prodrug ganciclovir (GCV). We investigated the anti-tumor effect of DC vaccination by introducing dying cells from HSV-TK gene treatment as an adjuvant. HepG₂-TK cell line was established by transfecting human hepatoma cell line HepG₂ (HLA-A₂ positive) with HSV-TK gene. Dying tumor cells were generated by culturing HepG₂-TK cells with GCV. After engulfed dying cells efficiently, immature DCs (imDC) derived from human monocytes were fully matured and elicited marked proliferation and cytotoxicity against HLA matched HepG₂ cells in autologous peripheral blood mononuclear cells (PBMC). It also implied that HepG₂ specific CTLs played an important role in the cytotoxicity which was primarily depended on Th1 responses. Given the feasibility of inducing dying cells by HSV-TK/GCV in vivo, our results suggest an effective method in clinical human hepatocellular carcinoma (HCC) treatment by an in vitro model of applying HSV-TK gene modified human tumor cells integrated with DC vaccination.     

Enzymatic relationship between human Tγ+ lymphocytes and thymocytes

The lactate dehydrogenase isoenzyme pattern has been determined in human thymocytes and in peripheral blood T lymphocytes, Tγ⁺, and Tμ⁺ lymphocytes. Thymocytes have a highly significant lower activity in LDH-1 and LDH-2 together with a highly significant higher activity in LDH-3 and LDH-4 than peripheral T lymphocytes. The same differences are seen when Tγ⁺ and Tμ⁺ cells are compared. On the contrary there are almost no differences in pattern between the peripheral T lymphocytes and Tμ⁺ cells on one hand and between thymocytes and Tγ⁺ cells on the other hand. These findings suggest that both thymocytes and Tγ⁺ cells exhibit a very immature LDH pattern with regard to the Tμ⁺ cell population.     

Early-Life Gut Bacteria Associate with IL-4?, IL-10? and IFN-γ Production at Two Years of Age

Microbial exposure early in life influences immune maturation and potentially also the development of immune-mediated disease. Here we studied early-life gut colonization in relation to cytokine responses at two years of age. Fecal samples were collected from infants during the first two months of life. DNA was extracted from the fecal samples and Bifidobacterium (B.) adolescentis, B. breve, B. bifidum, a group of lactobacilli (L. casei, L. paracasei and L. rhamnosus) as well as Staphylococcus (S.) aureus were detected with real time PCR. Peripheral mononuclear cells were stimulated with phytohaemagglutinin (PHA) and numbers of IL-4−, IL-10− and IFN-γ secreting cells were evaluated using ELISpot. We further stimulated peripheral blood mononuclear cells with bacterial supernatants in vitro and assessed the IL-4−, IL-10− and IFN-γ inducing capacity by flow cytometry and ELISA. Early S. aureus colonization associated with higher numbers of IL-4− (p = 0.022) and IL-10 (p = 0.016) producing cells at two years of age. In contrast to colonization with S. aureus alone, co-colonization with lactobacilli associated with suppression of IL-4− (p = 0.004), IL-10− (p = 0.004) and IFN-γ (p = 0.034) secreting cells. In vitro stimulations of mononuclear cells with bacterial supernatants supported a suppressive role of L. rhamnosus GG on S. aureus-induced cytokine responses. We demonstrate that the early gut colonization pattern associates with the PHA-induced cytokine profile at two years of age and our in vitro findings support that specific bacterial species influence the T helper cell subsets. This suggests that dysbiosis in the early microbiota may modulate the risk of developing inflammatory conditions like allergy.     

Morphologic alterations in cultured human synovial fibroblasts induced by blood mononuclear cells

Effects of peripheral blood mononuclear cells on cultured synovial fibroblasts were studied. When mononuclear cells from normal or rheumatoid blood were incubated on synovial fibroblast cultures, a part of the cells adhered to the fibroblasts. They were mainly T lymphocytes but also some B lymphocytes and monocytes. After a 10-hour incubation, adhered mononuclear cells induced morphologic alterations to synovial fibroblasts: appearance of stellate cells and thinning and branching of fibroblasts. No changes were seen when the cells were incubated in the presence of indomethacin. Cytotoxicity of peripheral blood mononuclear cells from 8 rheumatoid patients was also tested against three rheumatoid and three normal synovial fibroblast strains. Only 2 out of 48 combinations were cytotoxicity. The potentially cytotoxic mononuclear cells were bound equally well to rheumatoid and control synovial fibroblast cultures.     

Tumour necrosis factor-alpha and lipopolysaccharide enhance interferon-induced tryptophan degradation and pteridine synthesis in human cells

The capacity of recombinant interferon-alpha, -beta and -gamma, of bacterial lipopolysaccharide and of recombinant tumour necrosis factor-alpha to induce indoleamine 2,3-dioxygenase and synthesis of pteridines was studied in human peripheral blood mononuclear cells, human macrophages and normal dermal fibroblasts. The action of interferon-alpha and -beta on macrophages was supported by lymphocyte factors as indicated by the effect of these mediators in the absence or presence of lymphocytes. Tumour necrosis factor-alpha alone was ineffective in peripheral blood mononuclear cells and macrophages, but it significantly increased the action of all three interferon species on macrophages and fibroblasts. Lipopolysaccharide directly affected macrophages or dermal fibroblasts and enhanced the effect of interferon-gamma. However, in the presence of lymphocytes, the action of lipopolysaccharide was mediated via interferon-gamma.     

Imaging reactive oxygen species in asthma

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.