The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells

Ovarian carcinomas are thought to arise from cells of the ovarian surface epithelium by mechanisms that are poorly understood. Molecules associated with neoplasia are potentially immunogenic, but few ovarian tumor antigens have been identified. Because ovarian carcinomas can elicit humoral responses in patients, we searched for novel tumor antigens by immunoscreening a cDNA expression library with ovarian cancer patient serum. Seven clones corresponding to the homeobox gene HOXB7 were isolated. ELISAs using purified recombinant HOXB7 protein revealed significant serologic reactivity to HOXB7 in 13 of 39 ovarian cancer patients and in only one of 29 healthy women (P < 0.0001). Ovarian carcinomas were found to express HOXB7 at markedly higher levels than normal ovarian surface epithelium, suggesting that immunogenicity of HOXB7 in patients could be associated with its elevated expression in ovarian carcinomas. Overexpression of HOXB7 in immortalized normal ovarian surface epithelial cells dramatically enhanced cellular proliferation. Furthermore, HOXB7 overexpression increased intracellular accumulation and secretion of basic fibroblast growth factor, a potent angiogenic and mitogenic factor. These results reveal HOXB7 as a tumor antigen whose up-regulated expression could play a significant role in promoting growth and development of ovarian carcinomas.

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion

Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single “fully competent” LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an α-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/ΔD1–2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/ΔD1–2 and with an ≈2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/ΔD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/ΔD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, “fully competent” LFA-1-binding surface.

Cutin Monomers and Surface Wax Constituents Elicit H2O2 in Conditioned Cucumber Hypocotyl Segments and Enhance the Activity of Other H2O2 Elicitors1

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface and cut segments were conditioned to develop competence for H2O2 elicitation. Alkaline hydrolysates of cutin from cucumber, tomato, and apple elicited H2O2 in such conditioned segments. The most active constituent of cucumber cutin was identified as dodecan-1-ol, a novel cutin monomer capable of forming hydrophobic terminal chains. Additionally, the cutin hydrolysates enhanced the activity of a fungal H2O2 elicitor, similar to cucumber surface wax, which contained newly identified alkan-1,3-diols. The specificity of elicitor and enhancement activity was further elaborated using some pure model compounds. Certain saturated hydroxy fatty acids were potent H2O2 elicitors as well as enhancers. Some unsaturated epoxy and hydroxy fatty acids were also excellent H2O2 elicitors but inhibited the fungal elicitor activity. Short-chain alkanols exhibited good elicitor and enhancer activity, whereas longer-chain alkan-1-ols were barely active. The enhancement effect was also observed for H2O2 elicitation by ergosterol and chitosan. The physiological significance of these observations might be that once the cuticle is degraded by fungal cutinase, the cutin monomers may act as H2O2 elicitors. Corrosion of cutin may also bring surface wax constituents in contact with protoplasts and enhance elicitation.

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis.

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.

Essential requirement of an invariant V alpha 14 T cell antigen receptor expression in the development of natural killer T cells.

NK1.1+ T [natural killer (NK) T] cells express an invariant T cell antigen receptor alpha chain (TCR alpha) encoded by V alpha 14 and J alpha 281 segments in association with a limited number of V betas, predominantly V beta 8.2. Expression of the invariant V alpha 14/J alpha 281, but not V alpha 1, TCR in transgenic mice lacking endogenous TCR alpha expression blocks the development of conventional T alpha beta cells and leads to the preferential development of V alpha 14 NK T cells, suggesting a prerequisite role of invariant V alpha 14 TCR in NK T cell development. In V beta 8.2 but not B beta 3 transgenic mice, two NK T cells with different CD3 epsilon expressions, CD3 epsilon(dim) and CD3 epsilon(high), can be identified. CD3 epsilon(high) NK T cells express surface V alpha 14/V beta 8 TCR, indicating a mature cell type, whereas CD3 epsilon(dim) NK T cells express V beta 8 without V alpha 14 TCR and no significant CD3 epsilon expression (CD3 epsilon(dim)) on the cell surface. However, the latter are positive for recombination activating gene (RAG-1 and RAG-2) mRNA, which are only expressed in the precursor or immature T cell lineage, and also possess CD3 epsilon mRNA in their cytoplasm, suggesting that CD3 epsilon(dim) NK T cells are the precursor of V alpha 14 NK T cells.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists.

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.

Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets. Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery. Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs. Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA. This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site. Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.

Evidence for a preformed transducer complex organized by the B cell antigen receptor.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins.

We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B and C of Borrelia burgdorferi have limited surface exposure, finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer mem- branes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.