Characterization of a Helicobacter pylori neutrophil-activating protein

Antibody-blocking studies have demonstrated the role of CD6 in thymocyte-thymic epithelial (TE) cell adhesion. Here we report that CD6 expressed by COS cells mediates adhesion to TE cells and that this interaction is specifically blocked with an anti-CD6 monoclonal antibody (mAb) or with a mAb (J4-81) that recognized a TE cell antigen. We isolated and expressed a cDNA clone encoding this antigen and show that COS cells transfected with this cDNA bind a CD6 immunoglobulin fusion protein (CD6-Rg). This antigen, which we named ALCAM (activated leukocyte-cell adhesion molecule) because of its expression on activated leukocytes, appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. We prepared an ALCAM-Rg fusion protein and showed that it binds to COS cell transfectants expressing CD6, demonstrating that ALCAM is a CD6 ligand. The observations that ALCAM is also expressed by activated leukocytes and that both ALCAM and CD6 are expressed in the brain suggest that ALCAM-CD6 interactions may play a role in the binding of T and B cells to activated leukocytes, as well as in interactions between cells of the nervous system.     

Human NKR-P1A. A disulfide-linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes

In rodents, the NKR-P1 family of glycoproteins are preferentially expressed on NK cells and have been implicated in NK cell function. In this study, we describe the characterization and cloning of a human homologue. Human (h)NKR-P1A cDNA was cloned from a NK cell cDNA library by expression in COS7 cells with the use of the DX1 mAb. hNKR-P1A is a type II membrane glycoprotein with characteristic properties of the C-type lectin superfamily. Comparison of the predicted amino acid of human NKR-P1A with rat and mouse NKR-P1 indicates 46% homology. NKR-P1A is on human chromosome 12, the syntenic of mouse chromosome 6, where the murine NKR-P1 genes are located. All rat NK cells express NKR-P1; however, hNKR-P1A is present on only a subset of human NK cells. Although rodent T cells only infrequently express NKR-P1, hNKR-P1A is present on approximately 25% of adult peripheral blood T cells, including both CD4+ and CD8+ T cells, and is expressed preferentially on adult T cells with a "memory" antigenic phenotype. The anti-hNKR-P1A mAb failed to affect lysis of NK-sensitive targets; however, the spontaneous cytotoxicity mediated by certain NK cell clones against the murine P815 cell target was blocked by anti-hNKR-P1A mAb. Our findings demonstrate that NKR-P1A is a human homologue of the rodent NKR-P1 genes and suggest that this molecule may be involved in NK cell function.     

Fluorescent virions: dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells

T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-alpha and -beta chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471-94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-alpha chain even when it paired with a TCR-beta chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-alpha chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-alpha chains of human lupus Th clones. The lupus TCR-alpha chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-alphabeta+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes "Pantigens" (for promiscuous antigens).     

Requirement of CD28-CD86 co-stimulation in the interaction between antigen-primed T helper type 2 and B cells

The interaction between CD28 and its ligands, CD80 and CD86, is crucial for an optimal activation of antigen-specific T cells. However, the requirement of CD80 or CD86 co-stimulation in Th2 cell differentiation and activation is controversial. Freshly isolated murine CD4+ and CD8+ T cells were incubated with P815 transfectants expressing a similar level of either CD80 or CD86 in the presence of anti-CD3 mAb. Both CD80 and CD86 co-stimulated the proliferation of CD4+ and CD8+ T cells at comparable time-kinetics and magnitude, but CD86 alone was able to co-stimulate IL-4 and especially IL-10 production in CD4+ T cells. In typical Th2-dependent immune responses elicited by Nippostrongylus brasillensis infection, the anti-CD86 mAb treatment but not the anti-CD80 mAb treatment efficiently inhibited antigen-specific IgE and IgG1 production, which was accompanied with the reduced IL-4 production. Our results suggest that CD86 co-stimulation plays a dominant role not only in the primary activation of Th2 cells but also in the secondary interaction between antigen-primed Th2 cells and B cells.     

Induction of the 2B9 antigen/dipeptidyl peptidase IV/CD26 on human natural killer cells by IL-2, IL-12 or IL-15

Activation of human natural killer (NK) cells involves sequential events including cytokine production and induction of cell surface molecules, resulting in the enhancement of cytolytic activity. To delineate the activation process of NK cells, we generated murine monoclonal antibodies (mAbs) against YT, a human large granular lymphocyte/natural killer (LGL/NK) cell line. Among the mAbs reactive with YT cells, one mAb, termed 2B9, was noted because of the lack of reactivity with most of the human T- and B-cell lines tested. In fresh peripheral blood mononuclear cells (PBMC), however, the majority of cells expressing this antigen (Ag) were T cells but not CD16+ nor CD56+ NK cells. Since YT cells showed an activated phenotype expressing interleukin-2 (IL-2) receptor alpha chain, we examined whether 2B9 Ag could be induced on normal human peripheral blood NK cells by cytokines known to activate NK cells. The 2B9 Ag was induced on NK cells by IL-2, IL-12 or IL-15 while no induction was observed by interferon-gamma (IFN-gamma). Biochemical analysis showed that anti-2B9 mAb recognized a 115 kDa molecule in YT cells. A cDNA clone encoding the 2B9 Ag was isolated from a cDNA expression library of YT cells and its sequence was identical to CD26 cDNA although it was not of full length. Transient expression of the 2B9 cDNA on COS-7 cells revealed that this cDNA encodes the antigenic epitope(s) recognized by anti-2B9 mAb as well as Ta1, an anti-CD26 mAb. These results showed that the 2B9 Ag is identical to CD26, and demonstrated that CD26 is an activation antigen on CD16+ CD56+ NK cells inducible by IL-2, IL-12 or IL-15.     

Enhanced murine CD4+ T cell responses induced by the CD2 ligand CD48

The physiological functions of murine CD2 and its ligand CD48 are uncertain. We have examined the role of the CD2-CD48 interaction in murine T cell activation using a series of Chinese hamster ovary (CHO) cell transfectants. CHO cells expressing I-Ad together with CD48 induced more potent activation of OVA-specific, I-Ad-restricted DO11.10-transgenic T cells than CHO cells expressing I-Ad alone. CD48 augmented proliferation and IL-2 production in response to antigen. The enhancing effect of CD48 was of the same magnitude as that seen for CD80 (B7-1). Conjugate assays revealed the ability of CD48 to increase adhesion between T cells and CHO transfectants. The enhancing effects of CD48 on T cell-antigen-presenting cell adhesion and T cell activation were inhibited by anti-CD2 monoclonal antibody. This report provides the first evidence that the CD2 ligand CD48 contributes to the interactions of murine CD4+ T cells with antigen-presenting cells.     

T cell vaccination induces T cell receptor Vbeta-specific Qa-1-restricted regulatory CD8(+) T cells

Vaccination of mice with activated autoantigen-reactive CD4(+) T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8(+) regulatory T cells that are specific for pathogenic CD4(+) T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8(+) T cells emerge that preferentially lyse and regulate activated autologous CD4(+) T cells in a T cell receptor (TCR) Vbeta-specific manner. This TCR Vbeta-specific regulation is not observed in beta2-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vbeta8-specific Qa-1-restricted CD8(+) T cells are also induced by TCV with activated CD4(+) Vbeta8(+) T cells. These CD8(+) T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vbeta8. Further, CD8(+) T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4(+)Vbeta8(+) T cell clone specifically recognize other CD4(+) T cells and T cell tumors that express Vbeta8 and the syngeneic Qa-1(a) but not the allogeneic Qa-1(b) molecule. Thus, Vbeta-specific Qa-1-restricted CD8(+) T cells are induced by activated CD4(+) T cells. We suggest that these CD8(+) T cells may function to specifically regulate activated CD4(+) T cells during immune responses.     

The murine nonclassical class I major histocompatibility complex-like CD1.1 molecule protects target cells from lymphokine-activated killer cell cytolysis

Classical class I major histocompatibility complex (MHC) molecules, as well as the nonclassical class I histocompatibility leukocyte antigen (HLA)-E molecule, can negatively regulate natural killer (NK) cell cytotoxicity through engagement of NK inhibitory receptors. We show that expression of murine (m)CD1.1, a nonpolymorphic nonclassical MHC class I-like molecule encoded outside the MHC, protects NK-sensitive RMA/S target cells from adherent lymphokine-activated killer cell (A-LAK) cytotoxicity. Passage of effector cells in recombinant interleukin (rIL)-2 enhanced protection by mCD1.1, suggesting an expansion of relevant A-LAK population(s) or modulation of A-LAK receptor expression. Murine CD1. 1 conferred protection from lysis by rIL-2-activated spleen cells of recombination activating gene (Rag)-1(-/-) mice, which lack B and T cells, demonstrating that mCD1.1 can protect RMA/S cells from lysis by NK cells. An antibody specific for mCD1.1 partially restored A-LAK lysis of RMA/S.CD1.1 transfectants, indicating that cell surface mCD1.1 can confer protection from lysis; therefore, mCD1.1 possibly acts through interaction with an NK inhibitory receptor. CD1.1 is by far the most divergent class I molecule capable of regulating NK cell activity. Finally, mCD1.1 expression rendered RMA/S cells resistant to lysis by A-LAK of multiple mouse strains. The conserved structure of mCD1.1 and pattern of mCD1.1 resistance from A-LAK lysis suggest that mCD1.1 may be a ligand for a conserved NK inhibitory receptor.     

A T cell activation antigen, Ly6C, induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8+ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8+ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C+ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G0/G1 to S+G2/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4+ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4+ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.     

Differential expression of CD22 (Lyb8) on murine B cells

Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging IgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculating B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, in combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets.     

Nonclassical behavior of the mouse CD1 class I-like molecule

The mouse CD1 (mCD1) molecule is a class I-like molecule that is encoded outside of the MHC. We show here that mCD1 shares several properties with Ag-presenting class I molecules, including a requirement for beta2-microglobulin for stable cell-surface expression in T lymphocyte transfectants and thymocytes. mCD1 is also capable of binding to mouse CD8alphabeta heterodimers participating in the activation of CD8+ T cells in a manner similar to classical class I molecules. However, mCD1 surface expression is not decreased at high temperatures in cells that lack the transporter associated with Ag processing (TAP), including both RMA-S and Drosophila melanogaster cells. The data indicate that mCD1 does not require TAP to be expressed in a stable fashion at the cell surface. We speculate that the ability of mCD1 to reach the cell surface in transporter-deficient cells may reflect its ability to present a distinct set of ligands. The properties of mCD1 described here can account, in part, for the selection of the diverse populations of T cells that are known to be mCD1 reactive.     

Close phenotypic and functional similarities between human and murine alphabeta T cells expressing invariant TCR alpha-chains

Several studies have demonstrated the existence of a murine NK1.1+ alphabeta T cell subset expressing V alpha14+ TCR alpha-chains with highly conserved invariant junctional sequences and able to secrete Th2 cytokines when exposed to CD1+ stimulator cells. In humans, alphabeta T cells carrying invariant V alpha24+ TCR alpha-chains highly homologous to those expressed by murine NK1.1 cells have been recently described. Here we show that these cells (referred to as V alpha24inv T cells) and murine NK1.1+ alphabeta T cells resemble each other in several ways. First, like their murine counterparts, T cells expressing high levels of V alpha24inv TCRs can be either CD4- CD8- double negative (DN) or CD4+, but they never express heterodimeric CD8 molecules. Second, most V alpha24inv T cells are brightly stained by NKRP1-specific mAb but not by mAb directed against other type II transmembrane proteins of the NK complex. Third, DN and particularly CD4+ V alpha24inv T cells are greatly enriched for IL-4 producers. The concomitant expression of highly conserved TCRs of a particular set of NK markers and of Th2 cytokines in human and murine alphabeta T cells suggests a coordinate acquisition of these phenotypic and functional properties. Furthermore, the relatively high frequency of human V alpha24inv T cells, which are presently shown to represent on average 1/500 PBL, and the high interindividual variations of the size of this cell subset under physiologic conditions go for a major role played by alphabeta T cells carrying invariant TCR in a large array of immune responses.     

Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis, which is responsible for controlling deoxyribonucleoside triphosphate (dNTP) pool size. It has been shown that transfection of RR M2 cDNA in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool assays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cells. Total dNTP levels among the six cell lines rose in the following order: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resistant clone. The dCTP levels of the cells mimicked the total dNTP pools on a smaller scale. The significant increases in the dCTP pool sizes of the M2-D, X-D, and HUR clones were proportional to their respective increases in RR activity. Relative to all other transfectants, the M1-D clone demonstrated lower dCTP levels but increased dATP pools. The M1-D clone demonstrated a significant resistance to dNTP inhibition of RR activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhibition was observed in M2-D, X-D, and HUR clones. In summary, M2 cDNA transfectants and HUR clones had increased RR activity as well as expanded dNTP pools, particularly dCTP, when compared with wild-type KB cells. These data provide evidence for the intertwined relationship between RR activity and dNTP pools.     

Cutting edge: MHC class I triggering by a novel cell surface ligand costimulates proliferation of activated human T cells

BY55 is a human cell surface molecule whose expression is restricted to NK cells, a subset of circulating CD8+ T lymphocytes, and all intestinal intraepithelial T lymphocytes. Here, we report that BY55 is a novel NK receptor showing broad specificity for both classical and nonclassical MHC class I molecules, and that optimal binding requires a prior aggregation of MHC class I complexes. Using BY55 transfectants, we have identified functional consequences of MHC class I/ligand interactions for the class I-bearing cell. The triggering of MHC class I molecules on human T cell clones by BY55 delivered a potent proliferative signal in the presence of soluble CD3 mAb. The costimulatory signal provided by MHC class I ligation was only seen in activated, and not resting, peripheral blood T cells. This observation represents an additional and/or alternative pathway to CD28 costimulation and may be of particular relevance in memory T cells lacking CD28, such as intestinal intraepithelial T lymphocytes, which are CD28- but BY55+.     

Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions

Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors.     

Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the alpha 4 beta 7-integrin binding domains of murine MAdCAM-1, but extreme divergence of mucin-like sequences

The mucosal vascular addressin, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), is an Ig family adhesion receptor preferentially expressed by venular endothelial cells at sites of lymphocyte extravasation in murine mucosal lymphoid tissues and lamina propria. MAdCAM-1 specifically binds both human and mouse lymphocytes that express the homing receptor for Peyer's patches, the integrin alpha 4 beta 7. Functional expression cloning was used to isolate a cDNA from a macaque mesenteric lymph node library that encodes the homologue to murine MAdCAM-1. The macaque cDNA was subsequently used to clone the human homologue as well. Expression of human MAdCAM-1 RNA is restricted to mucosal tissues, gut-associated lymphoid tissues and spleen. Human MAdCAM-1 selectively binds both murine and human lymphocyte cell lines expressing alpha 4 beta 7. Human and macaque MAdCAM-1 have two Ig-like domains that are similar to the two amino-terminal integrin binding domains of murine MAdCAM-1. The conservation of sequences within the region homologous to the mucin/IgA domain of murine MAdCAM-1 is, however, much less apparent. These receptors exhibit considerable variation from murine MAdCAM-1 with respect to the length of the mucin-like sequence and the lack of a membrane proximal Ig (IgA-like) domain. The isolation of these different species of MAdCAM-1 demonstrates greater seleoffctive pressure for maintenance of amino acids involved in alpha 4 beta 7 binding than those sequences presumably involved in the presentation of carbohydrates for selectin binding.     

Signaling via major histocompatibility complex class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand

Activated T cells induce proliferation and differentiation of resting B cells in vitro through their CD40 molecules and lymphokine receptors. However, despite constitutive B cell expression of CD40 and lymphokine receptors, widespread nonspecific polyclonal B cell activation by activated T cells is seldom observed in vivo. The present study was designed to test the hypothesis that signals delivered via the B cell antigen (Ag) receptor (membrane immunoglobulin, mIg) and major histocompatibility complex (MHC) class II molecules enhance B cell responsiveness to CD40-mediated signals, providing specificity to the Ag-nonspecific, MHC-unrestricted CD40 signal. To test this hypothesis, both an Ag-specific mouse B cell clone CH12.LX, and freshly isolated resting splenic B cells were cultured with either soluble or membrane-bound forms of the T cell ligand for CD40 (CD40L), in the presence or absence of additional signals provided by Ag or anti-IgM, interleukin-4, and class II-specific monoclonal antibody (mAb). Differentiation of CH12.LX cells and proliferation of splenic B cells in response to both forms of CD40L was greatly enhanced by exposure to mIg-mediated signals, with greatest enhancement seen when cells were cultured with Ag prior to receiving other signals. Response to CD40L was further enhanced by concurrent culture with class II-specific, but not class I-specific mAb. Enhancement was greatest at limiting concentrations of CD40L. The ability of class II MHC-mediated signals to enhance Ag-specific B cell responsiveness to CD40-mediated signaling may selectively promote the activation of B cell clones capable of cognate interactions with helper T cells.