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MCL, Mincle and Dectin‐2 are C‐type lectin receptors expressed by subsets of myeloid cells, and their genes cluster together in the APLEC/Dectin‐2 gene complex. We have previously shown that MCL and Mincle form a heterodimer in the rat, and others have shown that MCL and Dectin‐2 form a heterodimer in the mouse. In the rat, Dectin‐2 is a pseudogene, but here, we examine the association of the three receptors in human. In co‐transfection experiments analyzed with flow cytometry and immunoprecipitation, we here show that human MCL and Mincle form a disulphide‐linked heterodimer that associates with the signalling adaptor molecule FcεRIγ, in accordance with our previous findings in the rat. In contrast to previous findings in the rat, data in this paper indicate a direct association of MCL with FcεRIγ, as previously shown for mouse MCL. We were unable to demonstrate the formation of a heterodimer between human MCL and Dectin‐2. Thus, despite similarities, there may be important differences in the conformation of these receptors between rat, mouse and human, and this may have functional consequences. [ABSTRACT FROM AUTHOR]

The subpopulation of parental-strain lymphocytes responsible for the recognition of a particular F1 hybrid strain as foreign has been shown to be subject to specific, reversible inactivation after its injection into neonatal rats of that F1 hybrid strain. Neonates born to mothers that were syngeneic with the parental-strain lymphocytes under test acquired the capacity to inactivate these lymphocytes at an earlier age than did the genotypically identical reciprocal F1 hybrids. Neonates had little capacity to inactivate completely allogeneic lymphocytes. It is inferred from the difference in behavior between reciprocal F1 hybrids that the augmented ability to inactivate anti-F1 hybrid maternal-strain lymphocytes follows exposure to such cells in utero and to antibodies with anti-F1 hybrid activity in colostrum. Specific inactivation of those marauding maternal lymphocytes with anti-fetal activity is envisaged as an important means of protection of the fetus from immunological attack by the mother. On the basis of the results presented in this and the preceding paper, it has been proposed that many of the sequelae of the transfer of immunocompetent parental-strain cells to F1 hybrid animals result not from graft anti-host activity but from an F1 hybrid anti-parental lymphocyte response that has eluded normal regulatory mechanisms. These experiments also raise the possibility that regulation of auto-immune responses may be achieved by the inactivation of lymphocytes with anti-self reactivity by other lymphocytes that respond to the recognition structure required for such reactivity. [ABSTRACT FROM AUTHOR]

The first ideas leading to The Two‐Signal Model of lymphocyte activation were published 50 years ago, but the model was not realized in one sitting. I describe the three phases that led to its contemporary formulations. A motivation underlying all these models was to generate a minimal description of what is required for antigen to inactivate and activate mature lymphocytes that, at the same time, accounts for how peripheral tolerance is achieved. I suggest the two signal model has not only provided a substantiated framework for understanding how antigen interacts differently with B cells and CD8 T cells, to result in their inactivation and activation, but its postulates are pertinent to contemporary issues concerning the inactivation and activation of CD4 T cells. [ABSTRACT FROM AUTHOR]

Antigen recognition by T cells is the key event for the antigen specific immune responses to be triggered. This recognition is initiated by the binding of the T cell receptor (TCR) to antigen peptide/major histocompatibility complex (MHC) on the surface of the antigen presenting cells. TCR on most of the T cells is a heterodimer composed of α and β chains which are associated with CD3 γδ∈ as well as ζ chains, the signal transmission molecules. The dynamics of this TCR complex upon antigen/MHC recognition, however, has not been well understood. In this paper the authors analyse the configuration of TCR complex on T cells from a TCR β chain gene transgenic mouse (TGM) strain. Unlike many other TGM strains reported, a considerable proportion of T cells from this TGM expresses both transgene-encoded (Vβ3) and endogenous TCR β chains on their surface. By immunoprecipitation and immunoblotting analysis of T cells stimulated with a superantigen, staphylococcal enterotoxin B (SEB), the authors found that Vβ3 was coprecipitated with Vβ8, demonstrating the clustering of TCR αβ upon superantigen/MHC recognition. [ABSTRACT FROM AUTHOR]

The mechanisms associated with the modulation of immune response in the chronic phase of human schistosomiasis mansoni infection are complex and involve many cell types. In the present paper the authors demonstrate that antigenic stimulation of peripheral blood mononuclear cells (PBMC) from chronic-intestinal schistosomiasis mansoni patients with polyacrylamide beads (PB) conjugated to Schistosoma mansoni soluble egg antigens (PB-SEA) or adult worm antigen preparation (PB-SWAP) were able to induce a statistically significant increase on the in vitro multinucleated giant cell (MGC) formation after the 15th day in culture. A correlation between an increase in the number of MGC and a decrease in in vitro granuloma formation index to PB-SEA and PB-SWAP was observed. Moreover, the authors demonstrated a down-regulation of lymphocyte proliferative responses to S. mansoni antigens, during the differentiation pathway of monocytes towards MGC formation, due to a decrease in the antigen-presenting capacity of these cells. These phenomena also correlate with a concomitant decrease in the expression of HLA-DR and CD54 adhesion molecules on the surface of MGC. The results suggest that differentiation of monocytes to MGC may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) prepared from a rough mutant of Salmonella typhimurium and deacylated enzymatically (dLPS) does not promote neutrophil adherence to human umbilical vein endothelial cells (HUVECs). This paper reports that similarly, a smooth form of LPS prepared from Yersinia enterocolitica O:3, a serotype known to trigger reactive arthritis in humans, and treated with alkali (yersinia LPS-OH) failed to augment neutrophil adherence to HUVECs. Studies of the mechanism underlying the poor augmentation revealed that neither enzymatically deacylated LPS from Escherichia coli J5 (J5 dLPS) nor yersinia LPS-OH stimulated expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1, whereas both intact J5 LPS and yersinia LPS were stimulatory. Impaired up-regulation could not be explained by decreased binding of yersinia LPS-OH to HUVECs. Furthermore, 51Cr-labelled HUVECs treated with different concentrations of yersinia LPS-OH released 51Cr in the presence of anti-yersinia anti-O antibody and complement. J5 dLPS and yersinia LPS-OH inhibited up-regulation of the adhesion molecules induced by J5 LPS and yersinia LPS but not that induced by tumour necrosis factor alpha. Taken together, the results suggest that although yersinia LPS-OH can depress development of acute inflammation by inhibiting up-regulation of endothelial-cell adhesion molecules, sufficient LPS-OH is bound to induce cell injury and thereby inflammation in the prescence of specific antibody and complement. The findings may have pathogenetic implications in yersinia-triggered reactive arthritis characterized by dissemination of yersinia LPS throughout the body. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (MoAb) to human leucocyte sialoglycoprotein, CD43, have been shown to deliver mitogenic signals to human T cells or to enhance T-cell proliferation induced by concanavalin A, anti-CD3 antibodies or phorbolester. In this paper, we studied the effects of anti-CD43 MoAb B1B6 on the activation of human B cells. Anti-CD43 MoAb B1B6 was not mitogenic by itself for human B cells. However, when added together with TPA, was not mitogenic by itself for human B cells, containing 5-10% and about 35% CD43+, respectively, responded with three- to fivefold higher proliferation compared to that obtained with TPA alone. A peak in the proliferative response was reached on day 3. Optimal proliferation was obtained when the antibody was present from the start of culturing. Addition of MoAb B1B6 together with a calcium ionophore, ionomycin, did not induced B-cell proliferation. Neither did mAb B1B6 sustain the growth of B cells that were already in the cell cycle, i.e. precultured with phorbol ester (PDB) and ionomycin for 3 days. The results are similar to those obtained with antibodies to CD22 and CD23 and show that early progression signals are delivered to resting B cells through CD43 in the presence of primary activation of protein kinase C. [ABSTRACT FROM AUTHOR]

The differentiation of Ig+ B cells into plaque-forming cells is dependent on antigen and factors produced by T cells and/or macrophages. We describe here the production of T-cell factors termed lymphocyte promotor factors (LPF). A foetal calf serum-specific T-cell line and its clones synthesize LPF, which is defined as factors that polyclonally stimulate normal spenic T cells to differentiate into cytotoxic T lymphocytes (T-LPF) and normal splenic B cells to differentiate into plaque-forming cells into (PFC) (B-LPF) in the apparent absence of specific antigen. The proliferation of and the B-LPF production of all T-cell clones tested were foetal calf serum-specific and I-Bb-restricted. Some of these clones produced only T-LPF, some clones produced only B-LPF, and some clones produced both T-LPF and B-LPF. B-LPF stimulate the polyclonal differentiation of Ig+ B cells into PFC without the apparent need for helper T cells, is different from T-LPF, and induces almost exclusively IgM PFC. The B-LPF described in the present paper are compared with previously described T-cell factors, which stimulate antigen-specific B-cell responses or bystander B-cell responses. The conclusion is that B-LPF are probably different from B-cell growth factors, T-cell replacing factors, allogeneic effector factors, and interleukin 2. [ABSTRACT FROM AUTHOR]

in this paper the results of primed lymphocyte typing (PLT) tests are reported which show the response to HLA-D-specific stimulator and to unrelated cells. The aim was to find out the sensitivity of the PLT method for evaluating not only qualitative HLA-D-region differences but also gene dosage effects. in addition, the primed cells were restimulated with cells from HLA-D-homozygous individuals mutually negative with the specific stimulator in the usual 5-day primary mixed lymphocyte culture incubation. No gene dosage effect in PLT experiments could be observed. [ABSTRACT FROM AUTHOR]

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell surface ligand for LFA-1 (CD1 Ia/18). Several cytokines, such as IL-1. TNF-α or IFN-γ, have been shown to increase the ICAM-1 expression on cultured endothelial cells. Here we show that IL-4 decreases the IFN-γ-induced ICAM-1 expression on an endothelial cell line (EA.hy 926), whereas IL-4 alone has practically no effects on ICAM- 1 expression. The down-regulative effect was also seen at the mRNA level suggesting that this regulation process is transcriptional. At the same time IL-4 was unable to alter the TNF-γ-induced ICAM-1 up-regulation. Both IFN-γ and TNF-γ are able to decrease the proliferation of the endothelial cell line, but IL-4 could not modify this response when added together with the other cytokines. Taken together we show in this paper that IL-4 decreases IFN-γ- but not TNF-α-induced up-regulation of ICAM-1 expression on EA.hy 926 cells. [ABSTRACT FROM AUTHOR]

This paper reports on the characteristics of killing by a human and a murine tuberculin (PPD)-specihc T helper clone of targets to which PPD was attached via the Iectin concanavalin A (Con A). The killing was specific for PPD from M. tuberculosis; and targets coupled to Con A alone or to PPD from M. paratuberculosis were not killed. Target cells carrying Con A-PPD were more effectively lysed than PPD-pulsed cells. This form of lymphocyte killing, though highly significant, was inefficient. Maximum killing of PPD carrying targets was 31-40% at effector to target ratios of 20:1 and at 16 h. Cells carrying 2 x 106 molecules of PPD and less than 1.5x106 molecules Con A per cell were killed most efficiently. A major distinction between this helper T cell killing and that mediated by cytotoxic T cells was that both TH clones displayed bystander lysis and killed PPD uncoupled targets when these were cultured with syngeneic PPD-bound targets. This suggests that the mechanism of cytotoxicity may involve soluble mediators. [ABSTRACT FROM AUTHOR]

The function of B-L (Ia-equivalent)-positive (B-L+) and -negative (B-L-) chicken peripheral blood lymphocytes (PBL) was studied in vitro and in vivo. The PBL were first stained in direct immunofluorescence tests with a fluorescein isothiocyanate-labelled anti-B-L alloantiserum and then separated by means of a fluorescence-activated cell sorter. In agreement with our previous findings, B-L- cells showed functional properties of T lymphocytes, responding to concanavalin A and phytohaemagglutinin-P in vitro and inducing a graft-versus-host (GVH) reaction when injected into allogeneic embryos. Sorted B-L+" gave no responses in any of these assays. Neither B-L+ nor B-L- cells, when tested alone, responded significantly to pokeweed mitogen, but mixtures of the two restored the responsiveness to that of the original unsorted suspension. Of the B-L+ PBL, 10% were T cells, which may account for the low GVH reactivity given by this population. [ABSTRACT FROM AUTHOR]

The average frequency of cytotoxic precursor cells in DBA/2 lymph node cell preparations reactive to the syngeneic tumour P815 has been determined as 1 in 2000. This frequency is similar to the precursor frequency for an allogeneic tumour EL-4. The normal lack of response of DBA/2 lymph node cells to the syngeneic tumour P815 in vitro cannot be attributed to a lack of cytotoxic precursor cells. We conclude that in this tumour-host system non-immunogenicity reflects a defect at the inductive step. [ABSTRACT FROM AUTHOR]

Primed LD typing cells were prepared against single HLA baplotypes within families and used to type a random panel of 48 individuals. PLT cells could be grouped on the basis of highly correlated responses with the panel test cells; the 21 different PLT cells could be used to define 6 different PL antigens. One of these, PL 3, was split by the responses of two of the 21 PLT cells which measured an antigen called PL 3.1. [ABSTRACT FROM AUTHOR]

A competitive inhibition assay was used to define the specificity of the in vitro cytotoxicity of activated lymphocytes. Human and mouse effector cells were produced by prestimulation with phytohtmagglutinin, pokeweed mitogen, or purified protein derivative for several days. The addition of increasing numbers of unlabeled Chang cells to a fixed number of stimulated human lymphocytes and 51Cr-labeled Chang cells gradually decreased chromium release. Admixture of unlabeled human bladder tumor cells, lung cells, or monkey kidney cells had a similar effect, whereas mouse L cells were not inhibitory. The reverse was found to be true when stimulated mouse lymphocytes and 51Cr-labeled mouse L cells were incubated. In this case, the addition of unlabeled L or rat tumor cells effectively inhibited 51Cr release, whereas human or monkey cells were less inhibitory. Both human and mouse cells inhibited the cytotoxicity of human effector lymphocytes towards mouse L cells. The cytotoxic effects of mouse lymphocytes on human cells were also decreased by addition of unlabeled human, monkey, or mouse cells, but in this case human cells were most inhibitory. The results indicate that activated lymphocytes recognize several surface structures on the target cells. Some of these structures may be shared by cells of different species origin, whereas others seem to be unique for cells from phylogenetically closely related species. [ABSTRACT FROM AUTHOR]

Psoriasis is an inflammatory disease that arises in genetically predisposed individuals. Chronic skin lesions that contain activated immune cells can persist for years. Systemic inhibition of TNF, IL‐17 and IL‐23 cytokines has revolutionized psoriasis care during the recent decades. Unfortunately, local relapse of disease is common at previously inflamed sites after cessation of treatment. This highlights that fundamental pathologic alterations of the affected tissues are not completely resolved during clinical remission. Here, we present arguments for a local disease memory located in both dermis and epidermis in psoriasis skin. We decipher different cellular components and intercellular crosstalk that sustain local disease memory and amplify disease relapse in human psoriasis. Decrypting the mechanisms underlying the establishment and persistence of pathogenic memory cells in resolved psoriasis may provide new therapeutic perspectives aimed at long‐term remission of psoriasis. [ABSTRACT FROM AUTHOR]

Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA), and adipose‐derived mesenchymal stromal cells (ADSCs) can inhibit experimental collagen‐induced arthritis model. This study aims to determine whether ADSCs also suppresses osteoclastogenesis and bone erosion in collagen‐induced arthritis (CIA). Osteoclasts were induced from bone marrow‐derived CD11b+ cells with receptor activator of nuclear factor‐κ B ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulation and assessed with tartrate‐resistant acid phosphatase (TRAP) staining. For human cells, osteoclasts were produced from human CD14+ cells. ADSCs were generated and added to cultures with different ratios with CD11b+ cells. Transwell and antibody blockade experiments were performed to define the mechanism of action. NF‐κB and RANKL expression were determined by Western blotting and RT‐qPCR. About 2 × 106 ADSCs or fibroblast cells were adoptively transferred to DBA1/J mice on day 14 after immunization with type II collagen/complete Freund's adjuvant (CII/CFA) while the onset and severity of the CIA were monitored. Adipose‐derived mesenchymal stromal cells but not fibroblast cells completely suppressed osteoclastogenesis in vitro for human and mice. ADSCs injected after immunization and before of onset of CIA significantly suppressed disease development. Treatment with ADSCs dramatically decreased the levels of NF‐κB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo. We have demonstrated that ADSCs can inhibit RANKL‐induced osteoclasts genesis via CD39 signals. Our findings also suggest that ADSCs can inhibit osteoclasts genesis without the involvement of regulatory T cells. ADSCs might represent a promising strategy for stem cell‐based therapies for RA. Thus, manipulation of ADSCs may have therapeutic effects on RA and other bone erosion–related diseases. [ABSTRACT FROM AUTHOR]

To understand functional role of PD‐1‐expressing MAIT cells during tuberculosis infection in humans, sorted PD‐1+ and PD‐1− MAIT cells from pleural effusions of patients with pleural tuberculosis were subjected to transcriptome sequencing. PD‐1‐expressing MAIT cells were analysed by flow cytometry and their phenotypic and functional features were investigated. Transcriptome sequencing identified 144 genes that were differentially expressed between PD‐1+ and PD‐1− MAIT cells from tuberculous pleural effusions and CXCL13 was the gene with highest fold difference. The level of PD‐1‐expressing MAIT cells was associated with extent of TB infection in humans. PD‐1‐expressing MAIT cells had increased production of CXCL13 and IL‐21 as determined by flow cytometry. PD‐1highCXCR5− MAIT cells were significantly expanded in pleural effusions from patients with pleural tuberculosis as compared with those from peripheral blood of both patients with tuberculosis and healthy controls. Although PD‐1highCXCR5− MAIT cells from tuberculous pleural effusions had reduced IFN‐γ level and increased expression of Tim‐3 and GITR, they showed activated phenotype and had higher glucose uptake and lipid content. It is concluded that PD‐1‐expressing MAIT cells had reduced IFN‐γ level but increased production of both CXCL13 and IL‐21. [ABSTRACT FROM AUTHOR]

Abstract: Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through 2 main pathways, immunoglobulin E‐dependent and E‐independent activation. In the latter pathway, mast cells are activated by a diverse range of basic molecules (collectively known as basic secretagogues) through Mas‐related G protein‐coupled receptors (MRGPRs). In addition to the known basic secretagogues, here, we discovered several endogenous protein and enzyme fragments (such as chaperonin‐10 fragment) that act as bioactive peptides and induce immunoglobulin E‐independent mast cell activation via MRGPRX2 (previously known as MrgX2), leading to the degranulation of mast cells. We discuss the possibility that MRGPRX2 responds various as‐yet‐unidentified endogenous ligands that have specific characteristics, and propose that MRGPRX2 plays an important role in regulating inflammatory responses to endogenous harmful stimuli, such as protein breakdown products released from damaged or dying cells. [ABSTRACT FROM AUTHOR]

MPT63 (Rv1926c), a major secreted protein of Mycobacterium tuberculosis, is immunoreactive in antibody assays in humans and animals and provides protection as a combined DNA vaccine in mice. This study was undertaken to determine the reactivity of MPT63 in T helper 1 (Th1) cell assays, i.e. antigen-induced proliferation and interferon-γ secretion, using peripheral blood mononuclear cells (PBMCs) obtained from 72 Mycobacterium bovis Bacille Calmette–Guérin vaccinated healthy subjects. PBMCs were tested with complex mycobacterial antigens and pools of synthetic peptides corresponding to MPT63, MPB70, MT24, PPE68, CFP10 and ESAT-6. The results showed that MPT63 induced moderate Th1 cell reactivity which was equivalent to the reactivity induced by other secreted antigens of M. tuberculosis, i.e. MT24 and MPB70. Furthermore, human leucocyte antigen (HLA) heterogeneity of the responding donors suggested that MPT63 was presented to Th1 cells promiscuously. Analysis of the MPT63 sequence and its peptides for binding to 51 alleles of 9 serologically defined HLA-DR molecules, using a virtual matrix-based prediction program (ProPred) showed that MPT63 sequence could bind to all the 51 alleles, whereas 9 of the 10 peptides of MPT63 were also predicted to bind promiscuously. When tested with PBMCs of HLA-DR heterogeneous donors that responded to MPT63 in interferon-γ assays, at least 9 of the 10 peptides of MPT63 were recognized by PBMCs from HLA heterogeneous donors. These results suggested that promiscuous Th1 cell reactive epitopes are scattered throughout the sequence of MPT63, and further support the inclusion of this protein in an antigen cocktail to develop a new anti-tuberculosis vaccine. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is one of the major causes of infertility and preventable blindness in the world. The organism is of particular interest from an immunological point of view because it is one of the few obligate intracellular bacterial pathogens. There is some evidence that repealed infections in humans stimulate protective immunity. However, until recently, it was unclear which components of the adaptive immune system give rise to protection. Studies in gene knockout mice reported here and elsewhere now give a coherent and cogent picture of the importance of the Th1 response, in particular IFN-γ, for the localization and eradication of C. trachomatis genital tract infection. The key questions still to be addressed are the identity of the IFN-γ responsive cells and whether the mouse is truly representative of host protection against chlamydiae in humans. [ABSTRACT FROM AUTHOR]

Histological investigations suggest that the size and number of lymphoid follicles of the adenoids and tonsils decrease during ageing. The mechanisms underlying the histological changes are unknown. The authors have analysed the frequency of lymphocyte subpopulations in the adenoids by flow cytometry. The proportion of B lymphocytes decreased and the proportion of T lymphocytes of all mononuclear cells increased with age. Of all B lymphocytes the proportion of CD38+, surface IgD- B lymphocytes representing the germinal centre cell phenotype, decreased and the proportion of CD38-, IgD- B lymphocytes representing the mature B lymphocyte phenotype, increased with age. The expression of CD23, a cell surface molecule associated with activation of follicular mantle IgD+ B lymphocytes, did not change with increasing age. The results imply that the involution of the adenoid is associated with a decreased germinal centre reaction and relative accumulation of mature B cells in the adenoidal tissue, as analysed by three-colour flow cytometry. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis synovial fluid (RA-SF) contains a factor that induces IgG2b antibody production in LPS-stimulated murine B cells and therefore is called IgG2b inducing factor (IgG2bIF). When LPS, together with crude RA-SF or semi-purified IgG2bIF, was added to highly purified LPS-stimulated B cells, the number of IgG2b-producing cells was substantially enhanced. This shows that IgG2bIF acts directly on activated B cells, presumably by binding to a receptor expressed on LPS-activated B cells. In vivo LPS-activated B blasts were not able to respond to RA-SF unless LPS was present in vitro, showing that LPS is needed to maintain cell viability and responsiveness to the IgG2bIF. To elucidate the mechanism for the IgG2bIF effect on highly purified B cells, the IgG2b response of LPS-stimuiated, Bruton's tyrosine kinase-defective, xidB blasts was studied. Purified B blasts from the btk-defective CBA/N mouse strain were sensitive to IgG2bIF. These findings show that IgG2bIF acts directly on B cells and activates cells through a btk-independent pathway. [ABSTRACT FROM AUTHOR]

Since the migration of immunologically active cells into perivascular tissue is an important step in acute and chronic inflammation, the authors studied the possible influence of age on the transendothelial migration of T cells in an in vitro model. After incubation on collagen gels coated with human endothelial cells, three populations of T cells of elderly and young subjects were harvested: non-adherent (NAD), bound (BND) and migrated (MIG) cells. The percentages of the BND and MIG fractions were similar in the young and elderly groups. Phenotypic analysis showed that in the starting population of the elderly subjects T-cell subpopulations that have an increased capacity of transendothelial migration (e.g. CD4+ memory cells, CD8+/CD57+ cells) were enriched: nevertheless ageing was not associated with an increase in the transendothelial migration of T cells. [ABSTRACT FROM AUTHOR]

Several reports have described polyclonal activation in mice acutely infected with Trypanosoma cruzi. The aim of this work was to analyse the participation of one T. cruzi antigenic fraction in this immunological event. The antigen selected was FI, an antigenic fraction of pI 7-9 obtained from T. cruzi cytosol separated by isoelectricfocusing. FI is constituted by molecules with molecular weights of around 60 and 20 KDa. The authors assayed the ability of this antigenic fraction to induce polyclonal activation of spleen mononuclear cells from normal (NSMC) BALB/c mice. NSMC showed a marked lymphoproliferative response measured by ³H-thymidine incorporation after 3 days of culture in presence of FI. The values reached by FI-stimulated cells were 10 times higher than the controls (non-stimulated cells). This effect was dose-dependent. Furthermore, the authors observed that a purified T-cell population in the presence of adherent cells was unaffected by FI. Additionally, in a culture of NSMC, FI stimulated the proliferation of B cells as observed by the increase of the percentage of B220+ cells determined by FACS using FITC-conjugated anti-mouse B220. The authors noticed that the percentage of B220+Ly1+(CD5) populations in the presence of FI did not change with respect to the control (non-stimulated cells), indicating that FI expanded both conventional and CD5+ B cells. The isotypic pattern of the antibodies produced after 6 days of culture of NSMC in the presence of FI was predominantly IgM, which reacted with highly conserved antigens such as actin, myosin, myoglobin, thyroglobulin and carbonic anhydrase, but did not react with FI. A slight increase of IgG1 and IgG3 with respect to the control was observed but no changes on the levels of IgG2 was noticed. These results indicate that FI promotes activation, proliferation and differentiation in antibody-secreting cells of normal murine B lymphocytes. [ABSTRACT FROM AUTHOR]

The concept that activation of MHC class I-restricted CD8+ cells entirely depends on help from MHC class II-restricted CD4+ T cells has recently been supplemented with an alternative model in which CD8+ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8+ T cells in two different in vitro models. Freshly isolated CD8+ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR+ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8+ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8+ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8+ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8+ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance. [ABSTRACT FROM AUTHOR]

Microencapsulated pancreatic Langerhan's islets in calcium alginate gels have been used us an implantable bio-artificial pancreas in the treatment of diabetes mellitus, but with limited success due to overgrowth of the capsule with fibroblasts and phagocytes. The authors earlier demonstrated that alginates enriched in mannuronic acid stimulate human monocytes to produce high levels of cytokines such as tumour necrosis factor (TNF), IL-1, IL-6. In this study the authors have measured the TNF production from peripheral blood mononuclear cells (PBMC) in different groups of insulin-dependent diabetes mellitus (IDDM) patients after stimulation with different alginates and lipopolysaccharide (LPS). It was found that high G-alginate did not induce TNF production in any of the groups. High-M alginate and LPS induced a dose-dependent TNF production in all groups and the production was significantly different from unstimulated cells. The highest TNF response was found in newly diagnosed IDDM patients and the lowest was in the controls. [ABSTRACT FROM AUTHOR]

Using precise and reproducible flow cytometric measurements, the surface densities of the cell adhesion molecules (CAMs) CD29, CD2 and CD11a were studied on the CAMhigh (primed) subsets of peripheral blood CD4+ and CD8+ lymphocytes in 56 healthy subjects; 18 patients with acute bacterial infections, 19 with acute viral infections and 18 with chronic inflammatory conditions. By Mann-Whitney analysis, with significant P values adjusted for multiple comparisons to <0.0007, patients with viral infections were found to have increased CD11a on CAMhigh cells (an increase in median values of 13.7% for CD4+ lymphocytes and 15.8% for CD8+ lymphocytes); patients with chronic conditions have increased CD29 on CD8+CAMhigh lymphocytes (an increase in the median of 19%); and patients with bacterial infections have increased CD2 on CD8+CAMhigh cells (an increase in the median of 8%). There were marked individual increases in CD29 density: eight (15%) patients had CD29 gender-adjusted density on CD4+ cells greater than the control mean + 3 standard deviations (SD). CD29 densities on CD8+ cells were elevated to > control mean + 3 SD in 12 (22%) patients. By multiple regression analysis CD11a density on CD8+ and CD4+ cells and CD2 density on CD4+ cells were found to be associated with HLA-DR expression, but not with CD25 expression. Using standardized intercepts the authors demonstrated that there are very few circulating CD11ahighCD25+ cells, suggesting that these are rapidly extravasated. This study demonstrates that in disease, lymphocyte adhesion molecules are not deployed in concert and there are characteristic deployment patterns for different types of immune response. [ABSTRACT FROM AUTHOR]

In this work, the authors analysed T and B lymphocyte subsets and cytokine production in the spleen of BALB/c mice during polyclonal lymphocyte activation (primary infection) and parasite-specific response to Plasmodium chabaudi chabaudi (secondary infection). The secondary response was evaluated in fully immunoprotected animals, 60 days after a chloroquine-cured infection. The authors observed that in polyclonal lymphocyte activation antibody-secreting cells of all isotypes increased, with predominance of IgG2a and IgG3 classes. At that time, IFN-γ was largely produced, but IL-4/IL-5 were just slightly enhanced. In mice re-infected after 60 days, the Ig-isotype pattern was restricted to IgG1 and only IL-4/IL-5 were produced. In both responses, however, the levels of IL-2 were greatly reduced, while those of IL-10 were enhanced to similar levels. The different involvement of Th1 and Th2 cells in both responses was confirmed through analysis of CD45RB expression by CD4+ cells. The authors observed that CD45RBhigh cells were the major CD4+ subpopulation in primary infected mice, while CD45RBlow cells predominated in 60 days re-infected animals. Moreover, the great majority of activated (large) CD4+ cells in the primary infection belonged to the CD45RBhigh subset, while alter re-infection most of the CD4+ large had a CD45RBlow phenotype. [ABSTRACT FROM AUTHOR]

Human membrane cofactor protein (MCP, CD46) is a receptor for the measles virus and serves as a complement regulator which protects host cells from autologous complement attack. MCP is highly polymorphic due to a variety of mRNA splice products. The levels of MCP expression on T and myeloid cell lines are usually two-eightfold higher than those on their normal counterparts, whereas Burkitt's lymphoma B cell lines express less MCP than B cell lineages carrying no EB virus. The molecule has a Ser/Thr-rich (ST) domain adjacent to the functional domain, namely short consensus repeats (SCR). The ST domain and a cytoplasmic tail (CYT) contribute to the MCP polymorphism. The ST domain is encoded by three exons (A, B and C) and major ST isoforms are STABC, STBC and STC. The authors investigated the relationship between the expression levels and isoform usage of MCP by flow cytometry using specific antibodies against STA and STC, by reverse transcriptase-polymerase chain reaction (RT-PCR) with size markers for each splice variant, and by RT-PCR/Southern blotting using a specific probe for STA. The results were (1) the profiles of mean shifts of myeloid and T cell lines were STC < STA on flow cytometry while those of B cell lines and normal blood cells were STA < STC; (2) all cell lines tested by RT-PCR expressed the messages for the isoforms STBC/CYT1, STC/CYT1, STBC/CYT2, and STC/CYT2. The band for STABC/CYT2 overlapped that for STC/CYT1, and the band for STABC/CYT1 was marginal in all cell lines examined; (3) semi-quantitative analysis of the STABC isoforms by Southern blotting indicated the presence of high levels of the STABC messages in myeloid and T-cell lines in comparison with B lymphoid cells and normal leucocytes. Thus, the quantity of MCP expressed parallels the STABC message level, which is up-regulated in T and myeloid leukaemia cell lines. [ABSTRACT FROM AUTHOR]

The evolution of cells of the monocyte-macrophage lineage (MML cells) in the spleen of Leishmania donovani (LD) infected BALB/c mice was studied. Spleen cells were fractionated on a discontinuous percoll gradient and adherent cells (AC) were purified from fractionated spleen cells by adherence steps that appeared at the interfaces of 25-35%, 35-40%, 40-45% and 45-50% percoll gradients. The AC were characterized as MML cells on the basis of positive staining for non-specific esterase. Adherent cells that appeared at the interfaces of 25-35% and 40-45% were defined as A and C, respectively, and both of them showed extreme variation in a progressive infection. It was observed that A supported parasite replication whereas C remained refractory when infected with LD in vitro. Furthermore, when A cells and C cells were used as antigen-presenting cells to stimulate mixed population of IFN-γ producing and IL-4 producing T-cells, it was observed that IL-4 and IFN-γ were the predominating cytokine in the T-cell supernatant, respectively. Both A and C were found to be increased hand-in-hand up to 5 months of infection and from then on A decreased and C increased in their numerical strength (A-C reciprocity). The evolution of A-C reciprocity coincided with the gradual reduction in the parasitaemia in the spleen suggesting that this may contribute to the acquisition of anti-leishmania immunity. [ABSTRACT FROM AUTHOR]

A biopsy specimen from a patient with follicular lymphoma was divided into two fragments. DNA was extracted from one fragment and a 1.2 kb region of the functional heavy chain (IgH) gene was amplified, cloned and sequenced (eight clones). From the other fragment a cell line (HF-1) was started. The IgH gene region was amplified from the cell line, and sequenced without cloning. The nine sequences obtained could be arranged into a genealogical tree where the individual sequences differed from the deduced ancestor by 16-29 single nucleotide changes, some also by an insertion and/or a deletion. It is apparent that the sequence alterations were caused by somatic mutations during the growth of the lymphoma. The comparison of the sequences with two published (allelic) germline sequences of the human JH region showed ∼20% non-homology. The differences included five additional multinucleotide insertion/deletion changes, the longest of them a 101-nucleotide insertion. Two long insertions were homologous to the adjacent germline sequences. We propose that most of the changes observed, including long deletions and insertions, represent or are linked to somatic hypermutation events of the Ig gene type. Although in a few cases large deletions and insertions (>2 bp) have been found in mutated immunoglobulin genes, our results, for the first time, firmly link these deletions/insertions to somatic hypermutations; their frequency was found to be 2.2% of the observed mutational events in the non-translated gene regions. HF-1 is the first follicular lymphoma line successfully established from a lymphoma known to have hypermutated its Ig genes during the malignant growth. It is a candidate cell line to be studied for its ability to generate mutations of B cell type in cell cultures. [ABSTRACT FROM AUTHOR]

Gangliosides modulate the expression of CD4 molecules on the cell surface of T lymphocytes. We report here that treatment of human peripheral blood lymphocytes with exogenous monosialoganglioside GM3 induces a rapid down-modulation of the CD4 molecules on the plasma membrane of CD4+ T lymphocytes, as assessed by cytofluorimetric analysis and quantitative immunoelectron microscopy. The CD4 down-modulation was ganglioside-dose dependent and was already evident after 5 min of treatment, reaching the maximum after 20 min. The expression of other surface antigens was not affected by GM3 treatment. The immunoelectron microscopic analysis showed that, following GM3 addition, gold labelled CD4 molecules were rapidly redistributed on the cell surface, clustered and internalized via endocytic pits and vesicles. These results indicate that CD4 down-modulation induced by GM3 occurs through an endocytic mechanism. A persistent low level of CD4 expression on the cell surface up to 24 h after GM3 treatment, compared with a stable expression of either CD4 in untreated cells and CD3 in GM3-treated cells, suggests intracellular degradation of the internalized CD4 molecules. [ABSTRACT FROM AUTHOR]

We have recently developed a model in which mice were treated with IL-4 after primary immunization, resulting in elevated total serum IgG1 and IgE levels, but decreased antigen-specific levels and memory formation for these isotypes. In this report, we describe that these effects of IL-4 are mediated at the B cell and not the T-cell level. Major changes occurred in the γ1ε-double positive B-cell population which is increased as a result of IL-4 treatment. Moreover, it is shown that γ1ε-double positive B cells can develop in vitro out of γ1-positive primed B cells and that these double positive cells can differentiate into IgG1- and IgE-secreting cells. The existence of γ1ε-double positive memory B cells can explain the differences in cytokine dependence of TNP-specific memory IgG1 and IgE responses found after adoptively transferring primed spleen cells into irradiated naive recipients. Whereas the IL-4 independent TNP-specific memory IgG1 responses could be blocked efficiently by neutralizing IL-5 and IL-6, TNP-specific memory IgE responses were virtually not susceptible to such treatment. These IgE responses were also not susceptible to IFN-γ, used in doses that could inhibit the primary IgE response. Inhibition of the TNP-specific memory IgG1 response by neutralizing IL-5 and IL-6 is accompanied by a 10-fold increase of the IL-4 independent TNP-specific IgE memory response. These data indicate that secondary IgE responses primarily result from B cells that are either switched to IgG1, or are double positive for IgG1 and IgE, thereby suggesting a minor role for c-single positive B cells in secondary IgE responses. [ABSTRACT FROM AUTHOR]

Beta-2 microglobulin (β2m) gene 'knock-out' mice (C1D) were primed with purified H-2Kb and H-2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2b binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2b, H-2d, as well as H-2k cells by FACS analysis. The MoAbs stained to two β2m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous β2m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous β2m suggesting that xenogeneic β2m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA-S, when cultured at 26°C, however, staining was reduced five-fold when RMA-S cells were cultured at 37°C. In total, these observations suggest that the MoAbs recognize conformational, presumably β2m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one-way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epitopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes. [ABSTRACT FROM AUTHOR]

We described previously the induction by RadLV infection of a lymphoma (NS8) expressing a cytolytic activity against an MCA-induced fibrosarcoma. We report here that the cytolytic activity of these immortalized CD3+, CD8+ T cells is non-MHC-restricted. We then determined the structure and expression of the TCR chains expressed by these cells. Only partial rearrangement of the β chain associated to an abnormally short transcript was detected in NS8 cells, whereas the γ chain is rearranged and normally transcribed. On the opposite, rearrangement and expression of these genes were found in the other RadLV-induced lymphomas analysed. Moreover, γδ TCR proteins were detected on the cell surface of NS8 cells only, whereas the αβ complex, presents on the other T cell lines, was not expressed by NS8 cells. The ability of NS8 cells or of cells obtained from activated lymph nodes (harvested from mice grafted with the T2 sarcoma used to induce the NS8 line) to lyse the T2 sarcoma cell line was analysed. With both types of lymphocytes, the cytotoxicity was partially inhibited by a preincubation of the effector cells with anti-γδ antibodies. These results demonstrate that γδ lymphocytes can mediate anti-tumour cytotoxicity and NS8 lymphoma line may be representative of the TCR γδ CD8+ T cell subpopulation expressing non MHC-restricted cytotoxicity and displaying antitumoral activity. [ABSTRACT FROM AUTHOR]

The new monoclonal antibodies (MoAbs) E401, E811, E907 and E919 were prepared and characterized. These recognized an extracellular domain (amino acids No. 292-370) on the human c-erbB-2 gene product. Utilizing MoAb E811 and MoAb E919, a double determinant immunoassay (DDIA) was established to detect the soluble and the shed forms of the c-erbB-2 molecule. The levels of circulating erbB-2 antigen in the sera of patients with benign diseases and healthy controls were very low. The incidence of positivity for shed c-erbB-2 antigen in gastric cancer, colonic cancer, gall-bladder cancer, pancreatic cancer and other cancers were 7.4%, 4.2%, 0%, 6.7% and 0%, respectively. Four of 54 patients with gastric carcinoma showed high levels of serum c-erbB-2 antigen. They belonged to clinical stage IV and their histological types were all well differentiated adenocarcinomas (two papillary and two tubular adenocarcinomas). Furthermore, the incidence of positive staining in gastric cancer was 34.6%; higher than that for shedding erbB-2 antigen. Most of the cases which showed erbB-2 expression on cells were well-differentiated adenocarcinomas. Meanwhile, the distribution of erbB-2 antigen was limited in normal tissues. The results suggest that the expression of erbB-2 antigen is largely restricted to adenocarcinoma cells. It may not shed easily from these cells, and therefore it may be a very useful target molecule for passive immunotherapy. [ABSTRACT FROM AUTHOR]

CD22 is a B cell lineage restricted cell-surface adhesion glycoprotein which recognizes ligands on human T and B cells and cell lines. A soluble recombinant form of human CD22 (hCD22Rg) has been used to identify and characterize CD22-specific ligands on human T cells, one of which has been shown to be the receptor-linked phosphotyrosine phosphatase CD45. Because CD45 plays a pivotal role in lymphocyte activation, we assessed whether human CD22 might display cross-species reactivity with CD45. In the study presented here we demonstrate that human CD22Rg recognizes several murine cell-surface sialoglycoproteins, including CD45, containing sialic acid in α2,6 linkage. Furthermore, hCD22Rg recognizes different ligands on functionally distinct T helper-cell subpopulations and selectively binds medullary thymocytes in vivo. Our results confirm and extend previous observations that CD22 is a sialic acid-binding lectin which interacts with CD45 and other glycoproteins capable of presenting α2,6-linked sialic acid in a manner that promotes high affinity binding. The cross-species reactivity of CD22 with its ligands underscores the potential physiologic importance of CD22-mediated lymphocyte interactions. [ABSTRACT FROM AUTHOR]

In the avian embryo the haemopoietic stem cells originate from the intra-embryonic area near dorsal aorta. The surface-marker expression of haemopoietic stem cells and their potential to produce different haemopoietic cells are still largely unknown. The surface antigen expression and particularly the MHC antigen expression on intra-embryonic haemopoietic cells was studied. Expression of B-F antigens, homologous to mammalian MHC class-I antigens, was found already on embryonic day (ED) 5. The first B-L antigens, analogous to mammalian MHC class-II antigens, were detected also from ED5 onwards. The appearance of surface antigens defined by MoAbs T10A6 and 3-298 during embryogenesis also was studied. The antigen defined with T10A6 was detected from ED4 onwards on endothelial cells but not on haemopoietic cells in the para-aortic region. The first 3-298+ haemopoietie cells were found on ED6, whereas endothelial cells were negative. These findings imply that some surface markers are shared with haemopoietic and endothelial cells indicating either a common embryonic origin or the importance of these molecules in embryonic stem-cell homing. [ABSTRACT FROM AUTHOR]

Interaction of ICAM-1 and its ligands plays an important role in the leukocyte binding to endothelium. The best characterized ICAM-ligands belong to the family of β2-integrins (CD11/CD18), but recently it has been suggested that CD43, a molecule with no structural resemblance to integrins binds ICAM-1 also. On the leukocytes the main regulatory pathway for ICAM-mediated binding is believed to be a short-term regulation of the avidity of CD11/CD18. In this study the authors investigated whether a quantitative increase in the surface expression of ICAM-ligands also can lead to enhanced binding to purified ICAM-1. PMA-treatment differentiates myelomonocytic cell lines into macrophages with a concomitant increase in the surface expression and mRNA-levels of the β2-integrin α- and β-chains as well as that of CD43, another ICAM-ligand. The binding of the PMA-treated THP-1 cells to ICAM-1 was increased simultaneously compared to non-treated cells. The binding was blocked completely with antibodies to CD18 and ICAM-1. It is concluded that in addition to the transient qualitative regulation, a long-term quantitative regulation of ICAM-1 ligands also plays a role in increasing the adhesiveness of myelomonocytic cells. This may be relevant in chronic inflammation episodes. [ABSTRACT FROM AUTHOR]

The cellular composition and VH-gene family repertoire were compared in different B-cell compartments from young adult (8-12 weeks) and old (18-24 months) C57BL/6 and BALB/c mice, Ageing mice were found to have a higher frequency of peripheral mature B cells utilizing genes from a single VH-gene family. While in each individual old C57BL/6 mice cells expressing the VH J558 gene family consistently were over-represented, a marked individual variation was observed in old BALB/c mice with increased frequency of either the VH J558, Q52 or J606 families. Aged mice were found also to have a reduced number of bone-marrow pre-B cells and an augmented number of splenic Ig-secreting cells. These results suggest that old mice express less diversified antibody repertoires possibly as a consequence of reduced input from precursors and increased peripheral selection, which may be responsible for the progressive establishment of immunodeficiency. [ABSTRACT FROM AUTHOR]

The expression ofa tryptic serine protease was detected in the cell line KU812 by Northern blot analysis with an oligonucleotide probe directed against a conserved region present in all of the five presently cloned human mast cell tryptases. PCR primers designed for the amplification of a nearly full-length copy of tryptase mRNAs were used to study the identity of the KU812 tryptase. Ten clones were characterized and all were found to be identical to one of the tryptases previously cloned from a human skin cDNA library. This tryptase has been thought to originate from mast cells of the skin. Two possible explanations may account for the observed identity between the presumed mast cell tryptase and the KU812 tryptase. Firstly, it is possible that the KU812 tryptase is a basophil-specific tryptase which has previously been cloned from a human skin cDNA library containing low levels of cDNA copies derived from basophils in the starting material. Secondly, the KU812cell line, and possibly normal basophils, express a tryptase which is identical to one of the tryptases expressed in normal skin mast cells. We cannot at present rule out any of the two possibilities, but we favour the second explanation as being the most likely. [ABSTRACT FROM AUTHOR]

The kinetic profile of cytokine gene expression in normal human peripheral mononuclear cells (MNC) activated by an anti-CD3 monoclonal antibody was studied. The presence or absence of 10 different cytokine mRNA were measured in a polymerase chain reaction (PCR) assisted mRNA amplification assay. After 2 h of stimulation the mRNA for interleukin-lα (IL-1α). interleukin-2 (IL-2). interleukin-3 (IL-3), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-y) were detcetable and remained present during the whole time period studied (22 h). lntcrleukin-6 (lL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were detected after 4 h. while interleukin-10 (IL-10) mRNA did not appear until after 7 h: they ail remained expressed at 22 h. A transient expression or interienkin-4 (IL-4) mRNA was observed between 4 and 7 h of stimulation. No gene expression of granulocyte colony stimulating factor (G-CSF) was detected at any time. These results show that anti-CD3 stimulation of M NC leads to a rapid sequential induction of different cytokine mRNA, some with a very transient expression. [ABSTRACT FROM AUTHOR]

Lymphoid follicles are the main B-cell areas in peripheral lymphoid tissues. These structures commonly consist of germinal centre (GC) and mantle zone (MZ) regions. In the present work, human tonsillar B cells belonging to these two compartments were purified by a combination of density centrifugation and separation techniques involving the recognition by monoclonal antibodies of specific surface molecules followed by panning and magnetic bead selection. These purified populations were identified as GC and MZ cells by three different criteria; (I) GC cells showed the phenotype IgO CD20-1bright CD38-1 CD44 and peanut agglutinin (PNA)-1, and MZ cells were IgD CD20-1, dim CD38 CD44-1 and PNA ; (2) morphologically, MZ cells appeared as small resting lymphocytes whereas GC cells consisted of large blastic cells of the germinal centre: (3) functionally, most GC, but not MZ. cells underwent apoptosis early in culture. The isolation of GC and MZ cells allowed the study of their proliferative response. As a result of these studies, GC cells were demonstrated to inhibit the proliferation of MZ cells in response to B-cell mitogens (Staphylococcus aureus Cowan I and anti-μ plus BCGF) in a concentration-dependent way; 50% inhibition was reached at a GC/MZ cell ratio of l.2. This effect did not require GC-cell DNA synthesis since similar results were obtained with irradiated GC cells. Neither was it due to a non-specific toxic effect since GC cells did not alter the proliferative response of autologous T cells to mitogens [phytohaemagglutin and anti-CD3), The inhibition required cellular contact between GC and MZ cells, and was not restricted by histocompatibility barriers. These data suggest the possible existence ofa new regulatory pathway wilhin peripheral B-cell areas. [ABSTRACT FROM AUTHOR]

To characterize the role of B lymphocytes in the pathogenesis of Multiple Sclerosis (MS), we have isolated mononuclear cells from cerebrospinal fluid (CSF) and stimulated them with a polyclonal B-cell mitogen (pokeweed mitogen). This study has been done with MS patients selected for the occurrence of an acute attack in the course of the disease and with patients hospitalized for other neurological diseases. Five of the 11 MS patients had B lymphocytes producing in vitro antibodies (Abs) directed against purified human myelin basic protein (hMBP), as revealed by Western blot analysis. None of the 20 patients with other neurological diseases showed such a reactivity. The produced Abs recognized only I or 2 hMBP peptides without dominance for a certain peptide. This result emphasizes the presence of B cells producing Abs against MBP in CSF of MS patients and shows the interest of studying mononuclear cells of CSF as a good marker ofthe pathogenesis. [ABSTRACT FROM AUTHOR]

We have hypothesized that functional maturation of T lymphocytes can be dissected into a series of discrete stages. For example. activation of T lymphocytes with the calcium ionophore A23187 drives CD8+ T cells to become dividing blasts, referred to as `pre-effector' cells in that these blasts do not express cytolytic function but are driven by IL-2 to do so. Here we characterize via Northern blots the functional maturation of CD8+ and CD4+ T-lymphocyte populations which have been activated via A23187 followed by stimulation with IL-2. Previously we have reported that no detectable !L-2 was found in the supernatants of A23187-activated pre-effector blasts. However, these cells do express levels of IL-2 mRNA very similar to those of OKT3-activated blasts, from which IL-2 is easily detected in the supernatant. Translational control may account for these findings. A23187-activated CD8+ pre-effector blasts do not respond to stimulation with IFN-γ nor do they express IFN-γ mRNA following stimulation with IL-2. These observations suggest that IL-2 may be sufficient to stimulate maturation of these cells. Activation via A23187 results in lower expression of the proto-oncogene c-myb relative to that found in OKT3 activation. C-myb mRNA levels are higher in CD8+ than in CD4+ A23187-activated pre-effector blasts and the c-myb level in the CD8+ pre-effector blasts falls in response to IL-2. This decrease in c-myb mRNA coincides with an increase in proliferation, and the expression of cytolytic activity. [ABSTRACT FROM AUTHOR]

Two monoclonal antibodies (MoAbs), F10.2 and F10.3, were selected for their ability to interfere in homotypic adhesion of human B cells. Precipitation studies and binding to intercellular adhesion molecule I (ICAM-1, CD54) cDNA transfected COS cells revealed that both MoAbs are directed against ICAM-1. The binding of MoAb F10.2 was inhibited by LB-2, a MoAb recognizing the NH₂-terminal immuglobulin-like domain of ICAM-I. This suggests that the epitope recognized by F10.2 is located on the first domain of the ICAM-1 molecule. Binding of the other MoAb, F10.3, was not inhibited by F10.2 nor by two other MoAbs mapping to the first domain of the ICAM-1 molecule. The ability of F10.3 to bind to ICAM-1 is influenced by glycosylation, suggesting that this epitope is located on one of the domains carrying possible glycosylation sites, i.e. domain 2, 3 or 4. The ICAM-1 epitopes recognized by F10.3 and LB-2 or F10.2 co-operated in homotypic adhesion of cells from the EBV cell line ML1. These results suggest that in addition to an epitope located on domain 1 of the ICAM-1 molecule, another epitope whose exposure can be regulated by glycosylation is involved in homotypic B-cell adhesion of cell line ML1. [ABSTRACT FROM AUTHOR]

Activation of T cells is a complex process which we have hypothesized involves a series of functional intermediates possessing some, but not all, of the characteristics of fully functional effector cells. We have identified two such functional intermediates, the poised T cell (poTc) and the pre-effector T cell (peTc). poTc do not proliferate or mediate cytolytic activity but are responsive to help in the form of interleukin 2 (IL-2): peTc are proliferating cells which are not cytolytic. Here the role of T-cell rcceptor/CD3 complex cross-linking in generating these functional intermediates is examined and it is shown that cells can be driven to different stages of functional maturation depending upon the sequence of antibody binding to CD3 and cross-linking. Highly purified T cells can be activated lo poTc stage if they are first labelled with the anti-CD3 monoclonal antibody OKT3 and then cross-linked with goat anti-mouse IgG-coated beads. If the OKT3 antibody is first bound to the IgG-coated beads and then added to highly purified Tcells, peTc are generated. In both cases the intermediates can be driven to become fully functional effeetor cells by the addition of IL-2. Finally, by removing the OKT3-bound beads during the activation process, were are able to show that poTc and peTc are sequential intermediates along the same pathway of activation. [ABSTRACT FROM AUTHOR]

Recombinant inlerleukin-4(rIL-4) caused a 65-70% reduction of the interferon-α (IFN-α) and -β production induced by Sendai virus (SV) in human peripheral blood monocytes in vitro. Significant inhibition was seen at concentrations as low as 0.1 ng/ml. The rIL-4 also reduced levels of IFN-α and -β mRNA by 60% and 67%, respectively, as well as the frequency of IFN-α and -β mRNA-containing cells by 65% and 54%, respectively. The frequency of IFN-α/β mRNA-containing cells was inhibited by rIL-4 throughout the whole course of induction by SV. IL-4 caused a shift of the grain count distribution towards less heavily labelled cells, suggesting an inhibitory effect of rIL-4 on most IFN-α/β mRNA-containing cells. Antibodies to rlL-4 did not influence the normal IFN-α/β response induced by SV, but abolished the inhibitory effect of the rIL-4. When rIL-4 was added to cell 4 h after start of stimulation by SV, at which time much mRNA has accumulated but little IFN-α/β has been secreted, no inhibition of the IFN-α/β production by rIL-4 was seen. IFN-γ had only a minor reversing effect on the rIL-4 inhibition, but if cells were precultivated in medium with or without IFN-γ for 6 h before SV induction, rIL-4 paradoxically enhanced the IFN-α/β response. Our results suggest that rIL-4 inhibits an early step of IFN-α/β induction in monocytes, at the level either of transcription of IFN-α/β genes or of the processing or stability of mRNA. The IL-4 effects may however depend on the state of activation of the monocytes. [ABSTRACT FROM AUTHOR]

Monocytes produce interferon-α (IFN-α) and -β when human peripheral blood mononuclear cells (PBMCs) are stimulated in vitro by Sendai virus (SV). We found that about 70% of the IFN-producing cells (IPCs) expressed both IFN-α and -β mRNA; the rest expressed only IFN-β mRNA. In the presence of the protein synthesis inhibitor cycloheximide(CMX), the frequency of IFN-α mRNA-containing cells, measured after 6h, was decreased by 85-90%. Results of nuclear run-on transcription assays showed that CHX inhibited IFN-α gene expression. The frequency of IFN-α mRNA-containing cells was not reduced by CHX. Actually, a threefold increase was observed at the lower CHX concentrations. Studies on the kinetics of IFN-α/β mRNA induction showed that CHX accelerated the appearance of IFN-β mRNA-containing cells, increased IFN-β mRNA levels, and delayed the normally occurring post-inductional decrease of IFN-β mRNA. Unexpectedly, an initially normal or even accelerated IFN-α mRNA response was seen in the presence of CHX during the first 3-4 h after SV stimulation. This occurred in a small proportion of the potential IPCs. However. CHX prevented the subsequent marked increase of IFN-α mRNA levels. Preincubation of PBMCs for 6 h in conditioned medium (CM) containing IFN and other cytokines prevented the CHX-mediated inhibition of IFN-α mRNA. Without preincubation this was not seen. The preincubation in CM caused an accelerated appearance of IFN-α mRNA. resembling that of IFN-β mRNA. The results suggest that IFN-α and -β genes are differentially regulated in the same monocytes, the former requiring de novo synthesis of intracellular protein(s) for efficient expression. [ABSTRACT FROM AUTHOR]