Internalization of Ia molecules by antigen-presenting B cells is limited

Our data demonstrate that the uptake of surface Ia into an intracellular compartment of B lymphoma or normal spleen cells is limited to about 20% after 2 to 3 h. The extent of internalization does not vary with several types of stimulation, including LPS, phorbol esters, anti-Ig-plus phorbol ester-stimulated EL-4 T cell supernatant, and Con A supernatant. Resting and activated B cells had similar rates of internalization. The rate and extent of uptake of surface Ia molecules into an intracellular compartment was monitored quantitatively through the use of a mAb radiolabeled with 125I. The internalization of Ia molecules was compared to that of transferrin receptor, a receptor that undergoes rapid internalization and recycling and accumulates in a intracellular pool that can be trapped by monensin. The internalization of Ia was not affected by monensin, although its synthetic pathway is disturbed by this drug. The potential use of internalized Ia for formation of T cell-triggering complexes of Ia and Ag fragments is not ruled out by these data, but it appears unlikely that internalization provides the major mechanism permitting Ia interaction with Ag.     

Activation of protein kinase C accelerates internalization of transferrin receptor but not of major histocompatibility complex class I, independent of their phosphorylation status.

Phosphorylation of membrane glycoproteins has often been invoked as a determinant of receptor internalization and receptor trafficking in a more general sense. Here we have studied the trafficking of major histocompatibility complex (MHC) Class I molecules and transferrin receptor (Tfr) related to their phosphorylation status in the human lymphoblastoid cell line JY. High resolution isoelectric focusing (IEF) allows the visualization of phosphorylated and non-phosphorylated protein species simultaneously, using protein backbone-labeling. Analysis on IEF was combined with a neuraminidase protection assay, in which sialic acid modification of the N-linked glycans present on Tfr and Class I molecules is used as a reporter group for cell surface expression. Phosphorylation of Class I heavy chains and Tfr was induced by exposure of cells to the phorbol ester tetradecanoyl phorbol acetate. We show that 1) phosphorylation of MHC Class I molecules is restricted to the cell surface fraction, 2) phosphorylation of MHC Class I molecules by protein kinase C (PKC) is not correlated with their internalization, as no internalization of Class I molecules, phosphorylated or non-phosphorylated, could be detected, 3) the initial rate, but not the final extent of the internalization of Tfr is affected by activation of PKC, and 4) phosphorylated Tfr behaves in a manner identical to non-phosphorylated Tfr in terms of internalization. The effect of activation of PKC on internalization of Tfr therefore most likely takes place at the level of the internalization machinery. Our data concerning the internalization of MHC Class I molecules contrast with earlier studies describing constitutive internalization in the B lymphoblastoid cell line A 46 and in HPB-ALL cells.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.     

Class I-restricted presentation occurs without internalization or processing of exogenous antigenic peptides

Previous studies have shown that glutaraldehyde-fixed cells can present fragmented, but not native, Ag to class II-restricted T cells. This presumably occurs via direct binding of peptides to class II molecules at the cell surface. More recently, it has been shown that viable target cells can present peptides and endogenous, but not exogenous, protein Ag in association with class I MHC molecules to CTL. We have derived CTL specific for a chicken OVA peptide (OVA258-276) recognized in association with H-2Kb. These CTL recognize target cells that endogenously synthesize OVA and cells "loaded" with native OVA but fail to recognize target cells in the presence of exogenous native OVA. Thus, OVA must be intracellularly located to be processed and presented for CTL recognition. It remains unclear, however, whether exogenous peptides require internalization and further processing by target cells or are able to associate directly with class I molecules at the cell surface for CTL recognition. We provide evidence that glutaraldehyde-fixed cells can present synthetic peptides to H-2Kb- and H-2Db-restricted CTL and that such presentation does not require internalization or processing. The peptides used range in size from 16 to 48 amino acids in length. In contrast, glutaraldehyde-fixed cells are incapable of presenting Ag to CTL specific for influenza nucleoprotein and OVA if the cells are fixed within 1 h of viral influenza infection or loading with OVA. Thus, CTL recognition of antigenic peptides appears to occur via direct binding of peptides to class I molecules at the cell surface and does not require any intracellular processing events.     

The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route

We studied the intracellular traffic and subcellular distribution of MHC class I and class II antigens in comparison with a recycling surface glycoprotein, the transferrin receptor (Tfr), in the human lymphoblastoid cell line JY. No internalization was detectable for class I molecules. Class II molecules were internalized but did not recycle. In contrast, Tfr was found to internalize and recycle. The biosynthetic pathway of class II molecules differ from that of class I molecules in that it shows a delay (1-3 hr) in transport from trans-Golgi to cell surface: here it intersects the endocytic route. Immunoelectron microscopy using anti-MHC antibodies revealed the existence of vesicular structures that were intensely labeled for class II molecules. It is proposed that at this site combination of class II molecules with processed antigen could occur.     

Physical association between MHC class I and class II molecules detected on the cell surface by flow cytometric energy transfer

The physical association of HLA class I and class II Ag in the membranes of PGF and JY lymphoblastoid cell lines was studied using flow cytometric energy transfer. This technique measures the proximity of cell surface molecules in the nm range and provides a distribution histogram of the average proximity of molecules on each cell of a population. HLA Ag were labeled with mAb conjugated to fluorescein, serving as donor, or tetramethylrhodamine, serving as acceptor molecules. Significant fluorescence energy transfer was detected between various combinations of class I and class II molecules indicating that these molecules are within 10 nanometers of each other. Specifically, energy transfer was observed between class I molecules and DR, DQ, or DP class II HLA molecules. In addition, energy transfer between all combinations of DR, DQ, and DP molecules was observed. No transfer was observed among class I molecules or among DR or among DP molecules. Among DQ molecules, subpopulations transferred fluorescence energy to each other. The close contact measured between class I and class II Ag correlates with previous reports of cocapping and may reflect an immunologically significant interaction or the reported tendency of class I Ag to associate with other cell surface receptors, including growth factor receptors. The energy transfer between fluorescent antibodies to class II Ag suggests the existence of heterodimers formed from the different locus products, as well as possible quaternary surface interactions between alpha/beta complexes from separate loci.     

Endocytosis of MHC molecules by B cell-B lymphoma and B cell-T lymphoma hybrids

Different cells and different cell surface determinants of the same cells take up liposomes, bound to them via monoclonal antibodies, with variable efficiency. We have previously reported that T and B lymphocytes differ in the extent to which they take up liposomes bound to MHC class I molecules; T cells endocytose class I molecules rapidly, but B cells endocytose class I molecules much less efficiently, although their endocytosis of class II molecules is rapid. An approach toward understanding the molecular basis for this difference was made by evaluating the internalization patterns of somatic cell hybrids of B and T cells. Hybrid cells were constructed between LPS-stimulated purified B cell blasts from C57BL/6 mice (H-2b) and the HAT-sensitive AKR T lymphoma BW 5147 (H-2k). Hybrids between the BALB/c B lymphoma M12.4.1 (H-2d) and B cell LPS blasts from B10.BR (H-2k) mice were also evaluated. In all cases, for hybrid tumor cells, liposomes that were bound to class I molecules encoded by genes from the B cell donor were endocytosed as efficiently as liposomes bound to the class I molecules of the recipient lymphoid cell. T cell tumors efficiently internalized their own class I molecules and those donated by B cells; B cell tumors internalized liposomes that were bound to their own and the donor B cell class I molecules poorly. Thus, our results suggest that the internalization of an MHC molecule is not an intrinsic property of the molecule, but rather of the cell in which it is found.     

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.     

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.     

Endocytosis and recycling of MHC-encoded class II molecules by mouse B lymphocytes

The movements of mouse MHC-encoded class II (H-2E) and class I (H-2K), transferrin receptor and surface Ig molecules of B lymphocytes were studied using radiolabeled mAb and electron microscopy. A total of 10 to 20% of antibodies specific for H-2E molecules were gradually internalized with a t 1/2 of 15 min, reaching a plateau after 30 min at 37 degrees C. Equivalent results were obtained either with the whole antibody or Fab' fragments, suggesting that the internalization of class II molecules was spontaneous. Similar results were obtained with antibodies specific for the transferrin receptor, of which 50% were internalized with t 1/2 of 5 min, reaching a plateau after 30 min. In contrast to antibodies specific for H-2E molecules and the transferrin receptor, antibodies specific for H-2K were not internalized. Reappearance of internalized H-2E-specific antibodies at the cell surface was observed at 37 degrees C. When compared to antibodies specific for surface Ig, degradation of antibodies specific for H-2E molecules was limited even after 5 h incubation. Neither ammonium chloride nor cycloheximide inhibited internalization and recycling. Electron microscopy showed that internalization of H-2E molecules occurred via coated pits/coated vesicles. These results indicate that class II molecules are spontaneously internalized and recycled by B lymphocytes.     

Fibronectin Receptor Internalization and AP-2 Complex Reorganization in Potassium-Depleted Fibroblasts

Potassium-depleted fibroblasts are unable to develop polarized morphology and lack coated pits. Experiments were carried out to measure internalization of fibronectin receptors (FNR) in potassium-depleted cells and possible association of FNR with AP-2 complexes after adding potassium back to the cells, which restores cell polarization. AP-2 complexes are the cell surface component of coated pits that contain both clathrin and membrane receptor binding domains. Potassium-depleted fibroblasts endocytosed antibody-tagged FNR and also internalized fluorescent fibronectin that previously had been adsorbed to the substratum. During cell polarization, antibody-tagged FNR reorganized into fibrillar structures along stress fibers beginning from nucleation sites at cell margins. Plasma membrane AP-2 complexes, which were undetectable in potassium-depleted cells, reappeared at the cell surface above the nucleus and then spread toward the cell margins. The results show that endocytosis of FNR can occur at least partially by a coated pit-independent mechanism.     

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr- 723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.     

The asialoglycoprotein receptor internalizes and recycles independently of the transferrin and insulin receptors

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface, followed by internalization of the complex into endocytic vesicles. We show that internalization of asialoglycoprotein by HepG2 hepatoma cells is accompanied by a rapid (t1/2 = 0.5-1 min) depletion of surface asialoglycoprotein receptors. This is followed by a rapid (t1/2 = 2-4 min) reappearance of surface receptors; most of these originate from endocytosed cell-surface receptors. The loss and reappearance of asialoglycoprotein receptors is specific, and depends on prebinding of ligand to its receptor. HepG2 cells also contain abundant receptors for both insulin and transferrin. Endocytosis of asialoglycoprotein and its receptor has no effect on the number of surface binding sites for transferrin or insulin. We conclude that binding of asialoglycoprotein to its surface receptor triggers a rapid and specific endocytosis of the receptor-ligand complex, probably due to a clustering in clathrin-coated pits or vesicles.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes.

Nef, a regulatory protein of human and simian immunodeficiency viruses, downregulates cell surface expression of both class I MHC and CD4 molecules in T cells by accelerating their endocytosis. Fibroblasts were used to study alterations in the traffic of class I MHC complexes induced by Nef. We found that Nef downregulates class I MHC complexes by a novel mechanism involving the accumulation of endocytosed class I MHC in the trans-Golgi, where it colocalizes with the adaptor protein-1 complex (AP-1). This effect of Nef on class I MHC traffic requires the SH3 domain-binding surface and a cluster of acidic amino acid residues in Nef, both of which are also required for Nef to downregulate class I MHC surface expression and to alter signal transduction in T cells. Downregulation of class I MHC complexes from the surface of T cells also requires a tyrosine residue in the cytoplasmic domain of the class I MHC heavy chain molecule. The requirement of the same surfaces of the Nef molecule for downregulation of surface class I MHC complexes in T cells and for their accumulation in the trans-Golgi of fibroblasts indicates that the two effects of Nef involve similar interactions with the host cell machinery and involve a molecular mechanism regulating class I MHC traffic that is common for both of these cell types. Interestingly, the downregulation of class I MHC does not require the ability of Nef to colocalize with the adaptor protein-2 complex (AP-2). We showed previously that the ability of Nef to colocalize with AP-2 correlates with the ability of Nef to downregulate CD4 expression. Our observations indicate that Nef downregulates class I MHC and CD4 surface expression via different interactions with the protein sorting machinery, and link the sorting and signal transduction machineries in the regulation of class I MHC surface expression by Nef.     

Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.     

CD4 and CD7 molecules as targets for drug delivery from antibody bearing liposomes.

We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation of d-[3H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4-targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly.     

Molecular cloning of bovine class I MHC cDNA

Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.     

Tumor necrosis factor-alpha up-regulates expression of secretory component, the epithelial receptor for polymeric Ig

Secretory component (SC) is the receptor that facilitates transcytosis of polymeric IgA and polymeric IgM through secretory epithelial cells and into exocrine fluids. The present study showed that rTNF-alpha enhanced the cellular pool, membrane expression, and secretion of functionally SC in a human colonic carcinoma cell line (HT-29m2) which is known to express and process SC like normal glandular cells. TNF-alpha also up-regulated membrane expression of the constitutive HLA class I molecules, whereas the cells remained HLA class II-negative.     

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.