Response Of Human Peripheral Blood Research Articles

The transcriptional response of mouse spleen B cells to IL-4: Comparison to the response of human peripheral blood B cells

The Th2 cytokine IL-4 triggers a signaling cascade which activates transcription by STAT6. The goals of the present study are to define the transcriptomic response of mouse spleen B cells (mSBC) to IL-4 used as single stimulus, its specificity compared to human peripheral blood B cells (hPBBC) and to mouse spleen T cells (mSTC), and the pathways affected. Oligonucleotide-based microarrays were performed using two references, the untreated sample and the cells cultured without IL-4, an experimental design which reduces the potential confounding effect of cellular stress during culture. Specificity was addressed by comparing the response of mSBC and our previously published study on hPBBC, of similar design, and a study by other authors on mSTC. We detected an mSBC-specific response (including novel genes, e.g., Sertad4, Lifr, Pmepa1, Epcam, Tbxas1; and common genes, e.g., Usp2, Cst7, Grtp1, and Casp6), an hPBBC-specific response (e.g., CCL17, MTCL1, GCSAM, HOMER2, IL2RA), and a common mSBC/hPBBC response (e.g., CISH, NFIL3, SOCS1, VDR, CDH1). In contrast, the mSBC and mSTC responses were largely divergent. Gene set enrichment analysis (GSEA) was applied for the first time to identify the pathways affected. Both in mSBC and hPBBC, IL-4 activated Myc, the transcriptional machinery itself, cell cycle, mitochondria and respiratory chain, ribosome, proteasome and antigen presentation, and Wnt signaling, and inhibited GPCR signaling. However, significant differences were found in histone demethylation, Nod signaling, and Rho signaling, which were downregulated in mSBC, and in chromatin condensation, which was downregulated in hPBBC. These findings may have therapeutic implications for the treatment of allergic diseases and parasitic infections.

Effects of Nitrogen-containing Bisphosphonates on the Response of Human Peripheral Blood Mononuclear Cells and Gingival Fibroblasts to Bacterial Components

Effects of Nitrogen-containing Bisphosphonates on the Response of Human Peripheral Blood Mononuclear Cells and Gingival Fibroblasts to Bacterial Components

In vitro studies of the genotoxicity of ionizing radiation in human G0 T lymphocytes

In an effort to mimic human in vivo exposures to ionizing irradiation, G(0) phase T lymphocytes from human peripheral blood samples were utilized for in vitro studies of the genotoxic effects of (137)Cs low-LET irradiation and (222)Rn high-LET irradiation. Both types of radiation induced mutations in the HPRT gene in a dose-dependent manner, with a mutant frequency (MF) = 4.28 + 1.34x + 7.51x(2) for (137)Cs (R(2) = 0.95) and MF = 4.81 + 0.67x for (222)Rn (R(2) = 0.51). Post (137)Cs irradiation incubation in the presence of cytosine arabinoside, a reversible inhibitor of DNA repair, caused an increase in the MF over irradiation alone, consistent with a misrepair mechanism being involved in the mutagenicity of low-LET irradiation. The spectrum of (137)Cs irradiation-induced mutation displayed an increase in macro-deletions (in particular total gene deletions) and rearrangement events, some of which were further defined by either chromosome painting or direct DNA sequencing. The spectrum of (222)Rn irradiation-induced mutation was characterized by an increase in small alterations, especially multiple single base deletions/substitutions and micro-deletions. These studies define the specific response of human peripheral blood T cells to ionizing irradiation in vitro and form a basis for evaluating the genotoxic effects of human in vivo exposure.

Heterologous desensitization of T cell functions by CCR5 and CXCR4 ligands: inhibition of cellular signaling, adhesion and chemotaxis.

T cells migrate into inflamed sites through the extracellular matrix (ECM) in response to chemotactic areas and are then simultaneously or sequentially exposed to multiple chemotactic ligands. We examined the responses of human peripheral blood T cells, present in an ECM-like context, to combinatorial signaling transduced by SDF-1alpha (CXCL12), and two CCR5 ligands, RANTES (CCL5) and MIP-1beta (CCL4). Separately, these chemokines, at G protein-coupled receptor (GPCR)-stimulating concentrations, induced T cell adhesion to fibronectin (FN) and T cell chemotaxis. However, the pro-adhesive and pro-migratory capacities of SDF-1alpha and RANTES or MIP-1beta were mutually suppressed by the simultaneous or sequential exposure of the cells to these CCR5 or CXCR4 ligands. This cross-talk did not involve the internalization of the SDF-1alpha receptor, CXCR4, but rather, a decrease in phosphorylation of ERK and Pyk-2, as well as inhibition of Ca(2+) mobilization. Strikingly, early CXCR4 signaling of phosphatidylinositol-3-kinase, detected by SDF-1alpha-induced AKT phosphorylation, was insensitive to RANTES-CCR5 signals. Accordingly, early chemotaxis to SDF-1alpha was not susceptible to CCR5 occupancy, whereas late stages of T cell chemotaxis were markedly down-regulated. This is an example of a specialized functional desensitization of heterologous chemokine receptors that induces GPCR interference with T cell adhesion to ECM ligands and chemotaxis within chemokine-rich extravascular contexts.

Ontogeny of the Response of Human Peripheral Blood T Cells to Glucocorticoids

The inhibitory effect of steroids such as dexamethasone on the immune response of adults has been widely documented. Little is known, however, about the effect of these agents on the developing immune system in newborns. The present study describes the ontogeny of the sensitivity of peripheral blood T cells to dexamethasone during the first year of life. Cord blood T cells of preterm or term infants appear to be extremely sensitive to inhibition of the proliferative response. This high sensitivity of cells to dexamethasone can still be observed in the first 2 weeks after birth. Subsequently, the sensitivity to dexamethasone inhibition of T-cell proliferation gradually decreases. At 1 year of age, the adult response pattern has been acquired.

Japanese Encephalitis Virus-Specific Proliferative Responses of Human Peripheral Blood T Lymphocytes

The T lymphocytes play an important role in prevention and recovery from viral infections. To characterize T lymphocyte responses to Japanese encephalitis (JE) virus infections, we analyzed JE virus-specific T lymphocytes in peripheral blood mononuclear cells (PBMC) obtained from seven JE patients and 10 vaccinees who had received a formalin-inactivated, purified JE virus vaccine (Biken vaccine). These PBMC were examined for proliferative responses against live JE virus, a glutaraldehyde-fixed lysate of cells infected with JE virus, and extracellular particles (EPs; subviral membrane vesicles released from cells infected with recombinant vaccinia viruses encoding the JE virus premembrane and envelope proteins). Japanese encephalitis virus-specific T cell proliferation was demonstrated with PBMC from both patients and vaccinees after stimulation with infectious JE virus or the lysate of JE virus-infected cells. Proliferating PBMC included CD4+ T lymphocytes and CD8+ T lymphocytes in responses to either form of JE viral antigens. Responses to EPs were observed only with PBMC from some American vaccinees whose PBMC also responded to the virus and lysate. These results indicate that JE virus infection and immunization with an inactivated JE vaccine induce JE virus-specific CD4+ and CD8+ T memory lymphocytes that can be induced to proliferate by infectious JE virus and noninfectious JE antigens.

The small heat-shock protein alpha B-crystallin as candidate autoantigen in multiple sclerosis.

The identification of key antigens in human autoimmune diseases is a crucial step towards the development of specific intervention. The autoantigen(s) relevant to multiple sclerosis (MS) probably reside in myelin of the central nervous system, the target of the disease. Here we examine proliferative responses of human peripheral blood T cells to the complete collection of myelin proteins fractionated by reversed-phase high-performance liquid chromatography. Myelin isolated from MS-affected brain contained a single protein fraction to which T cells from MS patients and from healthy controls showed dominant responses. This highly immunogenic protein was identified as alpha B-crystallin, a small heat-shock protein. Immunohistochemical examination of MS lesions revealed the presence of oligodendrocytes and astrocytes with raised alpha B-crystallin expression, which were not found in unaffected myelin. Our findings indicate that alpha B-crystallin serves as immunodominant myelin antigen to human T cells when expressed at the elevated levels found in active MS lesions.

The Interplay of Interleukin-10 (IL-10) and Interleukin-2 (IL-2) in Humoral Immune Responses: IL-10 Synergizes with IL-2 to Enhance Responses of Human B Lymphocytes in a Mechanism Which Is Different from Upregulation of CD25 Expression

The role of the interplay of interleukin-2 (IL-2) and interleukin-10 (IL-10) in responses of human peripheral blood B cells stimulated by ligation of antigen receptors was examined in detail. Highly purified peripheral blood B cells from normal human individuals were cultured with Staphylococcus aureus Cowan I (SA) in the presence or absence of IL-10 and IL-2. Although IL-10 alone could modestly induce Ig production by SA-activated B cells, IL-10 markedly enhanced the Ig production in synergism with IL-2. This synergistic effect between IL-2 and IL-10 was completely abrogated by addition of either anti-CD25 mAb or anti-IL-10 mAb, indicating signals through high-affinity IL-2 receptors are involved in the synergism. In fact, IL-10 upregulated the expression of α-chain of IL-2 receptors (CD25) on B cells stimulated by SA alone or SA + IL-2 for 72 hr. However, such upregulation of CD25 expression did not result in significant enhancement of subsequent responses of the activated B cells to IL-2. Of note, during the initial activation phase of B cells, IL-2 was much more effective than IL-10 in enhancing the subsequent responses to either IL-2 or IL-10. By contrast, IL-10 was much more effective than IL-2 in supporting the maturation of B cells following the initial activation. Finally, the synergy between IL-2 and IL-10 was not observed in the initial activation phase, but in the subsequent maturation phase of activated B cells. These results indicate that IL-10 and IL-2 synergistically enhance Ig production of SA-activated B cells in a mechanism which is different from the upregulation of IL-2 receptors. Moreover, the data emphasize the importance of the interplay of IL-2 and IL-10 in determining the outcome of humoral immune responses.

Minor myelin proteins can be majot targets for peripheral blood T cells from both multiple sclerosis patients and healthy subjects

T cell recognition of myelin is likely to play a role in the pathogenesis of multiple sclerosis. Predominant protein components of myelin, myelin basic protein (MBP) and proteolipid protein (PLP), have been considered as possibly relevant autoantigens, especially since both proteins are encephalitogenic in various laboratory animals. It has remained unclear, however, to what extent the numerous minor proteins contained in myelin may serve as targets for human T cell responses to myelin. In this study, the abilities of several minor myelin proteins to trigger proliferative responses of human peripheral blood T cells were compared to that of MBP. By using a water soluble collection of myelin proteins as an antigen, including MBP as the major component, short-term T cell lines were generated. Proliferative responses were determined against the various proteins after their fractionation by HPLC. Short-term T cell lines from both multiple sclerosis patients and healthy control subjects displayed significant responses to several minor myelin proteins but failed to respond to MBP. Only the use of purified MBP as trigger antigen allowed the selective expansion of MBP-specific T cell lines. These findings indicate that minor myelin proteins may act as relevant targets for autoreactive human T cells.

Inhibiting effects of serotonin antagonists on the proliferation of mercuric chloride stimulated human peripheral blood T lymphocytes.

The serotonin antagonists ketanserin and methiotepine were tested for a modulating effect on the proliferative response of human peripheral blood T lymphocytes to mercuric chloride. This response was inhibited by ketanserin at 8 x 10(-5) mol/l and by methiotepine at 8.0 x 10(-6) mol/l. There were no additive effects at these concentrations of antagonists and at 10(-5) mol/l of serotonin. Low concentrations of ketanserin eliminated the inhibiting effect of serotonin on mercuric chloride induced proliferation of T lymphocytes. It thus seems as if the inhibiting effect of serotonin on T lymphocytes is mediated by 5-HT1c or 5-HT2 receptors, while the mechanism for the intrinsic inhibiting effect of the antagonists at present is unknown.

Transfection of HLA-DR-expressing DAP.3 cells with a cDNA clone encoding the glycosyl phosphatidylinositol-linked form of lymphocyte function associated antigen-3: biochemical features and functional consequences

Structural and functional aspects of the accessory molecule lymphocyte function associated antigen (LFA)-3 (CD58) have been examined following the transfection of DAP.3 and P815 cells with a cDNA clone encoding the glycosyl phosphatidylinositol (GPI)-linked form of human LFA-3. Despite earlier observations that DAP.3 cells are deficient in GPI anchoring LFA-3 was expressed efficiently on DAP.3, as well as on P815 cells. Immunoprecipitation of LFA-3 from 35S-labelled cells revealed that the molecule expressed on the DAP.3 cells had a molecular weight intermediate between the transmembrane and GPI-linked forms expressed by human B cells. This suggests that the DAP.3 cells have a default pathway whereby the RNA transcript which encodes the GPI-linked form of the molecule can also encode an integral membrane protein. Functionally, expression of LFA-3 by DAP.3 which had previously been transfected with the genes encoding HLA-DR1 led to a marked augmentation of the proliferative response of five out of eight anti-DR1 human T cell clones. This effect was not reproduced when DR1 and LFA-3 were expressed by separate populations of DAP.3 cells, suggesting that the ligands for CD2 and for the T cell's receptor must be expressed on the same cell membrane. Expression of human LFA-3 also led to a substantial increase in the proliferative response of human peripheral blood T cells to a DR alloantigen. Separation of T cells into CD45RO+ and CD45RO- populations revealed that the augmentation was more marked for the memory than the virgin population. The mechanisms responsible for these differences are discussed.

Aging and lymphocyte function: a model for testing gerontologic hypotheses of aging in man

Advanced age in humans and experimental animal models has been consistently associated with declines in the in vivo and in vitro responsiveness of T lymphocytes. The declines in vitro responses cannot be explained by decrease in numbers of differentiated T cells or by age-associated changes in the proportion of CD4+ ‘helper’ to CD8+ ‘cytotoxic/suppressor’ T cells. However, recent studies have demonstrated a decline with age in numbers of what appear to be antigenically ‘naive’ or ‘virgin’ T cells, accompanied by a proportionate increase in ‘memory’ T cells which mediate anemnestic or recall responses to previously encountered antigens. The findings lead to the hypothesis that thymic involution leads to decline in input of newly differentiated ‘naive’ T cells, while T cell number in peripheral tissues is maintained by accumulation of longer lived ‘memory’ T cells due to lifetime antigen exposure. So far, limited data suggest that increasing average biological age of the memory T cells which accumulate with age is largely responsible for declines in vitro activation responses of human peripheral blood T cells and that a subset of these memory T cells becomes refractory to activation stimuli as a consequence of in vivo cellular aging. Approaches to testing this hypothesis are presented and the implications of these findings for use of memory T cells as a model for investigating the mechanisms of in vivo cellular aging, are discussed.

5-Azacytidine induced inhibition of mitogenic response by agents that activate or augment activation of human peripheral blood T lymphocytes

Previous work from our laboratory demonstrated the mitogenic response of human peripheral blood T lymphocytes by phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, or interleukin 2 (IL-2). Increasing levels of incorporated 5-azacytosine inhibited the action of the methyltransferase suggesting that incorporation of 5-azacytosine into DNA could be responsible for the inhibiting effect of 5-azacytidine (5-aza-CR) on DNA methylation. In this study, we first demonstrated the inhibition of mitogenic response by agents, such as PHA, PMA and ionomycin, or IL-2, that activate or augment activation of human peripheral blood T cells by treatment of the analog 5-azacytidine. Over 1 microM of 5-azacytidine, we detected significant inhibition of proliferative response and over 5 microM of 5-azacytidine toxic effect of cell viability. We found no significant change of T cell subsets after treatment of 5-azacytidine.

Different modulating effects of the monoamines adrenaline, noradrenaline, and serotonin on the DNA synthesis response of human peripheral blood T lymphocytes activated by mercuric chloride and nickel sulfate.

The influence of adrenaline, noradrenaline, and serotonin (5-hydroxytryptamine) was tested on the proliferative response of peripheral blood T lymphocytes of nickel-allergic subjects to mercuric chloride and nickel sulfate. With addition of the monoamines 1 h after mercuric chloride, there was a marked suppression with all the substances at 10(-4) M, while adrenaline also inhibited down to a concentration of 10(-5) M and serotonin down to 10(-7) M. At 3 days after mercuric chloride, adrenaline inhibited at 10(-4) M and serotonin also down to a concentration of 10(-5) M. With addition at 1 h after nickel sulfate, there was an inhibition by all the monoamines at 10(-4) M, while, in contrast, adrenaline at 10(-6) to 10(-8) M and serotonin at 10(-5) to 10(-6) M gave a stimulation of the response. These findings point at different modulating effects on the proliferative response induced by a polyclonal activator such as mercuric chloride and a monoclonal activator such as nickel sulfate.

Analysis of human peripheral blood T lymphocytes proliferating in response to sensitizing antigens: effect of interleukin-2 (IL-2)-responsive bystander cells.

The contribution of interleukin-2 (IL-2)-responsive bystander cells to the proliferative responses of human peripheral blood T lymphocytes to antigens used for sensitization such as Purified Protein Derivative (PPD), Tetanus toxoid (T T) and Influenza virus was investigated. Marked proliferation of the unfractionated peripheral blood mononuclear cells (PBMC) was observed following stimulation with these antigens to which the individuals were known to have been sensitized previously. Depletion of large granular lymphocytes (LGL) from PBMC resulted in substantial reduction in the response of the lymphoid cells in proliferating to the antigens. Proliferation of the T4+T8- (helper)-enriched population, or T4+T8- subset depleted of any IL-2 receptor (IL-2R)-bearing lymphoid cells to these antigens was comparable to that of LGL-depleted PBMC cultures. Cell titration experiments of the blast cells generated from these cultures revealed that PBMC-derived population contained fewer antigen-reactive lymphocytes. These results, therefore, suggested that IL-2-responsive LGL through expansion affected the concentration of antigen-proliferating T cells in the antigen-stimulated PBMC cultures.

Autologous mixed-lymphocyte reaction in man: XVII. In vitro effect of ion channel-blocking agents on the autologous mixed-lymphocyte response

The in vitro effect of ion channel-blocking agents verapamil (V), 4-aminopyridine (4AP), tetraethylammonium (TEA), and quinine (Q) was examined on the proliferative response of human peripheral blood T lymphocytes in the autologous mixed-lymphocyte reaction (AMLR). All the above channel blockers in a dose-dependent manner inhibited the AMLR. Tetramethylammonium (TMA), an analog of TEA that does not block K + channel currents, did not inhibit the AMLR. 4AP at 1 m M/ml concentration inhibited the expression of IL-2 receptors, as defined by monoclonal antibody anti-Tac, on T-cell activated in the AMLR. In vitro addition of recombinant interleukin 2 (rIL-2) completely corrected the inhibition of the AMLR by channel blockers. Furthermore, the concentrations of ion channel blockers required for blocking 50% response of T cells in the AMLR was much lower than that reported for 50% block of T-cell proliferation in response to phytohemagglutinin or in allogeneic mixed-lymphocyte culture (MLC). These data suggest a role of ion channels in T-cell functions and show that the AMLR provides a more sensitive system, as compared to lectin stimulation or MLC, to examine any immunosuppressive effects of ion channel-blocking agents in disease states where they are used as therapeutic modalities.

Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes.

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.

Influence of Beta-Endorphin, Somatostatin, Substance P and Vasoactive Intestinal Peptide on the Proliferative Response of Human Peripheral Blood T Lymphocytes to Mercuric Chloride

The influence of beta-endorphin, somatostatin, substance P (SP) and vasoactive intestinal peptide (VIP) was tested on the proliferative response to mercuric chloride of human peripheral blood T lymphocytes, cultured for 5 days. When beta-endorphin, 10(-8) M, was added 1 h after mercuric chloride, there was an enhancement of the response, while a slight suppression was obtained with a 10(-6) M concentration of SP and VIP. When beta-endorphin, 10(-7)-10(-9) M, somatostatin, 10(-6)-10(-9) M, and SP, 10(-11)-10(-12) M, were added 3 days after mercuric chloride, they enhanced the response. At 10(-6) M, SP gave a suppressive effect.

The T Cell Dependence of B Cell Differentiation Induced by Pokeweed Mitogen

The in vitro plasma cell response of human peripheral blood B lymphocytes to PWM was found to be dependent on the presence of T lymphocytes. B lymphocytes cultured alone with PWM did not respond with proliferation or differentiation into plasma cells. When T lymphocytes were added back to B lymphocytes before culture, the number of plasma cells of the IgM, IgG and IgA classes formed increased in proportion to the number of T lymphocytes returned. Allogeneic T lymphocytes from either peripheral blood or adult thymus could effectively substitute for autologous T lymphocytes. By comparing responses of B lymphocytes cultured with equal numbers of autologous or allogeneic T cells, it was found that the considerable variation in the magnitude of the plasma cell response from different normal donors was related to a quality of their T cells; T cells from high responders substantially enhanced plasma cell differentiation of B cells from low responders and vice versa. This T cell "helper function" was irradiation-resistant and was a property of thymocytes as well as peripheral T cells.