Showing papers in "International Immunology in 1995"

Up-regulation of monocytic IL-10 by tumor necrosis factor-α and cAMP elevating drugs

Abstract: It is well established that endotoxin [lipopolysacharide (LPS)] induces pro-inflammatory cytokine production in monocytes, which is followed by secretion of the anti-inflammatory cytokine, IL-10. IL-10 down-regulates inflammatory response [tumor necrosis factor (TNF)-alpha, IL-1, IL-6, IL-8] as well as IL-10 synthesis itself. We wondered whether pro-inflammatory cytokines such as TNF-alpha may be involved in the regulation of human IL-10 synthesis. TNF-alpha induced de novo IL-10 mRNA expression in a dose-dependent manner but no IL-10 protein in human peripheral blood mononuclear cells. Furthermore, LPS-induced IL-10 gene and protein expression was significantly inhibited by neutralizing anti-TNF-alpha mAb. On the basis of these results, we conclude that TNF-alpha is involved in the up-regulation of its antagonist IL-10. Paradoxically, drugs that effectively inhibit expression of TNF-alpha via the elevation of intracellular cAMP level (iloprost, pentoxifylline, prostaglandin E2 and N6,2-O-dibutyryl cAMP) augmented the endotoxin-induced IL-10 synthesis at both protein and mRNA levels. In order to provide a basis for the analysis of the transcriptional regulation of the human IL-10 gene, we isolated a fragment of the human IL-10 gene containing 1308 bp of the 5' non-coding sequence. It shows remarkable homology to the mouse IL-10 promoter in regions that have been associated with transcriptional regulation, including a cAMP responsive element which could explain the cAMP-mediated effects. The lack of a NF-kappa B-like binding site in the human sequence suggests a NF-kappa B-independent mechanism of TNF-alpha-induced IL-10 gene activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Predominant expression of invariant Vα14+ TCR α chain in NK1.1+ T cell populations

Abstract: A novel T cell subset characterized by cell surface NK1.1+ TCR alpha beta+ expression was investigated for its TCR alpha usage, particularly that of invariant V alpha 14 TCR, which was found to be preferentially used in peripheral CD4-CD8- T cells developed at extrathymic sites. We found that NK+ alpha beta T cell subsets account for 0.4% in thymocytes, 5% in the splenic T cells and 40.5% in the bone marrow T cells. Among these NK+ alpha beta T cells, two distinct subsets were detected; cell surface TCR V alpha 14+ and V alpha 14- subpopulations. Almost all of NK+ alpha beta thymocytes express V alpha 14 mRNA; however, only 80% were negative or undetectable for V alpha 14 TCR expression on the cell surface in the thymus. Similarly, approximately 50% of NK+ alpha beta T cells in spleen and bone marrow are V alpha 14+ as revealed by FACS. TCR repertoire analysis by nucleotide sequences on inverse PCR products demonstrated that most NK+ alpha beta T cells express an invariant TCR encoded by the V alpha 14J alpha 281 gene with a 1 base N-region in all tissues. Thus, invariant V alpha 14 TCR is uniquely expressed on NK T cells, and can be a marker to distinguish NK, NK T and T cells.

Cytolytic T lymphocytes displaying natural killer (NK)-like activity: expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine production

Abstract: Natural killer (NK) cells express surface receptors for defined groups of HLA class I alleles. The specific interaction between these receptors and HLA class I molecules expressed on target cells results in inhibition of NK-mediated target cell lysis. In this report, we analyzed whether similar mechanisms were operating in cytolytic T lymphocytes (CTLs) capable of lysing NK-sensitive target cells. T cell clones were screened for their ability to lyse K562 target cells. The selected clones expressed either gamma delta or alpha beta TCR. The majority of these clones failed to lyse the HLA class I+ R8/15375 cell line; however, upon addition of the previously described A6-136 (IgM) or 6A4 F(ab')2 anti-HLA class I mAbs, target cells were efficiently lysed. Lysis of autologous phytohemagglutinin blasts in the presence of anti-HLA class I mAbs occurred primarily with TCR gamma delta+ CTLs. Recognition of HLA class I molecules on target cells implies the expression of NK-related specific receptors in CTL clones. Indeed, phenotypic analysis of > 300 CTL clones with NK-like activity revealed that 28% expressed p58 molecules (specific for HLA-C alleles) while 30% expressed CD94 molecules (specific for the Bw6 specificity). These receptor molecules were found to function as inhibitory receptors, as revealed by the effect of anti-p58 or anti-CD94 mAbs (of IgG isotype) on the lysis of the Fc gamma R+ K562 target cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-γ production by anti-tumor T cells

Abstract: Unfractionated spleen cells taken from tumor-bearing mice 2 weeks after tumor implantation contained tumor-primed T cells which produced cytokines including IL-2 and IFN-gamma when cultured in vitro. With progressive tumor growth this initial lymphokine-producing capacity decreased. Here, we investigated the ability of IL-12 to (i) restore suppressed IFN-gamma production, (ii) cause tumor regression and (ii) induce anti-tumor protective immunity. Addition of rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearing mice resulted in a striking enhancement in the production of IFN-gamma compared with cultures of these cells in the absence of rIL-12 or of normal spleen cells in the presence of rIL-12. Five i.p. injections of rIL-12 into mice bearing s.c. tumors induced complete tumor regression. This was found when rIL-12 was given at early (1-2 weeks), intermediate (4-5 weeks) or even late (7 weeks) stages of tumor growth. Furthermore, IL-12-treated mice which rejected the primary tumor exhibited complete resistance to a rechallenge with the same tumor but did not reject a second syngenetic tumor. Immunohistochemical analyses following IL-12 treatment revealed that CD4+ and CD8+ T cells infiltrate the tumor. More importantly, IFN-gamma mRNA expression was observed in fresh tumor masses from tumor-bearing mice receiving IL-12 treatment. The importance of IFN-gamma was further demonstrated by the observation that the systemic administration of anti-IFN-gamma mAb prior to IL-12 treatment completely abrogated the anti-tumor effect of IL-12. Thus, these results indicate that administration of modest levels of rIL-12 to tumor-bearing mice results in tumor regression through mechanisms involving reversal of suppressed IFN-gamma production by anti-tumor T cells and the establishment of a tumor-specific protective immune response.

The status of Ig loci rearrangements in single cells from different stages of B cell development.

Abstract: Differential expression of c-kit, CD25 (TAC), surrogate L chain and cytoplasmic muH chain, and surface expression of IgM and IgD allows the separation of B220 (CD45+) B cell subpopulations. PCR analyses with DNA of single cells developed by others and by us have been used to monitor the conformation of the Ig H and L chain gene loci in these different B lineage subpopulations. The results of these analyses indicate that B220+/c-kit+/CD25- cells are the precursors of large B220+/CD25+/sIgM- which, in turn, are the precursors of small B220+/CD25+/sIgM- cells. The majority of B220+/c-kit+/CD25- cells are DHJH-rearranged, with L chain loci in germline configuration and are thus pre-B I cells. More than 90% of all large B220+/CD25+/sIgM- cells have at least one H chain locus VHDHJH rearranged; half of them have also the second locus VHDHJH rearranged and are thus large pre-B II cells. Rearrangements of at least one allele of the kappa L chain loci become detectable in 65% of the small B220+/CD25+/sIgM- cells, 67% of the immature B and > 75% of the mature B cells. The ratio of kappa L to lambda L gene rearrangements in all three subpopulations is approximately 10:1, indicating that the kappa L/lambda L ratio is established as soon as rearrangements are made.

Intra-epithelial lymphocytes. Evidence for regional specialization and extrathymic T cell maturation in the human gut epithelium

Abstract: The human gut epithelium is a unique immunological compartment, containing substantial amounts of intra-epithelial lymphocytes (IEL) with unknown functions. In this study we show that distinct and ...

Induction of the transcription factors NF-κB, AP-1 and NF-AT during B cell stimulation through the CD40 receptor

Abstract: To address elements that might uniquely characterize CD40 mediated signaling, the nuclear expression of three transcription factors was evaluated following B cell stimulation by CD40L and by anti-Ig antibody. Cross-linked CD40L was found to induce nuclear expression of NF-kappa B, AP-1 and NF-AT with a time course and intensity similar to that produced by anti-Ig. Examination of NF-kappa B in more detail demonstrated that the CD40 mediated expression of DNA binding complexes correlated with induction of trans-activating activity which again attained similar levels following cross-linking of CD40 and slg. Despite the marked similarity in transcription factor induction triggered through CD40 and slg, differences in the intracellular signaling pathways utilized were apparent in that protein kinase C (PKC) depletion did not affect CD40 mediated induction of NF-kappa B even as induction by anti-Ig was abolished. These results suggest that a 'final common pathway' or convergence of transcription factor induction may exist for two distinct receptors, each of which is individually capable of triggering cell cycle progression, despite the use of separate intracellular signaling pathways that differ at the level of PKC. Although transcription factor induction was similar for CD40L and anti-Ig early on, subtle differences in expressed NF-kappa B and AP-1 nucleoprotein complexes were apparent at 24 h. Such differences may play a role in determining the variant effects on B cells of stimulation through these two receptors.

BCMAp: an integral membrane protein in the Golgi apparatus of human mature B lymphocytes

Abstract: BCMA is a human gene expressed preferentially in mature B lymphocytes as a 1.2 kb mRNA, which encodes a 184 amino acid peptide (BCMAp). The study of BCMA mRNA expression, using human malignant B cell lines characteristic of different stages of B lymphocyte differentiation, demonstrated that the BCMA mRNA is absent in the pro-B lymphocyte stage. It is expressed faintly at the pre-B cell stage and its expression increases with B lymphocyte maturation. Polyclonal antibodies were used to show, by cellular fractionation and immunoprecipitation, that BCMAp is a non-glycosylated integral membrane protein. Furthermore, BCMAp inserts, in vitro, into canine microsomes, as a type I integral membrane protein. Cell surface labeling showed that BCMAp is not expressed in the plasma membrane of mature B lymphocytes. Immunofluorescence studies revealed that BCMAp lies in a cap-like structure near the nucleus, that was identified as the Golgi apparatus by co-localization of BCMAp with CTR433, a marker of the medial cisternae of the Golgi apparatus. Confocal scanning laser microscopy of U266 plasma cells labeled with markers of various Golgi apparatus subcompartments strongly suggests that BCMAp is located in the cis part of the Golgi apparatus. Thus, BCMAp is the first Golgi resident protein with a tissue specificity and whose expression is linked to the stage of differentiation of B lymphocytes. The location of BCMAp in the Golgi apparatus and its high expression in plasmocytes (secreting large amounts of Ig) suggest that BCMAp is implicated in the intracellular traffic of Ig.

Sharing of the IL-2 receptor γ chain with the functional IL-9 receptor complex

Abstract: The third subunit, the so-called common gamma (gamma(c)) chain, of the IL-2 receptor is shared among the receptors for IL-2, IL-4, IL-7 and IL-15, and dysfunction of the gamma(c) chain is thought to cause X-linked severe combined immunodeficiency (XSCID) ascribed to impairment of early T cell development. However, cytokines linked to XSCID are as yet unidentified. A mAb specific for the gamma(c) chain, TUGm2, profoundly inhibited cell proliferation in response to IL-9. Another mAb, TUGm3, immunoprecipitated [I-125]IL-9 cross-linked with either the IL-9 receptor or the gamma(c) chain. These results demonstrate that the gamma(c) chain is included in the functional receptor complex for IL-9, which was initially characterized as a T cell growth factor and is essential for IL-9-dependent growth signal transduction.

Stability of th1 and th2 populations

Abstract: Using an in vitro model for the development of IFN-gamma-producing (Th1) and IL-4-producing (Th2) cells from CD4+ T lymphocytes expressing a transgenic TCR, we show that IL-12 and IL-4 are the most potent stimuli for the differentiation of naive T cells to effector populations. When combinations of cytokines are present during T cell priming, the effect of IL-4 is dominant. Furthermore, differentiated Th1 cells can be converted into IL-4 producers by exposure to IL-4, but the Th2 phenotype is not reversible. The stability of Th2 populations may limit the ability to regulate Th2-dominant responses in pathologic situations.

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro.

Abstract: The interaction of CD28 and its ligands is critical for antigen-induced T cell activation. Recent studies have demonstrated the existence of at least two members of the B7 receptor family. In this report, the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2) were compared to CD28 ligation by mAb. We demonstrate that the kinetics of induction of T cell proliferation after anti-CD3 stimulation was similar regardless of the form of co-stimulation. Similarly, B7-1 and B7-2 could both maintain long-term expansion of CD4 cells. The co-stimulatory effects of both B7-1 and B7-2 were dependent on CD28 cross-linking, based on complete inhibition of proliferation by CD28 antibody Fab fragments. Co-stimulation with B7-1 and B7-2 induced high levels of cytokine secretion by resting T cells, and the effects of B7-1 and B7-2 could not be distinguished. This conclusion is based on analysis of the initial activation of CD28+ T cells, as well as T cell subpopulations consisting of CD4+ and CD8+ T cells. Both B7-1 and B7-2 could elicit IL-4 secretion from CD4+ T cells while anti-CD28 antibody induced substantially less IL-4 secretion. Furthermore, both B7-1 and B7-2 could stimulate high levels of IFN-gamma and IL-4 from CD4+CD45RO+ cells, while neither B7 receptor could co-stimulate IFN-gamma and IL-4 secretion from CD4+CD45RA+ T cells. B7-1 and B7-2 could, however, co-stimulate CD4+CD45RA+ T cells to secrete IL-2. By contrast, when previously activated T cells were tested, re-stimulation of CD4+ T cell blasts with B7-1 or B7-2 resulted in higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulation with anti-CD28 antibody maintained a higher level of secretion of IL-2 and IFN-gamma than B7-1 or B7-2. These observations may have important implications because they suggest that the manner of CD28 ligation can be a critical determinant in the development of cytokine secretion that corresponds to Th1- and Th2-like patterns of differentiation. Together these observations suggest that there are no intrinsic differences between B7-1 and B7-2 in their ability to co-stimulate the populations of cells that we have tested.

Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells

Abstract: Our recent studies on an autoantibody-transgenic mouse line demonstrated that peritoneal B-1 cells are responsible for autoimmune symptoms. However, whether B-1 cells in the peritoneum are generally involved in the pathogenesis of autoimmune disease remains controversial. To test the possible involvement of peritoneal B-1 cells in autoimmune symptoms of autoimmune-prone NZB mice, we eliminated the peritoneal cells by hypotonic shock with repeated i.p. injection of distilled water every 7 days into neonatal or 8-week-old NZB mice. By this treatment, B-1 cells, which self-renew within the peritoneal cavity, are expected to be preferentially eliminated, while other peritoneal cells can be easily supplied from bone marrows after this treatment. Indeed, in distilled water-treated old NZB mice, the number of B-1 cells decreased in spleen as well as in lamina propria of the gut but the numbers of conventional B cells and T cells did not change. Moreover, the production of autoantibodies against erythrocytes significantly decreased and the occurrence of autoimmune hemolytic anemia was reduced in 12-month-old treated NZB mice. Similarly, the elimination of peritoneal cells of NZB/NZW (NZB/W) F1 mice by water injection decreased anti-DNA IgG antibodies in the sera and reduced the pathological changes of the kidney. These results suggest that peritoneal B-1 cells may be a source of autoantibody-producing cells in autoimmune diseases of NZB and NZB/W F1 mice.

CD4+ but not CD8+ T cells are required for the induction of oral tolerance.

Abstract: It has been suggested that oral tolerance is mediated by CD8+ T lymphocytes, but the functional properties of these cells are unclear. Here we show that the induction of tolerance by feeding mice ovalbumin (OVA) does not prime antigen-specific class I MHC-restricted cytotoxic T cells in vivo. Indeed, such responses are markedly suppressed in mice fed OVA, and the induction of oral tolerance is abolished by depletion in vivo of CD4+ but not CD8+ T cells. These results indicate that CD8+ lymphocytes are unlikely to play a major role in the induction of oral tolerance and are the first demonstration that specific cytotoxic responses to an exogenous antigen can be suppressed by feeding antigen.

Fas (CD95) participates in peripheral T cell deletion and associated apoptosis in vivo

Abstract: Following exposure to some types of antigen (superantigens), responsive T cells expand and then decline in numbers, a phenomenon that has been called 'peripheral deletion'. This process may play a role in limiting autoimmune reactions and in the maintenance of immune homeostasis. Here we describe experiments on peripheral deletion in mice carrying the lpr/lpr defect, which has been shown to be due to defective production of the CD95/Fas molecule. Young lpr/lpr mice with no apparent immunologic abnormalities display a defect in bacterial superantigen-induced peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion in normal animals is dramatically reduced in the mutant mice. Further, the levels of Fas on responding cells in normal mice increases and decreases together with increases and decreases in cell numbers, suggesting that cells with the highest levels of Fas are preferentially deleted. These observations are consistent with the known ability of CD95 to transduce a signal leading to apoptosis, and they implicate this signal transduction pathway in peripheral deletion. In contrast, bacterial superantigen-induced deletion of thymocytes appears to be fully functional in these mice, and thus Fas/APO-1 does not appear to be required for this process. Further, antibody ligation of the TCR on activated T cells from normal or young lpr/lpr mice can induce apoptosis and therefore under some circumstances this phenomenon is not dependent upon CD95/Fas. Thus, to avoid autoreactivity and ensure immune homeostasis, several different apoptotic mechanisms exist in peripheral T lymphocytes, only some of which involve Fas.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of IL-13 and IL-4 production by human CD8+ and CD4+ Th0, Th1 and Th2 T cell clones and EBV-transformed B cells

Abstract: In the present study, the requirements and characteristics for the production of IL-13 by human T cells, T cell clones and B cells were determined and compared with those of IL-4. IL-13 was produced by human CD4+ and CD8+ T lymphocyte subsets isolated from peripheral blood mononuclear cells and by CD4+ and CD8+ T cell clones. CD4+ T cell clones belonging to Th0, Th1-like and Th2-like subsets produced IL-13 following antigen-specific or polyclonal activation. In addition, EBV-transformed B cell lines expressed IL-13 mRNA and produced small amounts of IL-13 protein. Expression of IL-13 mRNA and production of IL-13 protein by peripheral blood T cells and T cell clones was induced rapidly and was relatively long lasting, whereas IL-4 production by these cells was transient. In addition, IL-13 mRNA expression was induced by modes of activation that failed to induce IL-4 mRNA expression. IL-13 shares many biological activities with IL-4 which is compatible with the notion that the IL-13 and IL-4 receptors share a common component required for signal transduction. However, IL-13 lacks the T cell-activating properties of IL-4. Here we have shown that this is related to the fact that T cells fail to bind radiolabeled IL-13 and do not express the IL-13-specific receptor component. Taken together, these results indicate that the differences in expression and biological activities of IL-4 and IL-13 on T cells may have consequences for the relative roles of these cytokines in the immune response.

A CD4+ T cell line-secreted factor, growth promoting for normal and leukemic B cells, identified as thioredoxin

Abstract: In this study, a B cell growth stimulatory factor, constitutively secreted by a human CD4+ T cell hybridoma clone, MP6, has been purified and characterized. Serum-free 24 h culture media from MP6 cells were collected, concentrated by ultrafiltration and separated by gel chromatography. Fractions were analyzed for stimulatory activity using [3H]thymidine incorporation in normal and leukemic (B-CLL) B cells as target cells. Activity was present in a 12 kDa protein peak. Upon storage this lost activity indicating that the factor was sensitive to air oxidation, a well-known property of mammalian thioredoxins (Trxs). Treatment of the inactive fraction with dithiothreitol restored full activity. When culture medium was analyzed with a radioimmunoassay for human placenta Trx, the MP6 clone was shown to release 30-50 ng/ml per million cells during 24 h. The B cell stimulatory activity of the MP6 medium was removed by Sepharose-bound anti-human placenta Trx IgG and activity was recovered by elution from the antibodies. Furthermore, MP6 medium showed Trx activity with NADPH and Trx reductase using an insulin disulfide reduction assay. Starting from 5 l of serum-free MP6 conditioned medium, the Trx was purified approximately 100,000-fold. After gel electrophoresis banding, the material was analyzed by peptide sequencing and a full length sequence of an 104 amino acid long protein was obtained. This Trx sequence was identical to the previously published sequence of human Trx from HTLV-1 transformed T cells, adult T cell leukemia-derived factor/Trx.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term cultured CD40-activated B lymphocytes differentiate into plasma cells in response to IL-10 but not IL-4.

Abstract: We compared the effects of IL-10 and IL-4 on the functions of B lymphocytes triggered through their CD40. During the initial phase, IL-10 was as potent as IL-4 in inducing the expansion of viable B cells. Then, cellular expansion slowed down and after approximately 3 weeks the number of B cells started to decline. While the combination of IL-10 and IL-4 was synergistic during the first 2 weeks of culture, B cell recovery declined after 3 weeks, indicating that IL-10 prevails over IL-4. Those effects were not restricted to a specific B cell subset as both sIgD+ B cells and sIgD- B cells behaved in a similar way, though the latter population responded with a slightly accelerated kinetic. Inverted microscope examination and scanning electron microscopy showed that in response to IL-10, CD40-activated B cell cultures were heterogeneous with loose aggregates of cells as well as free floating large ovoid cells. In contrast, in the presence of IL-4, CD40-activated B cell cultures were essentially composed of tight cell clumps. IL-10 progressively induced all B cells to differentiate into non-replicating cells with intracytoplasmic Ig that secreted Ig at a high rate. Cytologic analysis indicated that IL-10 cultured cells display a basophilic cytoplasm with an arcoplasm and a low nucleus/cytoplasm ratio. Transmission electron microscopy demonstrated that when IL-10 was added to the culture, B cells displayed structures for excretion with extended endoplasmic reticulum and dilated cisternae containing paracrystalline structures, typical of plasmablasts cells. Taken together, these results indicate that IL-10 acts as a plasma cell differentiation factor for CD40-activated B cells.

Gene-targeted b-deficient mice reveal a critical role for b cells in the cd4 t cell response

Abstract: The role of B cells in promoting T cell responses is still controversial. In this study, we use JHD mice which have a targeted mutation in the JH gene and are thus rendered deficient in B cells to address this issue. We show here that immunization of JHD mice with soluble antigen fails to prime CD4 T cells, for either clonal expansion or delivery of immunological help for antibody responses. This lack of CD4 T cell priming in JHD mice corresponds to a 3- to 9-fold lower co-stimulatory activity of antigen-presenting cells (APC) from the JHD mice, as measured by anti-CD3-induced proliferative responses of CD4 T cells. This in turn is due to a defect of APC from JHD mice in response to T cell-mediated induction of co-stimulatory activity. As the development of macrophages and dendritic cells is unaffected in the JHD mice, our results demonstrate that B cells play a critical role in CD4 T cell priming, possibly by delivering a critical co-stimulatory activity for clonal expansion of CD4 T cells.

Novel mAbs reveal potent co-stimulatory activity of murine CD27

Abstract: Members of the tumor necrosis factor receptor (TNFR) family are emerging as important molecules implicated in the regulation of proliferation, differentiation and survival of T and B lymphocytes. Among these receptors is CD27, the function of which has thus far only been studied in the human system, where it amplifies the T cell proliferative response induced by TCR triggering. We report here the generation of mAbs to murine CD27, by an efficient method involving the use of transfected Armenian hamster fibroblasts. Previous analysis had already indicated that murine CD27 mRNA is uniquely expressed in lymphoid cells. As determined with one of the newly developed antibodies, murine CD27 is expressed on the great majority of both alpha beta and gamma delta T lymphocytes, on a small population of peripheral B cells, and on a very small subset of B220+ cells in the bone marrow. This distribution largely corresponds to that in the human system. However, unlike human CD27, which is primarily expressed in mature, medullary thymocytes, murine CD27 is found on all thymocytes, except a subset of CD4-CD8- precursors. Upon cross-linking, anti-CD27 mAb amplified the proliferative response of purified T lymphocytes to suboptimal stimulation with concanavalin A at least 4-fold. This indicates that such mAbs can mimick ligand binding and demonstrates that CD27 also acts as a potent co-stimulatory molecule in the murine system.

IL-7 transgenic mice: analysis of the role of IL-7 in the differentiation of thymocytes in vivo and in vitro

Abstract: We have generated a high copy number transgenic mouse line in which expression of mouse IL-7 cDNA is under the control of the mouse MHC class II E alpha promoter. These mice were generated in order to see if IL-7 over-production in the thymus altered either thymocyte differentiation or the process of negative selection. Using in situ hybridization, IL-7 transcripts could be detected in the thymic cortex and medulla as well as the spleen and lymph nodes of transgenic mice but was undetectable in normal controls. Phenotypic and molecular analysis of thymocytes from embryonic and adult transgenic mice failed to reveal a dramatic effect of IL-7 on thymocyte differentiation and negative selection of the TCR V beta repertoire appeared to be intact. In peripheral lymph nodes, there was a massive (30-fold) increase in the number of T cells (CD8+ > CD4+) and simultaneous presence of immature (B220+, Ig-) B cells. TCR repertoire analysis showed that the expansion of peripheral T cells was polyclonal. Using the polymerase chain reaction (PCR), transgene-specific IL-7 transcripts could be detected in the thymus from day 14 of fetal development. However, using semi-quantitative PCR, there was no dramatic increase in the degree of TCR beta or TCR alpha gene rearrangements during thymocyte ontogeny in vivo. Similarly, when fetal mouse thymus lobes were cultured with IL-7 in vitro, there was no dramatic increase in the degree of TCR beta or TCR alpha gene rearrangements. We conclude that IL-7 is probably not an important differentiation factor for immature mouse thymocytes.

De novo development and self-replenishment of B cells

Abstract: Previous studies distinguished two murine B cell lineages: the conventional lineage, which comprises the majority of B cells, and the Ly-1 B lineage (B-1a), which represents a small percentage of total adult B cells. A third subset, B-1b cells, shares many properties with B-1a cells, including the characteristic ability to self-replenish, but does not express Ly-1 (CD5). Reconstitution studies presented here show that (i) although the B220- population in adult spleen and bone marrow contains very little progenitor activity for B-1a cells, it can reconstitute roughly half the normal number of B-1b cells; (ii) B-1 progenitors present in adult bone marrow and spleen function at low levels in adult animals; (iii) peritoneal B-1 cells (principally B-1b) that develop following bone marrow transfer, like B-1 cells from normal animals, are capable of substantial self-replenishment; and (iv) conventional B cells do not expand (self-replenish) in adoptive recipients, although they can persist for long periods. Collectively, these progenitor and self-replenishment characteristics provide a developmental base for distinguishing B-1a, B-1b and conventional B cells.

The two novel MHC class II transactivators RFX5 and CIITA both control expression of HLA-DM genes

Abstract: MHC-encoded HLA-DMA and -DMB molecules are atypical MHC chains that play an essential role in antigen presentation by MHC class II molecules. They resemble both MHC class I and II molecules but are not expressed at the cell surface. From the study of MHC class II regulatory mutants, it was found recently that two novel transactivators, CIITA and RFX5, are essential for the control of MHC class II gene expression. We report here that CIITA and RFX5, although operating at different levels of transcriptional control, are also both essential regulators of HLA-DMA and -DMB genes. This is true for both the constitutive and the inducible mode of DM gene expression. Indeed, both CIITA and RFX5 cDNA can correct the HLA-DMA and -DMB gene expression defect in the respective regulatory mutants. The involvement of these two transcription factors accounts for the coordinate expression of MHC class II and HLA-DM, two sets of molecules that perform quite different functions in the overall process of antigen presentation.

Heat stable antigen (mouse CD24) supports myeloid cell binding to endothelial and platelet P-selectin

Abstract: P-selectin is a Ca(2+)-dependent lectin that participates in leukocyte adhesion to vascular endothelium and platelets. Myeloid cells and a subset of T lymphocytes express carbohydrate ligands at the cell surface. Previously, we suggested that heat stable antigen (HSA/mouse CD24), an extensively glycosylated cell surface molecule on many mouse cells, is a ligand for P-selectin. Here we show that HSA mediates the binding of monocytic cells and neutrophils to P-selectin. The monocytic cell lines ESb-MP and J774, peritoneal exudate cells, and bone marrow neutrophils could bind to lipopolysaccharide-activated bend3 endothelioma cells under rotation-induced shear forces and this binding was inhibited by mAb to P-selectin and HSA. Blocking was weak at room temperature but more efficient at 4 degrees C when integrin-mediated binding was decreased. Also the adhesion of neutrophils to stimulated platelets expressing P-selectin was blocked by HSA- and P-selectin-specific mAb. Latex beads coated with purified HSA from myeloid cells bound to activated endothelioma cells or platelets, and the binding was similarly blocked by mAb to P-selectin and HSA respectively. The HSA-coated beads were stained with P-selectin-IgG, very weakly with L-selectin-IgG but not with E-selectin-IgG. The staining was dependent on divalent cations and treatment with endoglycosidase F or neuraminidase indicated that sialylated N-linked glycans were recognized. The presence of these glycans was confirmed by biosynthetic labeling studies. Our data suggest that HSA, in addition to the recently identified 160 kDa glycoprotein ligand on mouse neutrophils, belongs to a group of monospecific P-selectin ligands on myeloid cells.

Role of macrophages and dendritic cells in primary cytotoxic T lymphocyte responses

Abstract: The successful induction of class I restricted cytotoxic T lymphocytes (CTL) responses with soluble non-replicating antigens relies upon vehicles which deliver antigen in vivo appropriately to antigen presenting cells (APC), which for CTL may be dendritic cells (DC). In this study, we have followed the distribution of liposomes and their incorporated antigen and compared the efficacy of splenic macrophages (Mo) and DC at inducing primary CTL responses in vitro. Our results show that whereas liposomes locate predominantly in the splenic red pulp and marginal zone locations, some of their soluble antigen contents redistribute to the central white pulp, comprising mainly DC and T cells. Such antigen redistribution was most apparent following administration of pH-sensitive liposomes. In comparisons of the APC activity of Mo and DC taken at various times post-injection, DC were always superior to Mo. However, if Mo were depleted prior to antigen exposure, DC were ineffective APC for CTL induction. Furthermore, addition of supernatant from OVA-liposome treated Mo to naive DC-T cell cultures in vitro resulted in OVA-specific T cell responses. These studies indicate a role for Mo in enhancing the antigen presenting function of DC.

Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes.

Abstract: The CAMPATH-1 (CD52) antigen is a 21-28 kDa glycopeptide which is highly expressed on lymphocytes and macrophages and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The function of this molecule is unknown. However, it is an extremely good target for complement-mediated attack and antibody-mediated cellular cytotoxicity. The humanized CAMPATH-1H antibody, which is directed against CD52, is very efficient at mediating lymphocyte depletion in vivo, and is currently being used in clinical trials for lymphoid malignancy and rheumatoid arthritis. It is therefore important to examine the functional effects of this antibody on different lymphocyte sub-populations. Because several other GPI-linked molecules expressed on the surface of T lymphocytes are capable of signal transduction resulting in cell proliferation, we have investigated whether the CAMPATH-1 antigen can also mediate these effects. In the presence of phorbol esters and cross-linking anti-Ig antibodies, mAbs specific for CD52 induced proliferation and lymphokine production in highly purified resting CD4+ and CD8+ T lymphocytes. The rat IgG2c YTH 361.10 anti-CD52 antibody, however, was able to activate resting CD4+ and CD8+ T cells directly without cross-linking or phorbol myristate acetate in the absence of Fc-bearing cells. Anti-CD52 antibodies also augmented the anti-CD3 mediated proliferative response of CD4+ and CD8+ T cells when the two antibodies were co-immobilized onto the same surface or cross-linked in solution by the same second antibody. Both CD4+ CD45RA and CD4+ CD45RO T cells were stimulated to proliferate by anti-CD52 antibodies in the presence of appropriate co-stimulatory factors. Anti-CD52 mAbs did not, however, synergize with anti-CD2 or CD28 mAb to induce CD4+ T cell proliferation. The activation of CD4+ T cells by anti-CD52 antibodies was inhibited by cyclosporin A, suggesting a role for the calcineurin-dependent signal transduction pathways. Although CD52 could transduce a signal in T cells, anti-CD52 antibodies did not inhibit antigen-specific or polyclonal T cell responses, suggesting this molecule does not play an essential co-stimulatory role in normal T cell activation.

IL-5 production by CD4^+ T cells of asthmatic patients is suppressed by glucocorticoids and the immunosuppressants FK506 and cyclosporin A

Abstract: IL-5 was produced in vitro by peripheral blood mononuclear cells (PBMC) of mite-sensitive atopic patients upon challenge with specific allergen, while PBMC of healthy controls produced essentially no IL-5. Stimuli delivered by the combination of phorbol ester and Ca2+ ionophore induced marked IL-5 production by PBMC obtained from atopic and non-atopic asthmatics, suggesting that both protein kinase C and Ca2+ influx are required for IL-5 production. CD2- or CD4-bearing cell depletion almost completely removed IL-5-producing cells while CD8-bearing cell depletion rather enriched them. These findings indicate that CD4+ T cells are the principal source of IL-5 in PBMC. The capacity of PBMC of atopic asthmatics, non-atopic asthmatics and healthy controls to produce IL-2, IL-4, IL-5 and IFN-gamma was compared, to find that cytokine-producing capacities other than that of IL-5 (IL-2, IL-4 and IFN-gamma) were not significantly different among the three groups. Dexamethasone, FK506 and cyclosporin A suppressed IL-5 production in vitro in a dose-dependent manner. Clear dose-dependent suppression of IL-5 gene expression by FK506 was also observed. Treatment of asthmatic patients with inhaled glucocorticoid (beclomethasone dipropionate) ameliorated clinical symptoms, improved lung function and markedly suppressed IL-5 production by PBMC, suggesting the essential role of IL-5 in the pathogenesis of bronchial asthma and the clinical importance of its regulation.

Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus.

Abstract: Listeria monocytogenes spends most of its intracellular life cycle in the cytosol of the infected eucaryotic cells. Within this cellular compartment originates the endogenous pathway of antigen processing and presentation. We thus assumed that recombinant L. monocytogenes expressing an heterologous protein, the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV), should be able to induce antigen-specific CD8+ T cells in vivo. The LCMV nucleoprotein gene was inserted in phase with the sequence coding for the putative signal sequence of the hemolysin of L. monocytogenes in order to target its secretion into the cytosol of the infected cell. The ability of this recombinant bacterium to induce LCMV-reactive CD8+ T cells was then monitored in BALB/c mice. The immune status of the immunized BALB/c mice was studied on the seventh day after a single i.v. injection of a sublethal dose of the recombinant bacteria: (i) cytotoxic CD8+ T cells were detected in liver; (ii) using in vitro re-stimulation with PMA and ionomycin, secondary cytotoxic CD8+ T cells were detected in spleen; (iii) an early inflammatory reaction dependent on the presence of CD8+ T cells occurred in the footpad after intraplantar inoculation of live LCMV; and (iv) mice were protected against an otherwise lethal intracerebral LCMV challenge; the protection was accompanied by elimination of the virus. When the immune status of the immunized hosts was monitored for a longer period post-immunization, the balance between immune protection and immunopathology described for the anti-LCMV immune responses was observed; two phases of protection were detected, flanking a transitory phase of exacerbation of the lymphocytic choriomeningitis disease (weeks 2-5).(ABSTRACT TRUNCATED AT 250 WORDS)

Are CD4+ Th1 cells pro-inflammatory or anti-inflammatory? The ratio of IL-10 to IFN-gamma or IL-2 determines their function.

Abstract: Human CD4+ T cells have, like their murine counterparts, been classified on the basis of their cytokine profile. Th1 cells produce IL-2 and IFN-gamma, but little or no IL-4. Th2 cells produce IL-4 but not IFN-gamma or IL-2, and Th0 produce IL-2, IL-4 and IFN-gamma. As IL-2 is the most potent T cell growth factor and IFN-gamma is the strongest activator of macrophages it is not surprising that CD4+ Th1 cells are considered to be pro-inflammatory. However, unlike results in the mouse, where IL-10 is only produced by Th2 cells, human IL-10 is produced by Th0, Th1 and Th2 cells. Hence some human Th1 cells are capable of producing both pro-inflammatory (IL-2, IFN-gamma) and anti-inflammatory (IL-10) cytokines, therefore the function of these cells may not be accurately encapsulated by the 'Th1' terminology. We thus investigated the correlation of cytokine production and function in human CD4+ Th1 clones. Cytokine production (IL-2, IFN-gamma, IL-10) was measured in supernatants by ELISA after stimulation with solid-phase anti-CD3. The capacity of these supernatants to activate or inhibit T cell proliferation or LPS induced TNF-alpha production by monocytes was assessed. The ratio of IL-2/IL-10 or IFN-gamma/IL-10 was of critical importance in determining the function of the supernatants. The inhibitory effects were verified to be due to IL-10, as they were neutralized by anti-IL-10 mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoblot analysis of cellular expression of Bcl-2 family proteins, Bcl-2, Bax, Bcl-X and Mcl-1, in human peripheral blood and lymphoid tissues

Abstract: The ability of Bcl-2 to inhibit apoptotic cell death is well established. Several homologues of the bcl-2 gene, such as bax, bcl-x or mcl-1, have recently been identified. Like Bcl-2, both Bcl-XL and Mcl-1 appear to function as repressors of apoptotic cell death, whereas Bax facilitates it, indicating possible interactions among them in the control of cellular survival. To investigate the in vivo role of expression of bcl-2 gene family products, immunoblot analysis using corresponding specific antisera was performed for peripheral blood cells and some lymphoid tissues in humans. We demonstrated that all Bcl-2 family proteins were expressed at various levels in hematolymphoid cell subpopulations isolated from peripheral blood, tonsil, spleen and thymus. Lymphoid expression of Bcl-2 family proteins tended to increase following activation, but declined with time in culture. Loss of Bcl-2 in cultured lymphoid cells was especially marked. Sole expression of Bax, but not other members of the Bcl-2 family, was observed on neutrophils, seemingly reflecting their shortest life-span among blood leukocytes. The results support the notion that a balance of expression of Bcl-2 family proteins may regulate the life and death of hematolymphoid cells at different stages of cell differentiation and activation.

Expression and function of fibronectin binding integrins on rat mast cells

Abstract: Adhesion molecules of the integrin family are implicated not only in leukocyte migration but also in leukocyte activation. Here we characterize the expression and function of fibronectin receptor integrins on rat mast cells. A rat basophilic leukemia cell line (RBL-2H3) and phorbol ester-stimulated rat peritoneal mast cells adhered to fibronectin (FN), vitronectin and fibrinogen. These mast cells expressed fibronectin receptor integrins, including very late antigen (VLA)-4, VLA-5 and vitronectin receptor (VNR), as estimated by immunofluorescent staining and inhibition of FN adherence by newly established mAbs reactive with the rat alpha 4 (MR alpha 4-1), alpha 5 (HM alpha 5-1) or beta 3 (HM beta 3-1) chains of the integrin molecules. The beta-hexosaminidase release, a marker for mast cell degranulation, triggered by high affinity IgE receptor (Fc epsilon RI)-mediated stimulation, was enhanced by adhesion of RBL-2H3 cells to either immobilized FN, MR alpha 4-1, HM alpha 5-1 or HM beta 3-1. This FN enhancement of beta-hexosaminidase release was inhibited by soluble MR alpha 4-1, HM alpha 5-1 and HM beta 3-1 as well as by GRGDSP and DELPQLVTLPHPNHLGPEILDVPST peptides which abrogate VLA-5/VNR and VLA-4 binding to FN respectively. In vivo, passive cutaneous anaphylaxis induced by IgE anti-DNP and DNP-BSA was inhibited by concurrent s.c. injection of MR alpha 4-1, HM alpha 5-1 and HM beta 3-1. These results demonstrate that FN receptor integrins expressed on rat mast cells play an important role in regulating mast cell activation both in vitro and in vivo.