Showing papers in "Journal of Immunology in 1995"

Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells.

Abstract: Dendritic cells are APCs that are unique in their potency to stimulate proliferation of primary Ag-specific responses in vitro and in vivo. In this study, we demonstrate that dendritic cells can produce IL-12, a dominant cytokine involved in the development of IFN-gamma-producing T cells. This finding resulted from our observations that dendritic cell-induced Th1 development from total CD4+ T cells upon neutralization of endogenous levels of IL-4 was IL-12-dependent. Furthermore, we demonstrate that dendritic cells can induce the development of Th1 cells from Ag-specific naive LECAM-1bright CD4+ T cells obtained from alpha beta-TCR transgenic mice, provided that CD4+ LECAM-1dull T cells, which produce significant levels of IL-4, are not present in the primary cultures. Production of IL-12 by dendritic cells was confirmed by positive immunofluoresence staining with Abs specific for the inducible IL-12 p40 subunit. This suggests that in addition to inducing proliferation and clonal expansion of naive T cells, dendritic cells, by their production of IL-12, play a direct role in the development of IFN-gamma-producing cells that are important for cell-mediated immune responses.

Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.

Abstract: The scid mutation was backcrossed ten generations onto the NOD/Lt strain background, resulting in an immunodeficient stock (NOD/LtSz-scid/scid) with multiple defects in adaptive as well as nonadaptive immunologic function. NOD/LtSz-scid/scid mice lack functional lymphoid cells and show little or no serum Ig with age. Although NOD/(Lt-)+/+ mice develop T cell-mediated autoimmune, insulin-dependent diabetes mellitus, NOD/LtSz-scid/scid mice are both insulitis- and diabetes-free throughout life. However, because of a high incidence of thymic lymphomas, the mean lifespan of this congenic stock is only 8.5 mo under specific pathogen-free conditions. After i.v. injection of human CEM T-lymphoblastoid cells, splenic engraftment of these cells was fourfold greater in NOD/LtSz-scid/scid mice than in C.B17/Sz-scid/scid mice. Although C.B-17Sz-scid/scid mice exhibit robust NK cell activity, this activity is markedly reduced in both NOD/(Lt-)+/+ and NOD/LtSz-scid/scid mice. Presence of a functionally less mature macrophage population in NOD/LtSz-scid/scid vs C.B-17Sz-scid/scid mice is indicated by persistence in the former of the NOD/Lt strain-specific defect in LPS-stimulated IL-1 secretion by marrow-derived macrophages. Although C.B-17Sz-scid/scid and C57BL/6Sz-scid/scid mice have elevated serum hemolytic complement activity compared with their respective +/+ controls, both NOD/(LtSz-)+/+ and NOD/LtSz-scid/scid mice lack this activity. Age-dependent increases in serum Ig levels (> 1 micrograms/ml) were observed in only 2 of 30 NOD/LtSz-scid/scid mice vs 21 of 29 C.B-17/Sz-scid/scid animals. The multiple defects in innate and adaptive immunity unique to the NOD/LtSz-scid/scid mouse provide an excellent in vivo environment for reconstitution with human hematopoietic cells.

Human IL-17: a novel cytokine derived from T cells.

Abstract: A cDNA encoding human IL-17 (hIL-17) was cloned from a CD4+ T cell library. The predicted 155-amino acids sequence contains an N-terminal signal peptide and exhibits 72% amino acid identity with HVS13, an open reading frame from a T-lymphotropic Herpesvirus saimiri, and 63% with murine CTLA8. High levels of hIL-17 were induced from primary peripheral blood CD4+ T cells upon stimulation. When expressed in CV1/EBNA cells, recombinant hIL-17 was secreted in both glycosylated and nonglycosylated forms. A hIL-17.Fc fusion protein and supernatants from cells transfected with hIL-17 induced IL-6 and IL-8 production and enhanced the surface expression of the intracellular adhesion molecule-1 (ICAM-1) in human fibroblasts.

Activation of telomerase in human lymphocytes and hematopoietic progenitor cells

Abstract: This is the first report describing up-regulation of telomerase activity in human normal cells. Telomerase, a ribonucleoprotein enzyme, has been thought to be involved in maintaining telomere length stability in germline and most cancer cells, but not in normal cells. However, in the present study, we demonstrate that telomerase activity is detectable at low levels in normal human T and B cells, increases by in vitro mitogenic stimulation, increases in hematopoietic progenitor cells upon their proliferation and differentiation, and decreases with aging. Understanding the regulation of telomerase activity in normal cells may provide important insights not only into the mechanisms of normal cellular senescence but also into the mechanisms of telomerase activity deregulation as part of cancer development.

Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein.

Abstract: Mannan-binding protein (MBP) is a serum lectin participating in the innate immune defense by opsonizing various microorganisms for phagocytosis. Opsonization defect due to MBP deficiency and low levels of the protein can partially be explained by the dominant effect of three different mutations in the structural part of the MBP gene. Large interracial differences in the frequencies of these variants have previously been described, but they cannot explain the large interindividual variation in MBP serum concentration. We describe the existence of additional polymorphisms at positions -550 (H/L variants) and -221 (X/Y variants) in the promoter region of the gene. The promoter haplotypes, HY, LY, and LX, show associations with high, medium, and low levels of MBP serum concentrations, respectively. Moreover, this represents a genetic system with additive effect of haplotypes in which a low producing LX haplotype in the homozygous state down-regulates the basal expression of MBP as effectively as a single structural variant. Populations of pure Eskimos, Caucasoids, and black Africans show marked interethnic differences in the frequencies of promoter haplotypes regulating the expression of the normal peptide, with the HY haplotype frequency varying from 0.83 in Eskimos via 0.33 in Caucasoids to 0.08 in Africans. The LY haplotype frequency varies from 0.04 in Eskimos via 0.39 in Caucasoids to 0.23 in Africans. The LX haplotype frequency varies from 0.03 in Eskimos via 0.24 in Caucasoids to 0.23 in Africans. The effect of the promoter variants can explain almost all of the ethnic differences not explainable by the structural variants alone.

Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily.

Abstract: Dendritic cells are potent APC that initiate primary T cell-dependent immune responses. The lack of lineage-associated cell surface Ags for human dendritic cells has made characterization of this lineage difficult. In this study, analysis of leukocyte subpopulations isolated from human blood revealed that circulating or cultured B and T cells, NK cells, and monocytes did not express CD83, whereas CD83+ cells were predominantly found in the dendritic cell-enriched metrizamide low density fraction of plastic nonadherent blood mononuclear cells. Blood CD83+ cells had a cellular morphology characteristic of dendritic cells and a cell surface phenotype that did not correlate with that of T cells, B cells, NK cells, or cells of the myelomonocytic lineage. Analysis of CD83+ cells with a panel of mAbs that identify 126 leukocyte cell surface Ags revealed the CD83+ cells to be a phenotypically homogeneous and unique population of cells that expressed the highest levels of MHC class II molecules when compared with other leukocyte lineages. CD83+ cells were also the most potent stimulator cells in an allogeneic MLR when compared with other leukocyte lineages. Functional analysis of CD83+ cells revealed that MHC class II, CD11a, CD40 and CD86 played functionally dominant roles, whereas CD80 contributed minimally to the specialized costimulatory activity of these potent APC. Thus, CD83 serves as a useful and specific marker for this unique population of human blood dendritic cells.

Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones.

Abstract: The effect of progesterone (P) on the cytokine production profile of Ag-specific human CD4+ T cell lines and clones was investigated. T cell lines specific for purified protein derivative or streptokinase (SK) derived in the presence of P exhibited significant increased ability to produce IL-5 in comparison with T cell lines derived in the absence of P. Moreover, IL-4 was significantly increased in SK-specific T cell lines derived in the presence of P in comparison with SK-specific T cell lines derived in the absence of this hormone. In addition, SK-specific T cell lines generated in the presence of P developed into T cell clones showing a Th0-, instead of Th1-like, cytokine profile. Furthermore, SK-specific T cell clones with an established Th1 profile of cytokine secretion did express mRNA for, and produced detectable amounts of, IL-4 when stimulated with P in combination with insoluble anti-CD3 mAb. Combined stimulation with P and insoluble anti-CD3 mAb also enabled Th1 clones to express CD30 on their surface membrane. These results indicate that P can favor the development of Th cells producing Th2-type cytokines and is an inducer of both transient IL-4 production and CD30 expression in established Th1 cells. Thus, P production at the placental level may be responsible, at least in part, for increased production of Th2-type cytokines which have been implied in fetal allograft survival and maintenance of successful pregnancy.

Effects of stress on immune cell distribution. Dynamics and hormonal mechanisms.

Abstract: Immune cell trafficking is crucial to the performance of the surveillance as well as effector functions of the immune system Because immune cells travel between tissues through the bloodstream, the numbers and proportions of leukocytes in the circulation provide an important representation of the state of leukocyte distribution in the body The studies described here examine significant and selective changes in numbers and percentages of peripheral blood leukocyte subpopulations in the rat These changes were rapidly induced under conditions of mild acute stress Stress-induced increases in plasma corticosterone were accompanied by a significant decrease in numbers and percentages of lymphocytes, and by an increase in numbers and percentages of neutrophils flow cytometric analysis revealed that B cell, NK cell, and monocyte numbers showed a greater stress-induced decrease than did T cells All stress-induced changes were observed during the light (inactive) as well as the dark (active) period of the animal's diurnal cycle Importantly, the stress-induced changes in leukocyte numbers and percentages were rapidly reversed upon the cessation of stress Furthermore, the effects of stress were largely dependent on adrenal hormones, because the magnitude of the stress-induced changes was significantly reduced in adrenalectomized animals Moreover, administration of corticosterone to adrenalectomized animals resulted in a close replication of stress-induced changes observed in adrenal-intact animals These results suggest that endocrine factors released during stress modulate leukocyte trafficking and result in the redistribution of leukocytes between the blood and other immune compartments Such a redistribution may significantly affect the ability of the immune system to respond to potential or ongoing immune challenge

Expression of the Fas ligand in cells of T cell lineage.

Abstract: Fas ligand (FasL) is a membrane-type cytokine belonging to the TNF family, and induces apoptosis through its cell-surface receptor, Fas. To determine the cell types that express FasL, various mouse tissues and cell lines were examined by Northern hybridization using a mouse FasL cDNA as a probe. Among tissues, lymphoid organs (thymus, lymph node, spleen), lung, and small intestine express low levels of FasL mRNA, suggesting the role of FasL in the general immune system and mucosal immunity. The testis expressed FasL mRNA most abundantly; however, the size of FasL mRNA in the testis was slightly shorter than those in other tissues. Distribution of FasL mRNA in a panel of cell lines indicated that the FasL expression is rather restricted to the cells of T cell lineage. Activation of the splenocytes with the T cell activators such as PMA and ionomycin, Con A, anti-CD3, or even IL-2 alone induced the expression of the FasL. CD8+ splenocytes expressed the FasL more abundantly than did the CD4+ splenocytes upon activation by Con A and IL-2. Among CD4+ CTL cell lines, the FasL was expressed in all Th1 and Th0, and some Th2 clones.

Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.

Abstract: Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage A proportion of these cells also express MHC class II constitutively in the normal CNS The role of microglia as APCs for CD4+ T cells extravasating into the CNS remains undefined In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45lowCD11b/c+ is shown to identify microglial cells specifically within the CNS Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45highCD11b/c+) have been obtained directly from the adult CNS, by using flow cytometric sorting Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct Of the two populations recovered from the normal CNS, it is the minority CD45highCD11b/c+ transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4+ myelin basic protein (MBP)-reactive T cells CD45highCD11b/c+ CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture, suggesting presentation of endogenous Ag This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated The heterogeneity of these populations in terms of APC function is clearly demonstrated Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4+ T cells to proliferate or secrete IL-2

Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.

Abstract: Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-gamma release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.

Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures.

Abstract: The mechanisms of bee venom immunotherapy (VIT) are largely unknown. The aim of this study was to follow the changes of T cell cytokine secretion during the course of VIT. Ten bee venom-allergic patients with a history of severe systemic reactions, positive skin tests, and bee venom (BV)-specific serum IgE Abs were treated as follows: on the first day, a cumulative dose of 111 micrograms, starting with 0.1 microgram, was administered s.c. under intensive care conditions. Further injections of 100 micrograms BV were given on day 7, day 21, and thereafter at intervals of 4 wk. Blood samples were obtained just before the initiation of VIT, after the last injection on the same day, and before the subsequent BV injections on days 7, 21, and 50 of VIT. Peripheral blood mononuclear cells (PBMC) were stimulated with phospholipase A (PLA), the major BV allergen, or with a control Ag tetanus toxoid (TT). Cytokine secretion was measured 24 h after restimulation of the cultures with solid-phase bound OKT3 F(ab')2 mAbs after 7 days of culture. In PLA-stimulated cultures, VIT resulted in decreased IL-4 and IL-5 and increased IFN-gamma secretion. In TT-stimulated cultures, we observed similar levels of cytokines before and during VIT. We conclude that ultra-rush VIT changes allergen-specific T cell reactivity.

T cell gelatinases mediate basement membrane transmigration in vitro.

Abstract: T cell homing into extravascular sites requires penetration across the subendothelial basal lamina, a specialized nonfibrillar connective tissue structure that anchors endothelial cells to parenchymal surfaces. Herein, we show that normal human T cells express gelatinases A and B, two matrix metalloproteinases active against the major basal lamina constituents, collagen types IV and V. Expression is confirmed at both the mRNA and protein levels. Gelatinase B is expressed constitutively, whereas gelatinases A and B expression is induced by T cell activation. In vitro migration of resting T cells across a basal lamina equivalent is mediated by gelatinase B, because it is specifically blocked by GM6001, a hydroxamic acid inhibitor of matrix metalloproteinases. Inhibition of T cell homing by interference with gelatinase function may represent a useful approach to the treatment of T cell-mediated autoimmune diseases.

CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL.

Abstract: Signals initiated through both the TCR complex and CD28 are required for optimal activation of T lymphocytes. Recently, it has been demonstrated that CD28 interacts with two different ligands, designated CD80 (B7/B7-1) and CD86 (B70/B7-2). We have produced stable transfectants that express CD80, CD86, or both ligands and have examined their ability to costimulate T cell proliferation, cytokine production, and the generation of CTL. When we used small, resting human peripheral blood T cells as responders, both CD80 and CD86 transfectants efficiently costimulated anti-CD3 mAb-induced proliferation and the secretion of IL-2 and IFN-gamma. Additionally, both CD80 and CD86 transfectants were able to generate functional CTL. The magnitude and kinetics of these responses were similar, which indicates that both ligands provide efficient costimulatory signals. Because many APCs coexpress both CD80 and CD86, we compared the ability of anti-CD80 and anti-CD86 mAbs to inhibit allogeneic MLR stimulated with B lymphoblastoid cell lines and showed that it is necessary to inhibit interactions with both ligands to optimally block CD28-dependent proliferation. Given the limited homology of CD80 and CD86, it was surprising that the binding of CD28-Ig fusion protein to CD80 and that to CD86 transfectants were essentially indistinguishable. Binding of CTLA-4-Ig fusion protein to both transfectants also was quite similar, but was of higher affinity than CD28-Ig binding. Results from these studies indicate that both CD80 and CD86 are potent and similar costimulators of T lymphocytes. Therefore, the role of CD80 and CD86 in an immune response may be determined primarily by their differential expression on APC.

Glucocorticoid treatment inhibits apoptosis in human neutrophils. Separation of survival and activation outcomes.

Abstract: We examined the direct effects of glucocorticoid treatment on neutrophil survival and function in vitro. Four different glucocorticoids caused a dose-dependent inhibition of apoptosis leading to increased survival of neutrophils. Maximal effects were found with dexamethasone at 10(-6) M, 16.6 +/- 6.2 vs 54.6 +/- 6.9, at 24 h (p < 0.05). Nonglucocorticoid steroids did not modulate apoptosis in neutrophils. Furthermore, the effect was inhibited in a dose-dependent manner by the glucocorticoid antagonist RU 486. Glucocorticoid-treated neutrophils produced significantly more superoxide in response to FMLP than untreated controls (p < 0.05). However, both basal and stimulated superoxide production were less than that found with freshly isolated cells. Such lack of priming or activation by glucocorticoids is in contrast to previous experience when increased survival was accompanied by cell activation. When compared with other stimuli, the effect of glucocorticoids at 24 h was similar to that of LPS but less than that of granulocyte-macrophage colony-stimulating factor (GM-CSF), 52 +/- 4, 57 +/- 6, and 70 +/- 4, respectively (p < 0.05). When added in combination, dexamethasone did not increase survival with LPS, but did augment the effect of GM-CSF, suggesting diversity in the mechanisms by which these stimuli regulate apoptosis. These data indicate that glucocorticoids can augment the effector potential of neutrophils by prolonging their survival and functional responsiveness, and such treatment might be detrimental in vivo because of delay in neutrophil apoptosis and in ultimate clearance of them from tissues.

An important role for the chemokine macrophage inflammatory protein-1 alpha in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis.

Abstract: Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated, inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for the human demyelinating disease, multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific T lymphocytes and Ag-nonspecific mononuclear cells into the CNS. In the present report we investigated the role of two C-C chemokines (macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemotactic protein-1) and a C-x-C chemokine (MIP-2) in the pathogenesis of EAE. Production in the CNS of MIP-1 alpha, but not that of MIP-2, a rodent homologue of IL-8, or monocyte chemotactic protein-1, correlated with development of severe clinical disease. Administration of anti-MIP-1 alpha, but not that of anti-monocyte chemotactic protein-1, prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the CNS initiated by the transfer of neuroantigen peptide-activated T cells. Ab therapy could also be used to ameliorate the severity of ongoing clinical disease. Anti-MIP-1 alpha did not affect the activation of encepahlitogenic T cells as measured by cytokine secretion, surface marker expression, and ability to adoptively transfer EAE. These results demonstrate that MIP-1 alpha plays an important role in directing the chemoattraction of mononuclear inflammatory cells in the T cell-mediated autoimmune disease, EAE.

Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.

Abstract: Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers.

Abstract: Costimulatory interactions between T and B lymphocytes are crucial for T cell activation and B cell proliferation and differentiation. We have compared the roles of CD40L and B7-2 in the initiation and maturation of humoral immunity by administering anti-CD40 ligand (L) or anti-B7-2 Ab during the early (days -1 to 3) or late (days 6-10) phases of primary responses to thymus-dependent (Td) and -independent (Ti) Ags. Germinal center (GC) formation in response to a Td Ag was inhibited completely by the early administration of anti-CD40L or anti-B7-2 Abs. Later in the response, established GCs remained sensitive to anti-CD40L but were resistant to treatment with anti-B7-2. However, Ig hypermutation was reduced dramatically in GCs of anti-B7-2-treated mice and humoral memory was impaired. Early administration of anti-CD40L reduced serum Ab levels to approximately 10% of controls, whereas early treatment with anti-B7-2 reduced Ab production by only 50%. Later treatments with either Ab had no effect on Ab production. Response to a type II Ti Ag was more resistant than Td responses to interruption of costimulatory interactions. Our findings suggest that the costimulatory roles of CD40:CD40L and B7-2:CD28/CTLA-4 differ in the GC; administration of anti-CD40L abrogates an established GC reaction, whereas Ab to B7-2 suppresses Ig hypermutation and entry into the B cell memory compartment. Once B cells have entered the differentiation pathway to Ab production, neither CD40L nor B7-2 is necessary for their continued differentiation and persistence.

Alpha and beta chemokines induce NK cell migration and enhance NK-mediated cytolysis.

Abstract: Chemokines have been shown to play an important role in both the adhesion and migration of numerous leukocytic cell types, including granulocytes, monocytes, mast cells, and T lymphocytes. However, the biologic effects of chemokines on NK cells remain to be defined. Chemotaxis studies using purified human NK cells and a panel of human recombinant chemokines revealed that macrophage inflammatory protein (MIP)-1 alpha and IFN-inducible protein-10 (IP-10) are potent NK cell chemoattractants in vitro. Modest but significant chemotactic (not chemokinetic) responses were also observed in response to RANTES, MCP-1, MCP-2, MCP-3, and MIP-1 beta. Chemokine receptor expression on human NK cells was determined through displacement and Scatchard analyses, using a panel of radiolabeled chemokines, and revealed the presence of both distinct and shared chemokine receptors with affinities similar to those previously described for other cell types. Functional studies have also revealed that the beta chemokines and IP-10 are capable of augmenting NK- but not LAK- or ADCC-specific cytolytic responses in both a dose- and donor-dependent fashion. Neutralization analysis using Abs specific for various adhesion molecules revealed that NK:tumor cell conjugate formation is required for chemokine-induced NK killing. In addition, NK cells incubated in the presence of beta chemokines and IP-10 for 4 h induced the release of granule-derived serine esterases, suggesting a possible mechanism for chemokine-mediated NK killing. These results suggest that chemokines not only play an important role in the recruitment of NK cells, but also may be important mediators of NK cell degranulation augmenting local tumor cell destruction.

Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction.

Abstract: The VH gene repertoire of human peripheral B cells was analyzed using PCR analysis of individual blood B cells. Because genomic DNA of single B cells was analyzed, data from both productive and nonproductive VDJ rearrangements were obtained. Nine out of 75 B cells contained both functional and nonfunctional rearrangement products, whereas 62/75 had a single productive VDJ rearrangement. The distribution of VH families was ordered in accordance with the germline complexity, although a bias toward VH3 and some of its members was found. This bias was noted in both the productively and nonproductively rearranged repertoires, indicating that it resulted from molecular and not selective processes. Evidence for negative selection of certain VH3 and VH4 family members was noted in that they were found less often as productive than nonproductive VDJ rearrangements. In addition, evidence for positive selection based on CDR3 was obtained, in that JH6 and DXP'1 were found at a higher frequency in the productively compared with the nonproductively rearranged repertoire. The CDR3 segments were markedly heterogenous, with a much greater degree of variability noted in the nonproductive VDJ rearrangements. Finally, a much greater frequency of mutations was noted in the nonproductively rearranged VH genes within individual B cells. These results have begun to delineate the human peripheral B cell repertoire and identify processes that shape it both positively and negatively.

Lymphotoxin-alpha-deficient mice. Effects on secondary lymphoid organ development and humoral immune responsiveness.

Abstract: Targeted mutagenesis in embryonic stem cells was used to generate mice deficient in lymphotoxin-alpha (LT-alpha). Mice lacking LT-alpha -/- (LT-alpha -/- mice) exhibit a phenotype dominated by defects in secondary lymphoid organ development. LT-alpha -/- mice lack lymph nodes and Peyer's patches, and possess spleens in which the usual architecture is disrupted. However, in a few of the mutants, abnormal lymph node-like structures were observed, mainly within the mesenteric fat. Abnormal clusters of lymphocytes were also found to accumulate in the periportal and perivascular regions of the liver and lung of LT-alpha -/- mice. Yet, lymphocytes from LT-alpha -/- mice appeared phenotypically normal, expressing the expected ratios of B and T cell surface markers as well as the lymphocyte homing marker, L-selectin. In addition, bone marrow cells from LT-alpha -/- mice were able to successfully reconstitute the lymphoid organs of severe combined immunodeficient mice. However, LT-alpha -/- mutant mice examined for humoral immune responsiveness were found to be impaired in their ability to respond to different Ag. These data illustrate the utility of this mouse model as a system for understanding lymphoid organ development and its effects on immune responsiveness.

IL-15 has stimulatory activity for the induction of B cell proliferation and differentiation.

Abstract: The identification and cloning of the novel cytokine IL-15 were recently described. IL-15 is produced by a wide range of cell types, with the highest levels of IL-15 mRNA being detected in epithelial lines, monocytes, muscle, and placenta. Although it has no sequence identity with IL-2, IL-15 shares many of the T cell-stimulatory activities described for IL-2. We have examined IL-15 for its ability to stimulate B cells and have compared its activity with that of IL-2. IL-15 costimulates proliferation of B cells activated with immobilized anti-human IgM or phorbol ester, but has no stimulatory effect on resting B cells. In combination with recombinant CD40L, IL-15 is a potent inducer of polyclonal IgM, IgG1, and IgA secretion, but does not cause production of IgG4 or IgE. The activity of IL-15 in B cell proliferation and differentiation assays is comparable with that of IL-2. Studies that used neutralizing Abs have demonstrated that, for signal transduction in B cells, IL-15 uses the beta-chain of the IL-2R complex but, unlike IL-2, does not require the alpha-chain. IL-2 is required for the generation of a human primary Ag-specific in vitro response using sheep erythrocytes as Ag. Of all cytokines examined, only IL-15 has the capacity to replace IL-2 in this system, although only partially. In summary, IL-15 has comparable activity with IL-2 for the induction of B cell proliferation and differentiation and uses at least some of the components of the IL-2R complex to mediate its effects.

Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity.

Abstract: IL-12, a heterodimeric cytokine, consists of two disulfide-linked subunits, p40 and p35. We investigated the role of p40 in ligand binding and signal transduction by expressing this subunit alone in COS cells. Culture media of the transfected COS cells exhibited specific dose-dependent binding to KIT225/K6 cells, a human T cell line that expresses IL-12R. Analysis of the culture media by SDS-PAGE and Western blotting demonstrated the presence of 40-kDa monomers and 80-kDa disulfide-linked homodimers. The two p40 species were purified and identified by N-terminal sequencing and proteolytic peptide mapping. Characterization of the p40 proteins for binding and bioactivity showed that both the p40 monomer and dimer inhibited 125I-labeled IL-12 binding to IL-12R, but the 80-kDa species, having a 50% inhibitory concentration (IC50) of 20 to 70 ng/ml, was at least 20-fold more effective than the monomer. Although neither the monomer nor the dimer stimulated human PHA-blast proliferation, the 80-kDa dimer inhibited IL-12-induced proliferation in a dose-dependent manner with an IC50 of 65 ng/ml. The results suggest that the IL-12 p40 subunit contains the essential epitopes for receptor binding. However, a proper conformation required for high affinity binding is achieved only when p40 is associated with a p35 subunit or another p40 subunit. When p40 is associated with a p35 subunit, the heterodimer acts as an agonist mediating biologic activity. However, when p40 associates with another p40, the homodimer behaves as an antagonist in vitro.

Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides.

Abstract: Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis.

Abstract: Infection of mice with the protozoan Leishmania major is an established in vivo model for the definition of factors that contribute to CD4+ T helper cell subset development In the current study, a central role for IL-12 in directing both the innate and adaptive immune responses to L major is established We show that in vivo neutralization of IL-12 eliminates the NK cell cytotoxic response and IFN-gamma production by lymph node cells from 2-day L major-infected C3H mice Moreover, anti-IL-12 treatment abrogated Th1 cell development and enhanced Th2 cell development Consistent with these results, elevated IL-12 p40 production and an increase in the number of IL-12 p40-producing cells were observed within 1 day of infection in C3H mice Because BALB/c mice lack an early NK cell response or a Th1-type immune response after L major infection, we investigated the possibility that they had a defect in the ability to produce IL-12 Surprisingly, L major infection stimulated IL-12 p40 production in BALB/c mice early after infection Further studies suggest that BALB/c mice are unable to generate an early IFN-gamma response because of the simultaneous production of IL-12 and cytokines that inhibit IL-12 function, such as TGF-beta, IL-4, and IL-10 Together, these data show that IL-12 regulates the immune response to L major, but that even when IL-12 is induced, Th1 cell development may be interrupted by simultaneous production of inhibitory cytokines

Mucosal imbalance of IL-1 and IL-1 receptor antagonist in inflammatory bowel disease. A novel mechanism of chronic intestinal inflammation.

Abstract: The etiology and pathogenesis of inflammatory bowel disease (IBD) are unknown Increasing evidence supports the theory that chronic IBD is the result of dysfunctional immunoregulation manifested by an inappropriate production of mucosal cytokines The aim of the present study was to test the hypothesis that a specific mucosal imbalance of IL-1 and IL-1 receptor antagonist (IL-1ra) production plays an important role in the perpetuation and chronicity of intestinal inflammation Total IL-1, IL-1ra, and the IL-1ra/IL-1 ratio were measured in freshly isolated intestinal mucosal cells, as well as in mucosal biopsies obtained from control, Crohn's disease, and ulcerative colitis patients IL-1 alpha, IL-1 beta, and IL-1 ra were measured by specific non-cross-reacting radioimmunoassays and ELISA A markedly significant decrease in the intestinal mucosal IL-1ra/IL-1 ratio was found in both Crohn's disease and ulcerative colitis patients when compared with control subjects (p < 001) The IL-1ra/IL-1 ratio correlated closely with the clinical severity of disease (r = -07846, p < 0001) Furthermore, the observed decrease in the IL-1ra/IL-1 ratio was specific for IBD because a decreased IL-1ra/IL-1 ratio was not found in patients with self-limiting colitis These results support the hypothesis that an imbalance between IL-1 and IL-1ra production is of pathogenic importance in chronic inflammatory diseases, including IBD

A randomized, controlled trial of IL-10 in humans. Inhibition of inflammatory cytokine production and immune responses.

Abstract: In vitro, IL-10 inhibits T cell proliferation and LPS-induced monocyte production of IL-1, TNF-alpha, IL-6, and IL-8. We studied the safety and immunomodulatory effects of IL-10 administration in humans. Seventeen healthy volunteers received a single i.v. bolus injection of either human IL-10 (1, 10, or 25 micrograms/kg) or placebo. Routine safety parameters, lymphocyte phenotypes, T cell proliferative responses, and stimulus-induced cytokine production were assessed before and 3, 6, 24, and 48 h after injection. There were no adverse symptoms or signs after IL-10 administration. A transient neutrophilia and monocytosis that peaked at 6 h (45-160% above base line) was observed. However, lymphocyte counts fell by 25% 3 and 6 h after the injection (p < 0.01). In particular, lymphocytes expressing the T cell surface markers CD2, CD3, CD4, CD7, and CD8 were significantly decreased. Mitogen-induced T cell proliferation was suppressed by up to 50% (p < 0.01) in the two higher dose groups. Significant dose-dependent inhibition (65-95%) of TNF-alpha and IL-1 beta production from whole blood stimulated ex vivo with endotoxin occurred after each dose of IL-10. In contrast, there was no reduction in the production of their respective antagonists, TNF soluble receptor p55 or IL-1 receptor antagonist. We conclude that a single intravenous injection of IL-10 is safe in humans, has inhibitory effects on T cells, and suppresses production of the pro-inflammatory cytokines TNF-alpha and IL-1 beta.

IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 alpha and STAT3 complexes in human T cells and monocytes.

Abstract: IL-10 affects monocytes and T cells by driving the progression of immune responsiveness such that Th2 lymphocyte-mediated effects predominate. In this report, we show that in monocytes and T cells IL-10 stimulates tyrosine phosphorylation of the signal transducers and activators of transcription, STAT1 alpha and STAT3, in a differential manner such that the relative formation of homo- and heterodimers varies between the two cell types. Moreover, monocytes express a novel IL-10-stimulated STAT protein with an M(r) of 70 kDa that is recognized by the anti-STAT3 Ab but is not observed in T cells. IL-10 treatment of both T cells and monocytes results in the ligand-induced tyrosine phosphorylation of tyk2 and Jak1, but not Jak2 or Jak3. Selective modulation of immune responsiveness by IL-10 in cells such as monocytes and T cells may result in part from the differential activation of STAT protein pairs.