Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus.

Several prior reports have identified peptides that are naturally associated with major histocompatibility complex (MHC) class II molecules on presenting cells. We have examined the delivery of a peptide from exogenous sources to MHC class II molecules. The peptide derives from the influenza virus hemagglutinin (HA) and activates a CD4+ T cell hybridoma. In functional assays of antigen presentation, this epitope is delivered effectively to T cells either in the context of influenza virus or chimeric immunoglobulin (Ig) molecules (Ig-HA) in which the peptide has replaced the CDR3 loop of the heavy chain. We find that the identical 11-mer peptide can be isolated from mouse MHC class II antigens whether the exogenous source of peptide is free HA peptide, the Ig-HA chimera, or ultraviolet-inactivated PR8 influenza virus. The Ig-HA chimera proves to be the most efficient vehicle for charging class II molecules via the exogenous route. Given the fact that self Igs represent natural long-lived carriers, we suggest that antigenized Igs have considerable potential for peptide delivery to MHC molecules in situ.

Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium.

A procedure has been developed to isolate dendritic cells to a high degree of purity from fresh blood. Prior enrichment methods have relied upon an initial 1-2-d culture period. Purified fresh isolates lack the characteristic morphology, phenotype, and immunostimulatory function of dendritic cells. The purified cells have the appearance of medium sized lymphocytes and express substantial levels of CD4, but lack the T cell molecules CD3, CD8, and T cell receptor. When placed in culture, the cells mature in a manner resembling the previously described, cytokine-dependent maturation of epidermal dendritic cells (Langerhans cells). The cells enlarge and exhibit many cell processes, express much higher levels of major histocompatibility complex class II and a panel of accessory molecules for T cell activation, and become potent stimulators of the mixed leukocyte reaction. Among the many changes during this maturation process are a fall in CD4 and the appearance of high levels of B7/BB1, the costimulator for enhanced interleukin 2 production in T cells. These changes are not associated with cell proliferation, but are dependent upon the addition of monocyte-conditioned medium. We suggest that the freshly isolated CD4-positive blood dendritic cells are recent migrants from the bone marrow, and that subsequent maturation of the lineage occurs in tissues in situ upon appropriate exposure to cytokines.

Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellular antigen of interdigitating dendritic cells and some B lymphocytes.

A monoclonal has been isolated that labels an intracellular antigen in dendritic cells and some B cells. The M342 hamster immunoglobulin was selected because it stained cells in the periarterial sheaths of spleen, the deep cortex of lymph node, and the thymic medulla--the same regions in which one finds interdigitating cells, the presumptive in situ counterparts of isolated lymphoid dendritic cells. M342 labeled an antigen within granules of isolated dendritic cells, but only in cells that had been cultured for a day and not in fresh isolates. This extends recent findings that most freshly isolated spleen dendritic cells are located in the periphery of the white pulp nodule and may serve as precursors for the periarterial pool of interdigitating cells, the site for M342 staining in situ. By electron microscopic immunolabeling, the M342 antigen was found exclusively in a type of multivesicular body. M342 staining was not found in mononuclear phagocytes from blood and peritoneal cavity. Peritoneal B cells expressed M342+ granules, and upon appropriate stimulation splenic B cells developed reactive granules as well. We conclude that M342 is a strong marker for interdigitating cells. Its existence reveals intracellular specializations in the vacuolar system of antigen-presenting cells including subsets of dendritic cells.

Tolerizing mice to human leukocytes: a step toward the production of monoclonal antibodies specific for human dendritic cells.

Despite several attempts to isolate a mAb specific for human dendritic cells, none currently exists. Recent attempts have utilized an improved dendritic cell purification method to prepare immunogens and a rapid two-color flow cytometric screening procedure that allows large numbers of hybridoma supernatants to be examined in each fusion. Yet these improvements have also failed, yielding only hybridomas that bind "shared" antigens expressed by both dendritic cells and other leukocytes. Dendritic cells express many shared antigens, including CD45 [leukocyte common antigen], CD40, leukocyte [beta 2] integrins CD11a and CD11c, CD54 [ICAM-1], CD44 [Pgp-1], CD58 [LFA-3], and the B7/BB1 antigen. Therefore, we are attempting to bias the immune response toward rarer, dendritic cell-specific clones by tolerizing or immunosuppressing our animals to shared antigens. In one approach, adult mice held in barrier cages are injected with "nondendritic" cells and cyclophosphamide [CP], in order to ablate responding "nonspecific" B cell clones. Fifteen days after the last dose of CP, they are challenged with nondendritic cells. A week later they are bled, and serum antibody titers against nondendritic cells are determined by FACS, in order to demonstrate tolerance compared to controls injected with CP alone. In the second approach, neonatal mice are injected with human T lymphoblasts at birth, followed by boosting at 1 week. In adulthood, they are challenged sequentially with sheep erythrocytes [sRBC], then with T blasts, to demonstrate that they can respond to unrelated cells but not to tolerogenic cells. One week after each kind of challenge, mice are bled and serum antibody levels are determined for treated and sham-injected mice. When these two approaches were compared, CP led only to nonspecific immunosuppression, while neonatal injections produced selective, antigen-specific nonresponsiveness to the tolerizing T blasts.

Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.

The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1(IIIB) virions at 1,200 x g for 2 h at 25 degrees C increases the number of particles that bind to CEM-SS T-cell targets by approximately 40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37 degrees C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, approximately 100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24(gag). Because the modest g forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.

Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145. II. Expression in situ in lymphoid and nonlymphoid tissues.

The rat monoclonal antibody NLDC-145, which has been utilized as a marker for mouse dendritic cells in numerous studies, binds an antigen that is more broadly distributed. This antigen is a unique 205-kDa integral membrane glycoprotein called DEC-205, which we have recently purified in quantities sufficient for basic biochemical studies, N-terminal sequencing, and immunization of rabbits. In cytofluorographic experiments, both the new polyclonal antibody and the original monoclonal detected DEC-205 on many classes of nondendritic murine leukocytes, particularly B cells. The quantities of DEC-205 on the surfaces of these cells were 10 to 50 times lower than those on epidermal and bone marrow dendritic cells. Here we utilize these reagents to reassess the tissue distribution of DEC-205 by immunohistochemical staining of frozen sections from a variety of organs, and by multiple-organ immunoblotting. Abundant expression of DEC-205 was confirmed histologically on thymic and intestinal epithelia and on dendritic cells in the T cell areas of peripheral lymphoid organs. In addition, DEC-205 was visualized in several other locations: B lymphocytes within B cell follicles, the stroma of the bone marrow, the epithelia of pulmonary airways, and the capillaries of the brain. Immunoblotting confirmed the presence of substantial levels of DEC-205 protein in lysates prepared from lymphoid tissues and from lung, marrow, and intestine. Thus, while DEC-205 is expressed at high levels by dendritic cells, it is also expressed by a number of other cell types in situ.

Eukaryotic initiation factor 4D. Purification from human red blood cells and the sequence of amino acids around its single hypusine residue.

Eukaryotic initiation factor 4D (eIF-4D) was purified from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A peptide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic character. Amino acid sequence determination on this peptide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.

DEC-205, a 205-kDa protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody NLDC-145: purification, characterization, and N-terminal amino acid sequence.

The monoclonal antibody NLDC-145 detects an antigen expressed at high levels by mouse dendritic cells (DCs) and thymic epithelial cells. Here we report on the purification and biochemical characterization of this antigen. The antigen detected by the NLDC-145 mAb is an integral membrane glycoprotein, with an apparent molecular mass of 205 kDa by immunoprecipitation and Western blotting. The isoelectric point is pH 7.5, and carbohydrates comprise about 7 kDa of the total mass. Lectin blotting and FACE analysis revealed heterogeneous biantennary N-linked glycans. O-linked glycans were not detected. N-terminal sequence analysis revealed that the antigen is a novel protein. Polyclonal antibodies prepared either to a synthetic N-terminal peptide or to whole purified protein bind the same 205-kDa protein recognized by NLDC-145. We refer to the protein as DEC-205, in view of its abundant expression by dendritic and thymic epithelial cells, and the revised molecular mass.

Integrin modulating factor-1: a lipid that alters the function of leukocyte integrins.

The avidity of integrin CR3 (also known as alpha M beta 2, Mac-1, Mo-1, and CD11b/CD18) may be reversibly altered without changes in the number of cell surface receptors. Here we describe a molecule termed integrin modulating factor (IMF-1), which controls CR3 avidity. Addition of IMF-1 to polymorphonuclear leukocytes (PMNs) or to purified CR3 causes enhanced binding of ligand. IMF-1 is not present in resting PMNs, but stimulation of cells results in a transient rise in IMF-1 content that parallels a transient rise in CR3 activity. We suggest that PMNs control adhesivity by controlling synthesis of IMF-1, which then acts as an allosteric activator of leukocyte integrins. IMF-1 is an acidic, amphiphilic molecule of Mr340 +/- 16 that does not contain ester, phosphate, amide, sialic acid, or glycosidic or vicinal hydroxyl functionalities, but does contain a carbon-carbon double bond. These results suggest that IMF-1 is an unsaturated fatty acid or an isoprenoid acid.

The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing.

Dendritic cells and thymic epithelial cells perform important immunoregulatory functions by presenting antigens in the form of peptides bound to cell-surface major histocompatibility complex (MHC) molecules to T cells. Whereas B cells are known to present specific antigens efficiently through their surface immunoglobins, a comparable mechanism for the capture and efficient presentation of diverse antigens by dendritic cells and thymic epithelial cells has not previously been described. We show here that their antigen-presentation function is associated with the high-level expression of DEC-205, an integral membrane protein homologous to the macrophage mannose receptor and related receptors which are able to bind carbohydrates and mediate endocytosis. DEC-205 is rapidly taken up by means of coated pits and vesicles, and is delivered to a multivesicular endosomal compartment that resembles the MHC class II-containing vesicles implicated in antigen presentation. Rabbit antibodies that bind DEC-205 are presented to reactive T-cell hybridomas 100-fold more efficiently than rabbit antibodies that do not bind DEC-205. Thus DEC-205 is a novel endocytic receptor that can be used by dendritic cells and thymic epithelial cells to direct captured antigens from the extracellular space to a specialized antigen-processing compartment.

Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature.

Two subsets of dendritic cells, differing in T-cell stimulatory function, have been purified directly from human blood. Both subsets are positive for major histocompatibility complex (MHC) class II expression and negative for lineage-specific antigens (e.g. CD3, CD14, CD16, CD19 negative), but are separated by exploiting differences in expression of the beta 2-integrin, CD11c. The CD11c-negative subset is functionally immature, requiring monocyte-derived cytokines to develop into typical dendritic cells. The CD11c-positive subset has potent T-cell stimulating activity and expresses the activation antigen CD45RO, unlike its immature counterpart. However, these mature cells only develop typical dendritic morphology and high levels of MHC proteins and adhesins after a period of culture independent of exogenous cytokines. Although the freshly isolated mature dendritic cells resemble monocytes in cytospin preparations, the former lack CD14 and have a much stronger primary T-cell stimulatory capacity. We hypothesize that the CD11c-negative immature cells are marrow-derived precursors to tissue dendritic cells, such as epidermal Langerhans' cells, while the CD11c-positive cells are derived from tissues where they have been activated by antigen, and are en route to the spleen or lymph nodes to stimulate T-cell responses there.

Contrasting efficacy of presentation by major histocompatibility complex class I and class II products when peptides are administered within a common protein carrier, self immunoglobulin.

Major histocompatibility complex (MHC) class I and II products are specialized to present antigens via different intracellular processing routes. Peptides originating from proteins in the cytoplasm can gain access to class I peptide-binding grooves, most likely in the rough endoplasmic reticulum. Peptides from proteins in acidic endocytic vacuoles gain access to class II. It has been proposed that MHC class I products also can capture peptides from "exogenous" or noninfectious sources, and this assumption underlies the use of intact proteins as vaccines for CD8+ cytotoxic T lymphocytes. Here we describe quantitative information comparing the efficacy of peptide presentation from exogenous proteins by administering a class I- and II-restricted peptide within the same context, the CDR3 loop of the VH domain of a self immunoglobulin. Antigen-presenting cells (APC), including primary dendritic cells, efficiently present an influenza hemagglutinin peptide from the immunoglobulin (Ig) carrier (50% maximal response at 10 nM Ig-HA) to an MHC class II-restricted T cell. In contrast, these same APC are unable to present an influenza nucleoprotein (NP) peptide from the same context (1 microM Ig-NP) to an MHC class I-restricted T cell. Ig-NP DNA transfectants do present the nucleoprotein viral peptide on class I. Thus, peptides within the complementarity-determining region loops of Ig carriers can be presented on class I or II MHC products, but the endocytic compartment, when offered MHC class I- and II-restricted peptides within the same carrier protein context, favors presentation by class II by at least 1000-fold.

An iron-carboxylate bond links the heme units of malaria pigment.

The intraerythrocytic malaria parasite uses hemoglobin as a major nutrient source. Digestion of hemoglobin releases heme, which the parasite converts into an insoluble microcrystalline material called hemozoin or malaria pigment. We have purified hemozoin from the human malaria organism Plasmodium falciparum and have used infrared spectroscopy, x-ray absorption spectroscopy, and chemical synthesis to determine its structure. The molecule consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme.

Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145. I. Expression on dendritic cells and other subsets of mouse leukocytes.

Prior studies by a variety of groups demonstrated that the mAb NLDC-145 reacted primarily with dendritic cells (DCs) and the epithelial cells of the thymic cortex. We recently reported that this mAb recognizes DEC-205, a 205-kDa integral membrane glycoprotein with a unique amino-terminal sequence, and raised a rabbit polyclonal antibody to purified DEC-205 with higher affinity for the blotted antigen than the original mAb. Here we utilize both the polyclonal and NLDC-145 to reassess the expression and function of DEC-205 on leukocytes. By cytofluorography, DCs derived from the epidermis (Langerhans cells) and from proliferating bone marrow progenitors (BMDCs) expressed high levels (2-3 logs) of DEC-205, while freshly isolated spleen DCs comprised two subsets, most (80%) staining at low levels (< or = 1 log), the remainder moderately (1.5 logs). DEC-205 epitopes were sensitive to trypsin, but were regenerated in culture. Resident and inflammatory peritoneal macrophages did not express the antigen, except for small amounts on thioglycollate-elicited cells. B cells from spleen, lymph node, bone marrow, blood, and peritoneal fluid expressed levels of DEC-205 that were 10- to 50-fold lower than those on BMDCs. Marrow pro- and pre-B cells did not express DEC-205. Polyclonal anti-DEC-205 failed to inhibit either stimulation of a primary mixed leukocyte reaction by DCs in vitro, or a local graft vs host response in vivo, where parental T cells were injected into F1 mice. DEC-205 is therefore more broadly expressed on leukocytes than previously appreciated, but its function remains unclear.