Abstract: MAJOR histocompatibility complex (MHC) class I molecules export peptides to the cell surface for surveillance by cytotoxic T lymphocytes1-3. Intracellular peptide binding is critical for the proper assembly and transport of class I molecules4–6. This mechanism is impaired as a result of a non-functional peptide supply factor gene (PSF) in several human mutant cell lines with genomic lesions in the MHC. We have now identified PSF in the MHC class II region by deletion mapping in mutants and chromosome-walking. PSF is homologous to mammalian and bacterial ATP-dependent transport proteins, suggesting that it operates in the intracellular transport of peptides.

Abstract: T lymphocyte activation requires recognition by the T cell of peptide fragments of foreign antigen bound to a self major histocompatibility complex (MHC) molecule. Genetic evidence suggests that part of the class II region of the MHC influences the expression, in trans, of MHC class I antigens on the cell surface, by regulating the availability of peptides that bind to and stabilize the class I molecule. Two closely related genes in this region, HAM1 and HAM2, were cloned and had sequence similarities to a superfamily of genes involved in the ATP-dependent transport of a variety of substrates across cell membranes. Thus, these MHC-linked transport protein genes may be involved in transporting antigen, or peptide fragments thereof, from the cytoplasm into a membrane-bounded compartment containing newly synthesized MHC molecules.

Abstract: WE describe a mutant human cell line (LBL 721.174)1,2 that has lost a function required for presentation of intracellular viral antigens with class I molecules of the major histocompatibility complex (MHC), but retains the capacity to present defined epitopes as extracellular peptides. The cell also has a defect in the assembly and expression of class I MHC molecules2,3,4, which we show can be restored by exposure of the cells to a peptide epitope. This phenotype suggests a defect in the association of intracellular antigen with class I molecules similar to that described for the murine mutant RMA-S (ref. 5), but in the present case the genetic defect can be mapped within the MHC locus on human chromosome 6.

Abstract: Models for antigen presentation have divided the world of antigens into two categories, endogenous and exogenous, presented to T cells by class I and class II major histocompatibility complex (MHC) encoded molecules, respectively. Exogenous antigens are though to be taken up into peripheral endosomal compartments where they are processed for binding to class II MHC molecules. Endogenous antigens are either synthesized or efficiently delivered to the cytoplasm before being partially degraded in an as yet undefined way, and complexed with class I MHC molecules. A useful phenotypic distinction between the two pathways has been the sensitivity to weak bases, such as chloroquine, which is a property only of the exogenous pathway. The fungal antibiotic brefeldin A (BFA), which blocks protein transport from the endoplasmic reticulum to the Golgi network, also blocks class I-restricted antigen-presentation, providing us with the corresponding marker of the endogenous pathway. Experiments with influenza virus antigens have supported the view that class II MHC molecules can present exogenous but not endogenous antigen, whereas the observation that class II MHC molecules present measles virus non-membrane antigens by a chloroquine-insensitive pathway suggests that this is not always the case. We show here that influenza A matrix protein can be effectively presented to class II-restricted T cells by two pathways: one of which is chloroquine-sensitive, BFA-insensitive, the other being chloroquine-insensitive and BFA-sensitive. Our results indicate that both class I and class II molecules can complex with antigenic peptides in a pre-Golgi compartment and favour a unified mechanism for MHC-restricted endogenous antigen presentation.

Abstract: Two prior studies with a small number of T cell lines have shown that the presentation of native protein antigens by epidermal Langerhans cells (LC) is regulated. When freshly isolated, LC are efficient antigen-presenting cells (APC), but after a period of culture LC are inefficient or even inactive. The deficit in culture seems to be a selective loss in antigen processing, since cultured LC are otherwise rich in major histocompatibility complex (MHC) class II products and are active APC for alloantigens and mitogens, which do not require processing. We have extended the analysis by studying presentation to bulk populations of primed lymph node and a T-T hybrid. Only freshly isolated LC can be pulsed with the protein antigens myoglobin and conalbumin, but once pulsed, antigen is retained in an immunogenic form for at least 2 d. The acquisition of antigen, presumably as MHC-peptide complexes, is inhibited if the fresh LC are exposed to foreign protein in the presence of chloroquine or cycloheximide. The latter, in contrast, improves the efficacy of antigen pulsing in anti-Ig-stimulated B blasts. In additional studies of mechanism, we noted that both fresh and cultured LC endocytose similar amounts of an antigen, rhodamineovalbumin, into perinuclear granules. However, freshly isolated LC synthesize high levels class II MHC molecules and express higher amounts of the class II-associated invariant chain. Fresh LC are at least 5-10 times more active than many other cells types in the level of biosynthesis of MHC class II products. These findings provide a physiologic model in which newly synthesized MHC class II molecules appear to be the principal vehicle for effective antigen processing by APC of the dendritic cell lineage. Another APC, the B lymphoblast, does not appear to require newly synthesized MHC class II molecules for presentation.

Abstract: Cytotoxic T lymphocytes (CTLs) recognize class I major histocompatibility complex (MHC) molecules associated with antigenic peptides derived from endogenously synthesized proteins. Binding to such peptides is a requirement for class I assembly in the endoplasmic reticulum (ER). A mutant human cell line, T2, assembles and transports to its surface some, but not all, class I MHC molecules. The class I molecules expressed on the surface of T2 do not present peptides derived from cytosolic antigens, although they can present exogenously added peptides to CTL. The transported class I molecules may interact weakly with an unknown retaining factor in the ER such that they can assemble despite the relative shortage of peptides.

Abstract: Jonathan W. Yewdell and Jack R. Bennink Laboratory of Viral Diseases National Institute of Allergy and Infectious Diseases Rockville, Maryland 20852 Two Types of Antigens Animals are confronted with numerous substances and organisms that threaten their existence. These may be present in extracellular body fluids (e.g., toxins, bacteria) or harbored in the animals’ own cells (e.g., viruses, on- cogene products). This distinction is critically important to thymus-derived lymphocytes (T cells), which constitute an essential component of immune responses to many agents. T cells have evolved parallel systems for recogniz- ing intracellular and extracellular antigens. In both sys- tems, antigens are recognized only when bound to mole- cules of the major histocompatibility complex (MHC). Two Antigen Binding Molecules The MHC encodes two types of cell surface molecules that act as receptors for protein antigens. Class I mole- cules consist of a highly polymorphic integral membrane glycoprotein a chain noncovalently bound to f3z-micro- globulin. Class II molecules consist of two noncovalently bound, highly polymorphic, integral membrane glycopro- teins. As with other cell surface proteins, MHC molecules are cotranslationally inserted into the endoplasmic reticu- lum (ER) and, following chain assembly, are transported to the plasma membrane via the Golgi complex and post- Golgi complex vesicles. Crystallographic analysis of the structure of class I mol- ecules reveals a groove at the top surface of the molecule formed by the two amino-terminal domains (Bjorkman et al., 1987). Functional studies strongly implicate the groove in antigen binding, and in the crystal structure the groove is indeed occupied by a ligand. Although the three-di- mensional structure of the class II molecule is not yet available, a similar antigen binding site can be modeled by aligning structural elements conserved between class I and II molecules (Brown et al., 1988). Two T Cells The recognition of class I versus class II molecules as an- tigen presenting ligands divides T cells into two classes, termed cytotoxic T cells (Tc) and helper T cells (TH), re- spectively. By directly lysing cells and secreting cytokines such as y-interferon, Tc eradicate intracellular pathogens and tumors. Although TH can also lyse cells, their pri- mary function is to secrete cytokines that promote the ac- tivities of B cells and other T cells. TH enhance antibody responses and thus help eradicate extracellular antigens susceptible to antibody-targeted immune mechanisms. The division of labor between Tc and TH correlates with the forms of antigen that are required for their activation. Whereas TH are activated by antigen presenting cells that have encountered exogenously added antigen, To generally require antigen presenting cells that are biosyn- thesizing antigen. The tissue distribution of MHC mole- cules is consistent with the different functions of Tc and TH in the immune response. Class I molecules are found on virtually all cell types. In contrast, class II molecules are constitutively expressed largely by cells of the im- mune lineage that either secrete cytokines necessary for TH stimulation (such as macrophages) or require TH cy- tokines for their own stimulation (B cells). Common Ground While differing widely in function, TH and Tc are similar with respect to the determinants they recognize and their interactions with antigen presenting cells that lead to acti- vation. Activation requires interaction between multiple T cell surface molecules and their respective ligands on an- tigen presenting cells. Specificity for foreign antigens is conferred by the

Abstract: The recognition of virus-infected cells by class I MHC-restricted cytotoxic T cells requires endogenous processing of antigen for presentation. It is still unclear whether endogenous processing of antigen can be utilized by class II MHC molecules for presentation. To test this possibility, a human B cell line expressing HLA-A2 and HLA-DR1 was infected with a recombinant vaccinia virus expressing the Influenza A virus M1 matrix protein (VAC-M1) and was assayed for lysis by different M1-specific cytolytic T cell lines, restricted by either HLA-A2 or by HLA-DR1. Class II-restricted lysis of VAC-M1-infected cells did occur. This lysis required de novo M1 synthesis and was not due to exogenous antigen. Several properties of the endogenous processing pathway for class II-restricted presentation were different from those of the pathway utilized by class I molecules. First, class II-mediated recognition of VAC-M1 infected cells was less efficient, requiring higher doses of virus and longer infection times, than the class I-mediated recognition. Second, chloroquine completely blocked presentation of endogenous M1 to class II-restricted T cells but had no effect on the class I-restricted presentation. Third, the class II-restricted presentation of M1 was only mildly affected by Brefeldin A, a drug that prevents transport from the endoplasmic reticulum to the Golgi, whereas the class I-restricted presentation of M1 was completely abrogated by this drug. These data demonstrate the existence of an endogenous processing pathway for the presentation of cytosolic antigen by class II molecules and show that this pathway is distinct from the one used for presentation by class I molecules.

Abstract: Freshly isolated epidermal Langerhans cells (LC) can actively process native protein antigens, but are weak in sensitizing helper T cells. During culture, when LC mature into potent immunostimulatory dendritic cells, T cell sensitizing capacity develops but antigen processing capacity is downregulated. Processing of exogenous antigens for class II-restricted antigen presentation involves acidic organelles. We used the DAMP-technique to monitor acidic organelles at the ultrastructural level in fresh, as well as cultured, mouse and human LC. We observed that the loss of antigen processing capacity with culture of LC was reflected by the disappearance of certain acidic organelles, namely endosomes (particularly early ones), and the hitherto enigmatic LC granules ("Birbeck Granules"). Our findings support the notion that endosomes are critical for antigen processing and suggest that LC granules might be involved as well.

Abstract: Cytotoxic T lymphocytes (CTLs) recognize foreign peptides bound to major histocompatibility complex (MHC) class I molecules. MHC molecules can also bind endogenous self peptides, to which T cells are tolerant. Normal mice contained CTLs specific for self peptides that were from proteins of ubiquitous or tissue-restricted expression. In vivo, these endogenous self peptides are not naturally presented in sufficient density by somatic cells expressing MHC class I molecules. They can, however, be presented if added exogenously. Thus, our data imply that CTLs are only tolerant of those endogenous self peptide sequences that are presented by MHC class I-positive cells in a physiological manner.

Abstract: Recent experiments have implicated intracellular events in the formation of the MHC class II-peptide complexes recognized by CD4-positive T cells. These data raise the possibility that the intracellular association of class II with the non-polymorphic glycoprotein, invariant chain (Ii), may regulate the interaction between processed antigen and MHC class II molecules. To address this possibility, we have generated a series of transfected fibroblast cell lines that express class II with and without Ii. Although the presence of Ii does not seem to affect the ability of the cells to process and present intact antigen, Ii-negative cells express an altered form of class II at the cell surface. This modified conformation of class II in Ii-negative cells is detectable by an increase in the ability to present antigenic peptides to T cells and a decrease in the binding of several class II-specific monoclonal antibodies.

Abstract: The effect of pH on functional association of peptide antigens with APC membranes was investigated by using aldehyde-fixed B cells and class II-restricted T cell hybridomas to assess antigen/MHC complex formation. The results indicated that the rate and extent of functional peptide binding was markedly increased at pH 5.0 as compared with pH 7.3. The pH dependence of binding was preserved after pretreatment of fixed APC with pH 5.0 buffer, suggesting that pH had a direct effect on the interaction of peptide with the APC membrane. Similar results were obtained by using several peptides and I-Ad- and I-Ed-restricted T cells, indicating that pH may be of general importance in regulating the formation of functional antigen/class II MHC complexes.

Abstract: The general thrust of this volume is to review the roles of accessory cells in regulating T and B lymphocytes To that end, we have summarized the evidence that indicates the crucial role that Langerhans cells play in the induction and expression of immunity to antigens that gain access to, or arise within, skin Langerhans cells accomplish this important goal by their abilities to (a) activate naive T cells to antigens not previously encountered by the host, and (b) activate memory/effector T cells specific for previously encountered antigens Arguments have been advanced to support the view that the functional properties of Langerhans cells used to present antigens to naive T cells differ substantially from the properties that equip Langerhans cells to activate effector T cells The arguments are based in part on the fact that Langerhans cells carry out these functions in two very different environments: in the epidermis, and in the draining lymph node The arguments are also based on results of in vitro experiments that reveal distinct differences in antigen processing and presenting properties of Langerhans cells freshly obtained from mouse and human skin as compared to Langerhans cells that have been cultured in vitro for 2-3 days We propose that freshly explanted Langerhans cells faithfully reflect the functional program of intraepidermal Langerhans cells, and are able to present antigen to memory/effector T cells that enter the epidermal compartment To accomplish this task, epidermal LC pick up environmental antigens, process them with great efficiency, and then present them in situ, without further upregulation of "accessory" signals (cell-adhesion molecules, secretion of additional cytokines) They can carry out this function, even in the presence of TGFB--a a cytokine which is constitutively made by keratinocytes, and which we have found to profoundly inhibit antigen presentation by most other types of "professional" antigen-presenting cells Intraepidermal Langerhans cells are also capable of carrying cutaneous antigens through the dermal epidermal junction and migrating to the draining lymph node We further propose that cultured Langerhans cells are fated to present antigens to unprimed/naive T cells, and thereby to initiate immune responses to new cutaneous antigens Cultured LC process antigens less efficiently than fresh cells, but their unique capacity to present antigen effectively to unprimed T cells rests chiefly on the fact that they have significantly upregulated cell surface adhesion molecules, expression of MHC molecules, and secretion of activating cytokines--the "accessory" signals that are required for arousing naive T cells(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract: Immune responses to proteins necessarily involve the recognition by T lymphocytes of a peptide or peptides derived from a protein complexed with a major histocompatibility antigen. The T-cell response of BALB/c mice to the bacteriophage lambda cI repressor protein (residues 1-102) is directed predominantly towards the epitope contained within a single peptide encompassing residues 12-26. Similar phenomena of immunodominance of a particular peptide have also been observed in other protein systems. The mechanisms that have been suggested to account for the focusing of the T-cell response are partial deletion in the T-cell repertoire, biased antigen processing, and competition for binding to the presenting molecule, the major histocompatibility complex encoded class II transplantation antigen. In a model system with a polypeptide containing two synthetically linked immunologically active epitopes, we now demonstrate the existence of a hierarchy between these epitopes, so that the immune response elicited is directed mainly towards the more immunogenic epitope, whereas the less immunogenic epitope elicits little or no T-cell reactivity. In addition, the same hierarchy of dominance is also apparent when the polypeptide is used to induce tolerance in the periphery in adult mice. The chimaeric peptide can induce tolerance only towards the more immunogenic epitope. These experiments indicate that the rules governing antigen processing and presentation that result in T-cell activation are apparently the same as the rules that govern the processes resulting in the induction of tolerance.

Abstract: Peptides bound to class I or class II major histocompatibility complex (MHC)-encoded molecules are ligands for the antigen-specific T-cell receptor of T-cells carrying the CD8 and CD4 antigens, respectively. MHC class I-restricted T cells generally recognize peptides derived from processing of endogenously synthesized cellular antigens, whereas class II-restricted T cells usually recognize peptides derived from exogenous antigens entering antigen presenting cells. Accordingly, two separate pathways of antigen processing and presentation have been proposed. The fungal metabolite brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum, inhibits presentation of endogenous antigens for MHC-restricted T-cell recognition. The selectivity of BFA activity has been inferred to reflect presentation of a given antigen processed through the cytosolic or the endocytic route. Here we show that BFA also greatly inhibits the presentation of exogenous protein antigens by MHC class II molecules to T cells, indicating a broader effect of this drug on antigen presentation and an additional similarity between the two processing pathways. As cycloheximide, a protein synthesis inhibitor, also inhibits presentation of protein antigens to class II-restricted T cells, the data indicate that peptides generated by processing of exogenous proteins binds to newly synthesized class II molecules for presentation to T cells.

Abstract: The in vitro polyclonal proliferative responses of peripheral blood mononuclear cells to whole blood stage parasites or fractionated antigens from the human malaria parasite Plasmodium falciparum were studied. Cells from healthy laboratory donors who had never been exposed to malaria antigens in vivo consistently proliferated to P. falciparum antigens, as did cord blood mononuclear cells. This response was only observed in sheep rosette-positive cells in the presence of adherent cells and was inhibited by NH4Cl, indicating a requirement for antigen processing. The proliferative response was strongest at day 6 and was dependent on the presence of cells expressing high levels of CD45 180-kD isomer (UCHL1 monoclonal antibody), a marker for activated or memory cells, but not for CD45R (SN130 monoclonal antibody) a marker for naive or unprimed T cells. This suggests a similarity to the recall response to tuberculin antigen. These results suggest that the proliferative response to malaria antigens observed previously and described as a nonspecific mitogenic response may be a cross-reactive response to epitopes shared between P. falciparum and other common immunogens. This would explain the establishment of T cell clones to malaria antigens from such donors, but might suggest that the epitopes to which such clones are specific may be of questionable protective or diagnostic use.

Abstract: Epithelial adaptations for antigen uptake and lymphoid organ cytoarchitecture differ according to the characteristics of specific sites throughout the mucosa but all facilitate antigen uptake. Whether surrounding mucosa is stratified squamous epithelium as in the oropharynx or columnar epithelium as in the intestine, organized lymphoid tissue in each area displays surface specializations which reduce local barriers and facilitate the approach and uptake of microorganisms, particles and macromolecules. In lymphoid tissue in each of these sites cellular defense mechanisms are arrayed for local containment, antigen processing and initiation of immune responses.

Abstract: in wvo. Marc H.V. van Regenmortel has expressed the feelings of many immunologists - that the science of immunology is replete with inductive inferences that are far from deduc- tively certain 1. 1he author referred to the philosopher of science, D.L. Hull, for his theorem "Science is not just the making of observations: it is the making of inferences on the basis of observation.: within the kamework of a theory. -2. The above opirion seems to be particularly apt for ~mmunolJgical research on antigen processing and its presentation to T cells. ,, ,e list of cell types that have this capability in

Abstract: The presentation of protein Ag with MHC class II proteins involves the uptake of the protein Ag by endocytosis followed by processing, probably proteolysis, in an intracellular acidic compartment However, there remains considerable controversy as to the precise route taken by the antigen and the MHC class II protein during this process The unusual stability of Ag-MHC class II protein complexes has led to speculation that antigen can only associate with newly synthesized MHC class II molecules An alternate possibility is that the MHC class II binding site can be regenerated within the cell during internalization and recycling of MHC class II proteins To address these possibilities, three different murine B lymphoma lines were tested for their ability to process and present native protein Ag in the presence of the protein synthesis inhibitor cycloheximide or the protein synthesis inhibitor cycloheximide or the protein export inhibitor, Brefeldin A Both agents blocked the presentation of native OVA or native hen egg lysozyme to Ag-specific T cell hybridomas No effect was seen on peptide presentation or on presentation to allo- or autoreactive T cells Inasmuch as Brefeldin A has been previously shown to block protein export without affecting protein internalization or protein degradation in the endocytic pathway, the simplest interpretation of these data is that antigenic fragments generated in the APC after uptake by the endocytic pathway, preferentially associate with newly synthesized rather than mature MHC class II proteins

Abstract: We have generated an alloreactive proliferative T cell clone that only is stimulated by HLA-DPw3+ antigen presenting cells (APC) that at the same time carry HLA-A1. The T cell clone is CD4+, and the proliferation is blocked by anti-DP monoclonal antibodies and not by antibodies towards other class II or towards class I molecules. Family studies show that APC with A1 and DPw3 on different haplotypes (trans) are able to stimulate the clone, and an HLA recombinant family gives evidence that the class I-carrying part of the haplotype is necessary for stimulation to occur. Stimulation is also observed with mixtures of APC expressing DPw3 and APC expressing A1, and likewise, DPw3+ APC become stimulatory when preincubated with supernatants from A1-positive cells. Our studies suggest that major histocompatibility complex (MHC) class I peptides presented by class II are allostimulatory and that APC can process MHC molecules that presumably are presented as allele-specific peptides in the context of other MHC molecules. We hypothesize that presentation of MHC peptides by MHC molecules constitutes an important part of alloreactive phenomena in vivo and in vitro.

Abstract: The immune response to antigens within the anterior chamber is deviant (anterior chamber associated immune deviation - ACAID) in that delayed hypersensitivity is deficient, whereas other immune effector modalities are intact. Experimental evidence indicates that the eye itself is critical to the induction of ACAID. We have examined the antigen processing and presenting potential of cells within the anterior segment of the eye, and have analyzed the potential immunoregulatory properties of these cells, their secretory products, and the aqueous humor itself. Evidence indicates that bone marrow-derived cells within the stroma of the iris and ciliary body inhibit antigen-driven T lymphocyte activation, although they themselves lack the capacity to present antigens to T lymphocytes. The mechanism is in part through secretion of immunosuppressive cytokines. Since aqueous humor contains similar cytokines, it is inferred that these molecules are constitutively secreted. We have determined that a major inhibitory molecule within normal aqueous humor is transforming growth factor-beta (TGFB), which inhibits antigen processing and presentation, and suppresses both T lymphocyte activation and certain aspects of non-specific inflammation. These effects also turn out to be properties of normal aqueous humor. These findings support the hypothesis that local features of the eye modify intraocular antigens such that an ACAID-inducing signal is produced. Experimental evidence suggests that these same properties may play a major role in suppressing efferent immune responses in the eye.