Inhibition of autoimmune diabetes by Fas ligand: the paradox is solved

Previous reports that diabetogenic lymphocytes did not induce diabetes in nonobese diabetic (NOD)-lpr mice suggested the critical role of Fas-Fas ligand (FasL) interaction in pancreatic beta cell apoptosis. However, recent works demonstrated that FasL is not an effector molecule in islet beta cell death. We addressed why diabetes cannot be transferred to NOD-lpr mice despite the nonessential role of Fas in beta cell apoptosis. Lymphocytes from NOD-lpr mice were constitutively expressing FasL. A decrease in the number of FasL+ lymphocytes by neonatal thymectomy facilitated the development of insulitis. Cotransfer of FasL+ lymphocytes from NOD-lpr mice completely abrogated diabetes after adoptive transfer of lymphocytes from diabetic NOD mice. The inhibition of diabetes by cotransferred lymphocytes was reversed by anti-FasL Ab, indicating that FasL on abnormal lymphocytes from NOD-lpr mice was responsible for the inhibition of diabetes transfer. Pretreatment of lymphocytes with soluble FasL (sFasL) also inhibited diabetes transfer. sFasL treatment decreased the number of CD4+CD45RBlow cells and increased the number of propidium iodide-stained cells among CD4+CD45RBlow cells, suggesting that sFasL induces apoptosis on CD4+CD45RBlow "memory" cells. These results resolve the paradox between previous findings and suggest a new role for FasL in the treatment of autoimmune disorders. Our data also suggest that sFasL is involved in the deletion of potentially hazardous peripheral "memory" cells, contrary to previous reports that Fas on unmanipulated peripheral lymphocytes is nonfunctional.     

Identification and analysis of a novel human surface CD5- B lymphocyte subset producing natural antibodies.

The production of "natural" autoantibodies or antibodies, i.e., Ig that bind a variety of self- and/or exogenous Ag and arise independently of known immunization, is though to be a feature of CD5+ B lymphocytes. To determine whether other lymphocyte subsets exist that might be committed to the production of natural antibodies, human peripheral blood B cells were sorted on the basis of surface CD5 expression and differential expression of surface CD45RA (CD5+CD45RAintermediate(int), CD5-CD45RAlow(lo), and CD5-CD45RAhigh(hi)), and analyzed for the type of Ig produced after EBV infection and culture. Like their CD5+ counterparts, most CD5-CD45RAlo B lymphocytes were precursors of cells producing IgM, a major proportion of which displayed the Ag-binding features of natural antibodies. In contrast, CD5-CD45RAhi B cells comprised a high frequency of IgG-producing cell precursors, possibly including memory B lymphocytes. Six of seven IgM mAb generated from sorted CD5-CD45RAlo B cells and three of four IgM mAb from sorted CD5+ B cells were polyreactive, binding with different affinities (Kd, 10(-5) to 10(-8) M) to two or more Ag; the remaining mAb from CD5-CD45RAlo and the mAb from CD5+ B cells each bound to a single Ag (Kd, 10(-7) to 10(-8) M), beta-galactosidase and ssDNA, respectively. CD5-CD45RAlo B cells account for 4.1 +/- 1.2% (mean +/- SD in 11 healthy subjects; CD5+ B cells, 23.3 +/- 6.9%) of total B lymphocytes and display the features of quiescent cells. In a given individual, the number of CD5-CD45RAlo B cells remains constant over time. CD5-CD45RAlo and CD5+ B cells bear surface CD11b and CD14, at densities and/or frequencies apparently higher than those of CD5-CD45RAhi B lymphocytes. Despite their surface CD5- phenotype, CD45RAlo B cells express CD5+ mRNA at levels comparable with those of CD5+ B lymphocytes, whereas CD5-CD45RAhi B cells express only trace amounts of CD5 mRNA. The commitment to natural antibody production and the degree of CD5 mRNA expression suggest that the newly defined CD5-CD45RAlo B cell subset is related to CD5+ B lymphocytes, and may constitute the human homologue of the mouse Ly-1-"sister" B cell population.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

CD40 ligand-deficient T cells from X-linked hyper-IgM syndrome carriers have intrinsic priming capability.

Deficiency in CD40 ligand (CD40L) expression is associated with impaired T cell immunity in mouse models and in humans. Previous studies have indicated that this is due to the failure of induction of extrinsic costimulatory molecules. However, other studies have suggested that CD40L is an intrinsic costimulatory molecule. The X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency caused by mutations in CD40L, resulting in impaired Ab production and T cell immunity. CD4+ T cells from female carriers of XHIM express a variable degree of normal CD40L based on random X chromosome inactivation. We have examined T cells from XHIM carriers to investigate whether CD40L supports T cell function by acting as an intrinsic costimulator or by induction of other costimulatory molecules by examining coexpression of CD40L and markers of T lymphocyte priming. These carriers provide a unique model for comparison of CD40L-expressing and -nonexpressing lymphocytes in that all factors, including immunological experience, are equivalent between the two populations. Our results show that compared with CD40L-deficient T cells, T cells that express CD40L normally have a minimal advantage in becoming primed, as defined by CD45 RO isoform expression and production of IFN-gamma and TNF-alpha. Conversely, CD40L-deficient T lymphocytes clearly were capable of becoming primed as defined by the same parameters. These findings imply that the intrinsic costimulatory activity of CD40L is not required for attaining primed status, and that CD40L primarily supports T cell function by inducing extrinsic factors that can be shared by CD40L-deficient cells.     

Differential effects of cytokines on proliferative response of human CD4+ T lymphocyte subsets stimulated via T cell receptor-CD3 complex.

We report that the subsets of CD4+ T cells characterized by differential expression of CD45RA (2H4) Ag showed significant differences in proliferative response to immobilized anti-CD3 antibody (Ab) and cytokines: IL-1, IL-2, IL-4, and IL-6. Most strikingly, CD4+/45RA+ but not CD4+/45RA- T cells responded to anti-CD3 Ab and IL-4. Similar difference in response to IL-4 occurred when the subsets were stimulated by two "alternative" T cell activation pathways via CD2 and GD3 Ag. The response of CD4+/45RA+ cells to anti-CD3 Ab and IL-4 was enhanced by the two monokines: IL-1 and IL-6. Further differences between the subsets included the preferential response of the CD4+/45RA+ cells to enhancing effect of IL-6 on proliferation mediated by the anti-CD3 Ab and IL-2. In contrast to IL-6, IL-1 was unable to increase this proliferation significantly. In turn, the CD4+/45RA- cells responded preferentially to a weak stimulation mediated by anti-CD3 Ab either alone, or together with IL-1 and IL-6. Existence of these significant differences in the response of CD4+ T cell subsets costimulatory effects of the cytokines, suggests that the in vivo events resulting in an accumulation of the cytokines in particular combinations may lead to selective activation of one of the CD4+ T cell subsets during the immune response.     

Coreceptor function of CD4 in response to MHC class I molecule

Specificity of T cell receptor (TCR) and its interaction with coreceptor molecules play decisive role in successful passing of T lymphocytes via check-points during their development and finally determine the efficiency of adaptive immunity. Genes encoding alpha- and beta-chains of TCR hybridoma 1D1 have been cloned. The hybridoma 1D1 was established by the fusion of BWZ.36CD8alpha cell line with CD8+ memory cells specific to MHC class I H-2Kb molecule. Exploiting retroviral transduction of thymoma 4G4 cells with TCR genes and coreceptors CD4 and CD8, variants of this cell line expressing on the surface CD3/TCR complex and coreceptors, separately or simultaneously have been obtained. The main function of CD4 is stabilization of interaction between TCR and MHC class II molecule. Nevertheless, we have found that CD4 could successfully participate in the activation of transfectants via TCR specific to MHC class I molecule H-2Kb. Moreover, coreceptor CD4 dominates CDS, because the response of transfectants CD4+CD8+ is blocked by antibodies to CD4 and MHC Class II Ab molecule but not to coreceptor CD8. The response of CD4+ cells was not due to cross-reaction between TCR 1D1 with MHC class II molecules, because transfectants do not respond to splenocytes of H-2b knockouted mice with impaired assembly of TCR/beta2-microglobulin/peptide complexes resulting in their absence on the cell surphace. The effect of domination was not due to sequestration of kinase p56lck, because truncated CD4 with the loss of binding motif for p56lck remained functional in 4G4 cells. Results obtained can explain the number of features of intrathymic selection and represent experimental basis for development of new methods of cancer gene therapy.     

Calcium signaling capacity of the CD11b/CD18 integrin on human neutrophils.

The CD11b/CD18 integrin is a major cell adhesion molecule of myelomonocytic cells. Exposure of human neutrophils in suspension to CD11b or CD18 monoclonal antibodies (mAbs)2 does not affect the resting level of cytosolic free Ca2+ in these cells; however, a subsequent cross-linking of either of these antibodies triggers a prompt and significant cytosolic-free Ca2+ transient lasting about 10 min. The rise in cytosolic-free Ca2+ (from 130 +/- 2 to 414 +/- 12 nM or 111 +/- 12 to 331 +/- 22 nM caused by cross-linking of CD11b or CD18 subunits, respectively) is due to both mobilization of Ca2+ from intracellular stores and influx of Ca2+ across the plasma membrane. Cross-linking of the common leukocyte antigen (CD45) did not alter the basal level of cytosolic free Ca2+. In accordance with other adherence-induced phenomena and with CD11/CD18-mediated phagocytosis, these Ca2+ signals were only modestly affected by pertussis toxin. Thus, the present data clearly indicate that the CD11b/CD18 integrin on human neutrophils is capable of inducing a prompt cytosolic-free Ca2+ signal. These findings directly support the recent suggestion that the CD11b/CD18 integrin is responsible for the "spontaneous oscillations" of cytosolic-free Ca2+ observed in adherent neutrophils and, at least partially, also explain how integrin-mediated adherence can modify the functional responsiveness of neutrophils to a subsequent agonist stimulation.     

Expression of variable exon A-, B-, and C-specific CD45 determinants on peripheral and thymic T cell populations.

A mAb (I/24) has been generated that is specific for a determinant on mouse CD45 molecules. Reactivity of this mAb with a panel of CD45 transfected cell lines demonstrated that the determinant recognized is dependent upon expression of one or more CD45 variable exons and that exon C is sufficient for its expression. The exon C-specific epitope detected by I/24 is expressed at high density on essentially all B lymphocytes and at an intermediate density on the vast majority of CD8+ splenic T cells. Two distinct subpopulations of CD4+ splenic T cells were detected, a minor subpopulation that expresses this exon determinant at high density and a major subpopulation that expresses it at a much lower density. This first identification of a CD45RC-specific reagent allowed a comparison of the expression of exon A-, exon B-, and exon C-specific determinants on peripheral and thymic lymphoid populations. When splenic lymphocytes were analyzed for expression of CD45RA (reactive with mAb 14.8), CD45RB (reactive with mAb 23G2 or mAb 16.A), and CD45RC (reactive with mAb I/24) determinants, it was found that each of these CD45 determinants had a distinct pattern of expression on CD4+ and CD8+ T cells and B cells. CD45RB and RC epitopes were also detected at high density on a small proportion (0.7 to 4.1%) of thymocytes. Both CD45RB and RC epitopes were found predominantly on CD4-CD8- and CD4-CD8+ thymocytes but were also found on small numbers of CD4+CD8+ and CD4+CD8- cells. The population of thymocytes that expressed CD45RB and CD45RC determinants displayed a novel TCR CD3 phenotype characterized by a level of expression that was intermediate between that seen in the larger CD3 bright and CD3 dull populations of thymocytes.     

T cell activation via CD2 [T, gp50] molecules: accessory cells are required to trigger T cell activation via CD2-D66 plus CD2-9.6/T11(1) epitopes

Binding monoclonal antibodies (MAb) both to D66 and 9.6/T11(1) epitopes on the CD2 [T,gp50]-defined molecule produces a high level of T cell mitosis. This was observed with a battery of MAb of different isotypes. In contrast, none of the anti-D66 or anti-9.6/T11(1)Ab could trigger T cell proliferation in combination with anti-T11(3). Moreover, all anti-D66-9.6/T11(1) pairs of MAb tested required monocytes to activate T cells which were recruited through their Fc receptors. Variations among normal individuals were observed in the level of response to anti-D66-9.6/T11(1) pairs of Ab, 75% of a population of French Caucasians giving a high response. The level of response of a given individual was determined by his accessory cells. However, the level of response of an individual appeared to be minimally influenced by the isotype of a peculiar anti-D66 or anti-9.6/T11(1) Ab. The addition of exogeneous IL 2 could overcome the removal of accessory cells or the modulation of CD3 molecules. In contrast, IL 2 receptor appearance was not overcome by removal of monocytes. Thus, T cell activation via CD2 seems to be produced by "touching" several definite regions of this molecule which trigger a cascade of events similar to those produced by mitogenic lectins. One can assume that the appropriate conformational changes of the CD2 molecule induced by anti-D66-9.6/T11(1) pairs of Ab are solely produced when they are presented by accessory cells. This leaves open the question of whether accessory cells would also play a more active role.     

Identification and characterization of gp TFA-1, a guinea pig T cell surface antigen associated with T cell function

Monoclonal antibodies (Ab) were produced that specifically recognized guinea pig T cells. FACS analysis revealed that Ab 188 bound to the majority of peripheral T lymphocytes of strain 2 and strain 13 guinea pigs and to a minor population of thymocytes. It failed to react with the Ia-bearing guinea pig B cell leukemia line EN-L2C, with macrophages, bone marrow cells, erythrocytes, or thrombocytes. Treatment of T cells with Ab 188 and complement prevented T cell activation. Culturing primed T cells with antigen- or mitogen-pulsed syngeneic or with allogeneic macrophages in the continuous presence of Ab 188 produced a marked, dose-dependent inhibition of T cell proliferation. The antigen defined by Ab 188 was therefore designated guinea pig T lymphocyte function-associated antigen-1, gp TFA-1. The magnitude of inhibition by Ab 188 varied between 65 and 85% whereas three other antibodies to guinea pig T cells had no inhibitory effect on T cell proliferation. Time course experiments revealed that gp TFA-1 is critically involved in an early phase of T cell activation. Maximal inhibition was achieved only if the antibody was present from the beginning of the cell culture; the addition of antibody after 24 hr of culture no longer had an inhibitory effect. Ab 188 did not induce T cell mitogenesis. Two-dimensional analysis (one-dimensional, IEF; two-dimensional, SDS-PAGE) of immunoprecipitates obtained from NP40 lysates of [35S]methionine-labeled T cell blasts indicated that a molecule was specifically precipitated that consisted of two noncovalently associated polypeptide chains with apparent m.w. of 43,000 and 38,000. Both subunits displayed extensive charge heterogeneity focusing at an average isoelectric point of 5.0 and 6.5, respectively. The gp TFA-1 molecule exhibits striking similarities in its functional and structural properties to recently described clonotypically expressed T cell glycoproteins, which were shown to be involved in antigen recognition by T cells in the murine and human systems.     

The dendritic cell-specific CC-chemokine DC-CK1 is expressed by germinal center dendritic cells and attracts CD38-negative mantle zone B lymphocytes

DC-CK1 (CCL18) is a dendritic cell (DC)-specific chemokine expressed in both T and B cell areas of secondary lymphoid organs that preferentially attracts CD45RA(+) T cells. In this study, we further explored the nature of DC-CK1 expressing cells in germinal centers (GCs) of secondary lymphoid organs using a newly developed anti-DC-CK1 mAb. Immunohistochemical analysis demonstrated a remarkable difference in the number of DC-CK1 expressing cells in adjacent GCs within one tonsil, implicating that the expression of DC-CK1 in GCs depends on the activation and/or progression stage of the GC reaction. Using immunohistology and RNA analysis, we demonstrated that GCDC are the source of DC-CK1 production in the GCs. Considering the recently described function of GCDC in (naive) B cell proliferation, isotype switching and Ab production, we investigated the ability of DC-CK1 to attract B lymphocytes. Here we demonstrate that DC-CK1 is a pertussis toxin-dependent chemoattractant for B lymphocytes with a preference in attracting mantle zone (CD38(-)) B cells. The findings that GCDC produce DC-CK1 and attract mantle zone B cells support a key role for GCDC in the development of GCs and memory B cell formation.     

Humoral response of cynomolgus macaques to human soluble CD4: antibody reactivity restricted to xeno-human determinants.

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4+ cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other non-human primates may localize to this regions.     

Expression of lymphocyte adhesion receptors for high endothelium in primates. Anatomic partitioning and linkage to activation

Glycoproteins of 90 to 95 kD Mr are involved in adhesion to high endothelium and migration into secondary lymphoid tissues in several species. Recent evidence indicates that in primates, one type of these molecules may be subsumed under the CD44 grouping of lymphocyte differentiation Ag. Flow cytometric analysis of circulating macaque lymphocytes for expression of these structures revealed a prominent bimodal distribution, defining two subpopulations with a 5- to 10-fold difference in immunofluorescence staining intensity. Studies of lymphocytes from different anatomic compartments revealed marked differences in the relative numbers of these two phenotypes: splenic and peripheral blood lymphocytes were mostly CD44hi, whereas thoracic duct lymphocytes, which have recently exited lymph nodes and Peyer's patches, were predominantly CD44lo, suggesting that these subsets may have different migratory behavior in vivo. Activated lymphocytes, as defined by light scatter measurements, expression of IL-2-R, and CD45R levels, were all CD44hi. Simultaneous three parameter flow cytometric determination of CD44 and CD45R expression, and cell-cycle analysis with 7-amino actinomycin D further demonstrated that intrinsically cycling cells were contained in the CD44hi, CD45R+ subset. After stimulation of CD44lo lymphocytes in vitro with cross-linked anti-CD3 antibodies and PMA, CD44 expression markedly increased. These data indicate that CD44 up-regulation is an early event in T lymphocyte activation in vivo, and support the hypothesis that patterns of lymphocyte traffic are regulated in concert with cell activation, possibly by differential expression of CD44.     

Crosslinking of the CD5 antigen on human T cells induces functional IL2 receptors.

CD5 is a 67-kDa antigen that is expressed on the membrane of the majority of human T cells, and on a subset of B cells. Previous studies have demonstrated that anti-CD5 monoclonal antibodies (mAb) can provide a helper signal for T cell activation through the TCR/CD3 complex. We now demonstrate that when CD5 is crosslinked by immobilized anti-CD5 mAb in the absence of other activating stimuli, the T cells proliferate in response to recombinant interleukin 2 (rIL2) (but not to rIL4). Four different anti-CD5 mAb (anti-Leu1, 10.2, anti-T1, and OKT1) had a similar effect. IL2 responsiveness could be induced with immobilized anti-CD5 mAb in cultures of purified T cells, but was enhanced by the addition of monocytes, by monocyte culture supernatant, or by the combination of IL1 and IL6. Staining with an anti-IL2 receptor (p55) mAb demonstrated expression of IL2 receptors on about 10% of the anti-CD5-stimulated T cells. Both virgin (CD45RA+) and memory (CD45RO+) T cells were responsive. Our data provide further evidence for the involvement of CD5 in T cell activation.     

Immobilization of immunoglobulins on silica surfaces. Stability.

The development of new immunosensors based on surface-concentration-measuring devices requires a stable and reproducible immobilization of antibodies on well-characterized solid surfaces. We here report on the immobilization of immunoglobulin G (IgG) on chemically modified silica surfaces. Such surfaces may be used in various surface-oriented analytical methods. Reactive groups were introduced to the silica surfaces by chemical-vapour deposition of silane. The surfaces were characterized by ellipsometry, contact-angle measurements and scanning electron microscopy. IgG covalently bound by the use of thiol-disulphide exchange reactions, thereby controlling the maximum number of covalent bonds to the surface, was compared with IgG adsorbed on various silica surfaces. This comparison showed that the covalently bound IgG has a superior stability when the pH was lowered or incubation with detergents, urea or ethylene glycol was carried out. The result was evaluated by ellipsometry, an optical technique that renders possible the quantification of amounts of immobilized IgG. The results outline the possibilities of obtaining a controlled covalent binding of biomolecules to solid surfaces with an optimal stability and biological activity of the immobilized molecules.     

Tumor-selective cytolysis is executed exclusively by CD45R+ CTL whereas allo-specific cytotoxicity can be executed also by CD45R- CTL

Naive and memory CD4+ T helper cells can be distinguished on the basis of expression of the CD45R molecule. Whether this dichotomy applies also to CD8+ T cells has not yet been established. In the present investigation the cytolytic activity of peritoneal CD8+CD45R+ and CD8+CD45R- T cells from tumor- and allo-immunized rats has been studied. More than 90% of the CD8+ peripheral blood T lymphocytes expressed the CD45R molecule, whereas in the peritoneal cavity about 60% of the CD8+ T cells displayed the CD45R+ phenotype. Analysis of cytotoxicity of sorted peritoneal cells of W439 tumor-immunized donors demonstrated selective cytolytic activity of the CD5+CD4-CD8+CD45R+ subpopulation to W439 lymphoma target cells but no effect of CD5+CD4-CD8+CD45R- lymphocytes. None of these lymphocyte populations exhibited cytolytic activity to the NK-sensitive cell line YAC-1, whereas the CD5-CD45R+ population showed strong cytotoxicity to YAC-1 cells. In allo-immunized rats both CD5+CD4- CD8+CD45R+ and CD5+CD4-CD8+CD45R- peritoneal cells exhibited strong allo-specific cytolytic activity, but no activity to YAC-1 cells. Both CD5+CD4-CD8+CD45R+ and CD5+CD4-CD8+CD45R- cells from tumor-immunized rats proliferated in response to Con A and rIL-2. This is the first study demonstrating that tumor-selective cytolytic CD8+ T cells express the CD45R molecule and that allo-specific cytolytic CD8+ T cells are found in both the CD45R+ and CD45R- populations.     

16. Environmental specimen cryobanking

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines

BACKGROUND: Identification of human T-helper cell subsets is possible by measurement of intracellular cytokines after coincubation of lymphocytes with phorbol myristate acetate (PMA), calcium ionophore, and brefeldin A for up to 20 h. However, exposure to PMA leads to internalization of membrane CD4 and to loss of resolution of the CD4+ cells. Detection of CD3+CD8- cells or preselection of CD4+ cells prior to stimulation is more cumbersome than direct measurement of CD4+ cells. We report the use of the Leu3a/Leu3b multiclone for the accurate determination of CD4 cells after PMA stimulation. METHODS: Peripheral blood lymphocytes were isolated from healthy normal donors and the proportion of CD3+ / CD4+ T cells was determined by flow cytometry before and after incubation with PMA, calcium ionophore, and brefeldin A for 20 h using a variety of anti-CD4 monoclonal antibodies. RESULTS: The Leu3a/3b multiclone reagent was the only anti-CD4 monoclonal antibody capable of resolving more than 98% of the initial CD4+ events after incubation with PMA. CONCLUSIONS: The higher signal-to-noise ratio associated with Leu3a3b reagent, compared with other CD4-specific antibodies available, allows the direct and accurate identification of the CD4 subset even after PMA treatment of cells.     

Separation of mesenchymal stem cells with magnetic nanosorbents carrying CD105 and CD73 antibodies in flow-through and batch systems

The aim of this study is to develop magnetically loaded nanosorbents carrying specific monoclonal antibodies (namely CD105 and CD73) for separation of mesenchymal stem cells from cell suspensions. Super-paramagnetic magnetite (Fe3O4) nanoparticles were produced and then coated with a polymer layer containing carboxylic acid functional groups (average diameter: 153 nm and polydispersity index: 0.229). In order to obtain the nanosorbents, the monoclonal antibodies were immobilized via these functional groups with quite high coupling efficiencies up to 80%. These nanosorbents and also a commercially available one (i.e., microbeads carrying CD105 antibodies from Miltenyi Biotec., Germany) were used for separation of CD105+ and CD73+ mesenchymal stem cells from model cell suspension composed of peripheral blood (97.6%), human bone marrow cells (1.2%) and fibroblastic cells (1.2%). The initial concentrations of the CD105+ and CD73+ cells in this suspension were measured as 5.86% and 6.56%, respectively. A flow-through separation system and a very simple homemade batch separator unit were used. We were able to increase the concentration of CD105+ cells up to about 86% in the flow-through separation system with the nanosorbents produced in this study, which was even significantly better than the commercial one. The separation efficiencies were also very high, especially for the CD73+ cells (reached to about 64%) with the very simple and inexpensive homemade batch unit.