B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties.

A gene, called m-mb-1, was isolated from a murine pre-B-minus T lymphocyte subtracted library. It was found expressed as mRNA at low to medium abundance in early progenitors of the B lineage, in pre-B and mature B lineage cell lines, in normal resting B lymphocytes and in polyclonally activated B cell blasts. The gene was not expressed in plasmacytomas, in cell lines of the monocyte/macrophage, the T lymphocyte or the fibroblast lineages, nor in thymus, liver, heart, kidney, lung or brain. The nucleotide sequence of the m-mb-1 gene encodes a putative membrane glycoprotein with 220 amino acids, which includes a leader sequence, a putative extracellular domain with two potential N-glycosylation sites, a transmembrane portion and a putative intracellular domain. The partial sequence of a human homologue, h-mb-1, shows nearly 90% homology in nucleotide as well as amino acid sequences to the murine form of a stretch of the putative intracytoplasmic domain. Antibodies raised against a fusion protein of m-mb-1 with protein A, affinity purified for their m-mb-1 specificity, stained pre-B and mature B cell lines on their surface, but did not stain T cell lines and fibroblasts. Antibodies raised against a stretch of 20 amino acids of the putative intracellular domain with 90% homology between the mouse and human protein did not stain the surface of any cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the murine T cell receptor for IgE (Fc epsilon RII). Demonstration of shared and unshared epitopes with the B cell Fc epsilon RII

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.     

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

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

Genomic structure and chromosomal mapping of the murine CD40 gene.

The B cell-associated surface molecule, CD40, is likely to play a central role in the expansion of Ag-stimulated B cells, and their interaction with activated Th cells. In our study we have isolated genomic clones of murine CD40 from a mouse liver genomic DNA library. Comparison with the murine CD40 cDNA sequence revealed the presence of nine exons that together contain the entire murine CD40 coding region, and span approximately 16.3 kb of genomic DNA. The intron/exon structure of the CD40 gene resembles that of the low affinity nerve growth factor receptor gene, a close homolog of both human and murine CD40. In both cases the functional domains of the receptor molecules are separated onto different exons throughout the genes. Southern blot analysis demonstrated that murine CD40 is a single copy gene that maps in the distal region of mouse chromosome 2.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture

Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.     

The murine homologue of the T lymphocyte antigen CD28. Molecular cloning and cell surface expression

The human T lymphocyte Ag CD28 (Tp44) is a homodimeric glycoprotein expressed on the surface of a majority of human peripheral T cells and thymocytes. Although exposure of T cells to anti-CD28 mAb does not activate T cells, stimulation of CD28 can synergize with signals transmitted through the TCR or other stimuli to augment proliferation and lymphokine production. We have used a portion of the human CD28 cDNA to isolate a homologous murine cDNA from an EL4 T lymphoma library. The murine clone has 61% nucleotide identity with the human cDNA. Both human and murine sequences exhibit homology with members of the Ig supergene family and CTLA-4, a T cell specific murine gene. Many characteristics of the human CD28 molecule are conserved within the putative murine CD28 polypeptide. The murine cDNA sequence encodes a polypeptide of 218 amino acids that has 68% identity with the human sequence. Both the murine and human molecules are integral membrane glycoproteins with hydrophobic signal peptide sequences and transmembrane region. All five potential N-linked glycosylation sites are conserved and six of the seven cysteine residues of the mouse protein are found in the human CD28 polypeptide. The murine cDNA is encoded by a single copy nonrearranging gene whose expression at the mRNA level is restricted to T cells. A rabbit antiserum was raised against a synthetic peptide corresponding to a hydrophilic portion of the translated murine cDNA sequence. This antiserum identifies an 80-kDa homodimer consisting of disulfide-bonded subunits of 40 kDa that is expressed on splenic T cells, thymocytes, and several T cell tumors, but not on B cells. deglycosylation studies indicate that four of the five N-linked glycosylation sites are used and that the mature core protein has a molecular mass of 25 kDa, close to that predicted by the cDNA sequence. Transfection of the murine cDNA into Chinese hamster ovary cells resulted in the expression of an 80-kDa dimeric molecule that was immunoprecipitated by the antipeptide antiserum. Taken together, these data provide strong support that we have identified the murine homologue of CD28.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.     

Use of monoclonal antibodies for flow cytometric detection of intracellular Toxoplasma gondii in murine splenic lymphocytes

Various monoclonal antibodies (mAbs) against Toxoplasma gondii RH tachyzoites were used for flow cytometric detection of intracellular parasites in murine splenic lymphocytes. Tg110 and Tg563 (reacting with the major surface protein SAG1), Tg505 (with another surface protein SAG2), Tg695 and Tg786 (with rhoptry proteins), Tg507, Tg621, and Tg317 (with dense granule proteins), Tg536 (with a microneme protein), and Tg685 (with a cytosol antigen) were the mAbs used. After an in vitro infection of lymphocytes with tachyzoites and reactions with the different mAbs, flow cytometry was performed using an indirect immunofluorescent technique. The proportions of whole infected lymphocytes and of each infected lymphocyte phenotype, CD4+ T cells, CD8+ T cells, and B cells, were determined, and their fluorescent intensities were quantified. The best reaction was seen when Tg110 or Tg695 was used as the mAbs. The results suggest that mAbs against surface or rhoptry proteins are highly useful for the flow cytometric detection of intracellular T. gondii in host cells.     

CD4 expression decrease by antisense oligonucleotides: inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.     

Scaffold Protein JLP Is Critical for CD40 Signaling in B Lymphocytes

CD40 expression on the surface of B lymphocytes is essential for their biological function and fate decision. The engagement of CD40 with its cognate ligand, CD154, leads to a sequence of cellular events in B lymphocytes, including CD40 cytoplasmic translocation, a temporal and spatial organization of effector molecules, and a cascade of CD40-induced signal transduction. The JLP scaffold protein was expressed in murine B lymphocytes. Using B lymphocytes from jlp-deficient mice, we observed that JLP deficiency resulted in defective CD40 internalization upon CD154/CD40 engagement. Examination of interactions and co-localization among CD40, JLP, dynein, and Rab5 in B lymphocytes suggested that CD40 internalization is a process of JLP-mediated vesicle transportation that depends on Rab5 and dynein. JLP deficiency also diminished CD40-dependent activation of MAPK and JNK, but not NF-κB. Inhibiting vesicle transportation from the direction of cell periphery to the cell center by a dynein inhibitor (ciliobrevin D) impaired both CD154-induced CD40 internalization and CD40-dependent MAPK activities in B lymphocytes. Collectively, our data demonstrate a novel role of the JLP scaffold protein in the bridging of CD154-triggered CD40 internalization and CD40-dependent signaling in splenic B lymphocytes.     

Cloning and expression of murine IL-12.

Human IL-12 (NK cell stimulatory factor, cytotoxic lymphocyte maturation factor) is a heterodimeric cytokine that can act as a growth factor for activated human T and NK cells, enhance the lytic activity of human NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting human PBMC. Because in our hands, human IL-12 did not elicit similar responses in murine lymphocytes, we have cloned and expressed the murine IL-12 subunit cDNA in order to obtain recombinant protein for murine studies. Comparison of the predicted amino acid sequences of the murine subunits with their human counterparts revealed that the p40 subunits are more highly conserved than the p35 subunits (70% vs 60% identity, respectively). The sizes of the p35 and p40 subunit mRNA were estimated to be 1.5 kb and 2.6 kb, respectively. RNA blot analysis showed that p35 mRNA was expressed in lymphoid tissues (spleen, thymus) and nonlymphoid tissues (lung, brain), whereas p40 mRNA expression was only detected in lymphoid cells. Incubation of splenocytes with pokeweed mitogen did not significantly affect p35 mRNA levels, however, it resulted in a decrease of p40 mRNA. Coexpression of the murine p35 and p40 cDNA clones in COS cells resulted in the secretion of IL-12, which was active in human and mouse T cell proliferation, murine NK cell activation, and murine IFN-gamma induction assays. Transfection of each subunit cDNA alone did not result in measurable secreted IL-12 activity. A hybrid heterodimer consisting of murine p35 and human p40 subunits retained bioactivity on murine cells; however, the combination of human p35 and murine p40 was completely inactive on murine cells. These results indicate that the observed inability of human IL-12 to act on murine cells is largely determined by the p35 subunit.     

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.     

CD4+ T lymphocytes with constitutive CD40 ligand in preautoimmune (NZB x NZW)F1 lupus-prone mice: phenotype and possible role in autoreactivity

Lupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black x New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.     

Class I major histocompatibility proteins are an essential component of the simian virus 40 receptor.

The class I molecules encoded by the major histocompatibility complex (MHC) present endogenously synthesized antigenic peptide fragments to cytotoxic T lymphocytes. We show here that these proteins are an essential component of the cell surface receptor for simian virus 40 (SV40). First, SV40 binding to cells can be blocked by two monoclonal antibodies against class I human lymphocyte antigen (HLA) proteins but not by monoclonal antibodies specific for other cell surface proteins. Second, SV40 does not bind to cells of two different human lymphoblastoid cell lines which do not express surface class I MHC proteins because of genetic defects in the beta 2-microglobulin gene in one line and in the HLA complex in the other. Transfection of these cell lines with cloned genes for beta 2-microglobulin and HLA-B8, respectively, restored expression of their surface class I MHC proteins and resulted in concomitant SV40 binding. Finally, SV40 binds to purified HLA proteins in vitro and selectively binds to class I MHC proteins in a cell surface extract.     

A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II

The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor-ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low- affinity receptor-ligand interactions has been difficult. Utilizing a novel chelator-lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA), we have developed a convenient liposome system for directly detecting low-affinity receptor-ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or 'engrafted' directly onto fluorescently labelled liposomes via a metal-chelating linkage with NTA-DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence- activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4-6-fold above control cells. Such liposomes could detect directly the low-affinity interaction of murine CD40 and B7.1 with CD154- and CD28-expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes--a molecule related to nerve growth factor receptor.

The antigen recognized by the monoclonal antibody (mAb) MRC OX40 is present on activated rat CD4 positive T lymphocytes but not other cells. cDNA clones were isolated from an expression library using the MRC OX40 mAb and the protein sequence for the OX40 antigen deduced. It contains a typical signal sequence and a single putative transmembrane sequence of 25 predominantly hydrophobic amino acids giving an extracellular domain of 191 amino acids and a cytoplasmic domain of 36 amino acids. The sequence of the extracellular domain includes a cysteine-rich region with sequence similarities with the low affinity nerve growth factor receptor (NGFR) of neurons and the CD40 antigen present on human B cells. Within this region three cysteine-rich motifs can be recognized in OX40 compared with four similar motifs in both NGFR and CD40. OX40, CD40 and NGFR constitute a new superfamily of molecules with expression including lymphoid cells (OX40, CD40) and neuronal cells (NGFR). This is reminiscent of the immunoglobulin superfamily whose molecules are variously found at the surface of lymphoid or brain cells or both.     

Inhibition of lymphocyte proliferation by synthetic peptides homologous to human plasma apolipoproteins B and E

Apolipoproteins B and E of the human plasma lipoproteins are known inhibitors of lymphocyte proliferation. In this report, two synthetic peptide amides, apoB3358-3372 and apoE141-155, showed a dose-dependent inhibition of both the murine mixed lymphocyte culture reaction and the anti-T3 induced proliferation of lymphocytes. Their structures and antiproliferative potencies were similar to that of the heptadecapeptide CKS-17, a consensus peptide of a highly conserved region among HTLV-I, -II and C-type human retroviral proteins. SP-9-2-amide, a peptide homologous to the amino-terminal half of CKS-17, also suppressed lymphocyte activation. In contrast, a peptide homologous to the gp41 protein of HTLV-III that is sequence related to CKS-17 (approximately 35% homology) showed little antiproliferative activity. Neurotensin, a control peptide, showed no activity. The results suggest that a basic tetrapeptide sequence common to CKS-17-amide, SP-9-2, apoB3358-3372 and apoE141-155, but not HTLV-III-amide may account, in part, for the antiproliferative activities of these peptides.