Macrophages present pinocytosed exogenous antigen via MHC class I whereas antigen ingested by receptor-mediated endocytosis is presented via MHC class II

Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.     

Macrophages in the interstitial tissue of the rat testis

Summary Macrophages were identified in the intertubular tissue of the rat testis by loading animals with a particulate vital dye (trypan blue or India ink) and by localizing immunocytochemically a macrophage membrane antigen (MRC W3/25). Leydig cells were identified by the histochemical staining reaction for 3-hydroxysteroid dehydrogenase activity and by a monoclonal antibody. Macrophages were scattered in the interstitial tissue closely attached to and mixed with the Leydig cells. They were never found in the seminiferous tubules. The macrophages comprised about 25% of all the cells in the interstitium. Double staining with a vital dye and a marker antibody showed that all the phagocytosing cells were macrophages and that the Leydig cells did not take up vital dyes. Double staining for the demonstration of the 3-hydroxysteroid dehydrogenase activity and the macrophage antigen likewise revealed two distinctly different cell populations. Crude Leydig cell preparations obtained by collagenase treatment of the testis contained macrophages (12–14%). Macrophages were present throughout the postnatal prepuberal development of the testis. Their density was increased in the cryptorchid and irradiated testis.     

Immunolocalization of clusterin in the ram testis, rete testis, and excurrent ducts

Clusterin, a glycoprotein that elicits cell aggregation, has previously been isolated from ram rete testis fluid, and has been partially characterized. In experiments reported, we have used monoclonal antibodies against clusterin in combination with indirect immunofluorescence microscopy to investigate the distribution of clusterin in the adult ram testis, rete testis, and excurrent ducts. Tissue blocks (5 mm3) were fixed in periodate/lysine/paraformaldehyde containing 0.1% glutaraldehyde and, after embedding, 5-microM sections were prepared for immunolocalization. In the testis, 2 basic patterns were observed: 1) strong to moderate staining for clusterin in the adluminal region with little staining in the basal region of the seminiferous epithelium and germinal cells; and 2) moderate staining throughout the seminiferous epithelium between germinal cells. In the rete testis, strong clusterin staining was localized intracellularly in the rete epithelial cells, most often associated with the luminal surface. In the epididymis, intracellular clusterin was localized in some principal cells of the caput epididymidis. The luminal surfaces and spermatozoa within the lumen were strongly positive. In the vas deferens, clusterin staining was associated with the luminal surface only. The presence of clusterin was clearly detected in unwashed isolated epididymal spermatozoa, but not in spermatozoa washed with phosphate-buffered saline containing 0.05% Tween 20.     

Processed antigen binds to newly synthesized MHC class II molecules in antigen-specific B lymphocytes

We describe the direct detection of radiolabeled antigen fragments bound to class II MHC molecules following immunoglobulin-mediated endocytosis and processing of native antigen in B lymphoblastoid cells. Tris-Tricine SDS gels revealed six distinct iodinated processing products that could be detected on class II MHC 1 hr after antigen endocytosis and persisted for at least 20 hr. These physiological processed antigen-class II complexes were remarkably stable, as judged by the fact that class II alpha beta dimers, which remain associated in SDS, became labeled with the same set of processed peptides. Using a lectin-binding assay, we show that these physiological processing products bind to the newly maturing population of MHC molecules rather than binding to the preexisting cell surface population; in contrast, an exogenous peptide binds predominantly to the latter population. A direct T cell-independent assay for processed peptide-MHC complex formation should facilitate additional studies on the exogenous antigen processing pathway.     

Class II MHC molecules and antigen enter the same vesicles during internalization by resting B lymphocytes

Efficient presentation of Ag by a B cell to a T cell requires that Ag bind to the Ag receptor (Ig) on the B cell, after which it is internalized into an acid compartment where it is modified and returned to the cell surface in the context of class II MHC molecules. It remains uncertain whether processed Ag binds to class II which has been internalized and recycled with Ag, or to nascent class II inside the cell. To determine if cell surface class II enters the same vesicles as Ag, or is excluded during internalization of Ag which is bound to the B cell receptor, 5- and 16-nm gold particles were labeled with anti-class II and anti-Ig, respectively. Cells were incubated at 37 degrees C and internalization of these particles was observed using electron microscopy. By 10 min, 60-75% of the B cell sections contained vesicles with gold particles inside them. Between 40 and 64% of these vesicles had both 5- and 16-nm particles. Maximum internalization occurred by 30-60 min, and by 2 hr the number of small and large particles on the B cell surface became constant or increased, respectively. Both kinds of particles moved from electron-lucent to electron-dense vesicles as the incubation time increased, although a portion of the anti-class II particles remained in electron-lucent vesicles. These data clearly show that labeled, cell surface class II is not selectively excluded from Ag-containing vesicles during Ag internalization. Thus, cointernalization of Ag and class II may represent a mechanism by which processed Ag meets class II.     

ATGs help MHC class II,but inhibit MHC class I antigen presentation

We have recently shown that the LC3/Atg8 lipidation machinery of macroautophagy is involved in the internalization of MHC class I molecules. Decreased internalization in the absence of ATG5 or ATG7 leads to MHC class I surface stabilization on dendritic cells and macrophages, resulting in elevated CD8⁺ T cell responses during viral infections and improved immune control. Here, we discuss how the autophagic machinery supports MHC class II restricted antigen presentation, while compromising MHC class I presentation via internalization and degradation.     

Lipid metabolism in the testis of the ram

1. Analysis of rams testes revealed that phosphatidylcholine was the major phospholipid and accounted for about 40% of the total. Only small amounts of choline plasmalogen were present. 2. The ratio of phosphatidylcholine to choline plasmalogen in the testis was very different from that occurring in the spermatozoa. This result was in contrast with those for rat testis and rat spermatozoa (obtained from the head of the epididymis), where the ratio of the two lipids was very similar. 3. Infusions of [(32)P]orthophosphate into the testicular artery of rams resulted in incorporation of radioactivity into most phospholipids; phosphatidylinositol labelling accounted for 68% and 39% of the radioactivity after infusions lasting 3hr. and 5hr. respectively. 4. With the exception of phosphatidic acid the specific radioactivity of phosphatidylinositol was higher than that of any other lipid. 5. After the infusion of [U-(14)C]glucose, triglycerides accounted for about 60% of the radioactivity in testicular neutral lipids, whereas diglycerides had only about 15% of the radioactivity. 6. Palmitic acid (16:0) was the major component both in neutral lipids and phospholipids of ram testes. 7. The effects of gonadotrophic hormones (luteinizing hormone and follicle-stimulating hormone) on the incorporation of [(32)P]orthophosphate into total testicular phospholipids in vivo were also examined.     

MHC class II invariant chains in antigen processing and presentation

Most protein antigens cannot elicit a T-cell response unless they are processed to peptides, which are then presented to T lymphocytes by surface MHC class II molecules. Recent evidence supports an essential role of the invariant chain associated with class II MHC polypeptides in antigen processing.     

Class II MHC self-antigen presentation in human B and T lymphocytes

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.     

Towards a systems understanding of MHC class I and MHC class II antigen presentation

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.     

The N-acetylhexosaminidase components of the ram testis and epididymis

Three and four N-acetylhexosaminidase components, from ram testis and epididymis respectively, have been separated by ion-exchange chromatography on DEAE-cellulose. Although they all have the same molecular weight (approx. 140000) and very similar catalytic properties towards the synthetic substrates, 4-methylumbelliferyl N-acetyl-beta-glucosaminide and N-acetyl-beta-galactosaminide, isoelectric focusing of the individual components showed that each had a distinct pI value. Isoelectric focusing has also been used to demonstrate the occurrence of multiple forms in ejaculated ram semen.     

The class II genes of the rat MHC

Genes that encode class II Ag from the MHC of the rat, the RT1 region, have been isolated as a series of cosmid clones. The cosmids define two clusters, each of which contains three identifiable sequences; one homologous to alpha-chain and two to beta-chain genes. Both the serologically identified rat class II Ag have been expressed in mouse L cell fibroblasts after the introduction of each alpha-chain gene along with a beta-chain gene from the same cluster. There are substantial homologies to the I region of the mouse H-2 complex in the presence, location, orientation, and expression of the six identified sequences from the rat RT1, supporting the view that the overall organization of the two gene complexes has remained conserved since the species separated.     

Serum and testis selenium concentrations and testis morphology in ram lambs of varying ages

Serum and testis selenium (Se) concentrations, body and testes weights, seminiferous tubule height and width measurements and percent of tubules containing luminal spermatozoa were determined in Se-treated (SSe) and control (NSe) crossbred ram lambs at 60, 90, 120, 150 and 180 days of age. With IM injections, SSe lambs received 3 mg of Se as selenite and NSe lambs received 0.9% saline at 30-day intervals throughout the study. For each age group, lambs were weighed, jugular vein blood collected and testes removed at the designated age. Serum and testis tissue samples for each lamb were assayed for Se, and testis tissue was also evaluated for histological parameters. For all parameters, only serum Se concentrations were affected (P<0.0001) by Se treatment; however, all other parameters were affected (P<0.0001) by age. For combined groups, mean testis Se concentration (0.33 ppm), testes weights, seminiferous tubule measurements and percent of tubules (82.2) containing luminal spermatozoa were greatest (P<0.05) at 180 days of age, and mean testis Se concentrations were significantly correlated with these testicular parameters. These data lend support to the hypothesis that the increase in concentration of testicular Se to adult concentrations (>0.3 ppm) around the time of puberty is associated with rapid testicular development and production of spermatozoa.     

Primary structural sequence polymorphism in the human class II MHC antigen 33.1

Monoclonal antibody 33.1 defines a non-DR, class 11, human major histocompatibility complex antigen, 33.1, which appears to be distinct from other class II antigens in its cellular distribution and primary structure. To characterize the structure more fully and to determine the degree of polymorphism within 33.1, a comparative N-terminal sequence study has been undertaken using a series of ten B lymphoblastoid cell lines with different DR and MB types. The results confirm that both the and chains of 33.1 are homologues of the corresponding chains of the murine I-A antigen and indicate that while 33.1 does not appear to be identical with MB, it is closely related. Sequence analyses revealed two major variants of 33.1, corresponding to cells with specificities MB1 and MB 3, respectively. Within each MB type, other polymorphisms have been detected. Cells that are MB2 do not react with monoclonal antibody 33.1. Suggestive evidence is presented that monoclonal antibody 33.1 reacts predominantly with the chain of the antigen. The preferential expression of 33.1 on activated B cells suggests that expression of at least the 33.1 chain gene is greatly enhanced in the course of B-cell activation, but the specific function of 33.1 remains to be determined.Abbreviations used in this paper McAb monoclonal antibody - BLCL B lymphoblastoid cell lines - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - NP-40 Nonidet P-40 Fellow of the Arthritis Foundation     

HAGE,a cancer/testis antigen with potential for melanoma immunotherapy: identification of several MHC class I/II HAGE-derived immunogenic peptides

There remains a need to identify novel epitopes of potential tumour target antigens for use in immunotherapy of cancer. Here, several melanoma tissues and cell lines but not normal tissues were found to overexpress the cancer-testis antigen HAGE at the mRNA and protein level. We identified a HAGE-derived 15-mer peptide containing a shorter predicted MHC class I-binding sequence within a class II-binding sequence. However, only the longer peptide was found to be both endogenously processed and immunogenic for T cells in transgenic mice in vivo, as well as for human T cells in vitro. A different class I-binding peptide, not contained within a longer class II sequence, was subsequently found to be both immunogenic and endogenously processed in transgenic mice, as was a second class II epitope. These novel HAGE-derived epitopes may contribute to the range of immunotherapeutic targets for use in cancer vaccination programs.     

MHC class II molecules on the move for successful antigen presentation

Major histocompatibility complex class II (MHC II) molecules are targeted to endocytic compartments, known as MIIC, by the invariant chain (Ii) that is degraded upon arrival in these compartments. MHC II acquire antigenic fragments from endocytosed proteins for presentation at the cell surface. In a unique and complex series of reactions, MHC II succeed in exchanging a remaining fragment of Ii for other protein fragments in subdomains of MIIC before transport to the cell surface. Here, the mechanisms regulating loading and intracellular trafficking of MHC II are discussed.