Importance of the COOH terminal of angiotensin in antigenicity and in the formation of an antigen-containing complex with cellular membrane structures

To more carefully determine how a peptide antigen interacts with the antigen-presenting cell (APC), we have begun an analysis of the fate of APC-associated peptide antigens. These studies have shown that a stable cell-bound form of APC-associated peptide exists, which is a complex of the peptide with surface membrane structures (peak A). In the experiments described here, we have begun to examine the chemical mechanism of this peak A complex formation. By modifying either the carboxyl terminal or amino terminal group of the octapeptide antigen angiotensin II we have established that the terminal carboxyl group, but not the terminal amino group, was critical for forming the peak A complex with APC membrane structures. In addition, blocking the carboxyl but not the amino terminal dramatically reduced the antigenicity of the peptide for AII-immune T cell in vitro proliferation. These results show that the carboxyl terminal of AII is essential for both peak A formation and antigenicity, and suggest that peak A is critical for antigen presentation to T cells.     

In vivo treatment with anti-I-A antibodies: differential effects on Ia antigens and antigen-presenting cell function of spleen cells and epidermal Langerhans cells

The in vivo activation of T cells by a variety of antigens can be inhibited by the administration of anti-I-A antibodies (Ab) at the time of antigen priming. This inhibition can partially be explained by the temporary loss of Ia molecules from Ia-bearing antigen-presenting cells (APC) in the spleen. In this study, the effects of i.p. injected monoclonal Ab specific for I-A glycoproteins of different H-2 haplotypes on Ia antigen expression and APC function of spleen cells and epidermal Langerhans cells were compared. It was found that anti-I-A Ab quickly bound to both spleen cell and Langerhans cell Ia antigens. Although spleen cell Ia antigens were modulated and thus temporarily disappeared, Ia antigen expression by epidermal Langerhans cells was not modulated. In functional studies, the capacity of spleen cells and epidermal cells from anti-I-A Ab treated vs control animals to function as APC for antigen-specific, I-A- or I-E-restricted T cell clones was tested. A single injection of anti-I-A Ab completely abolished the APC function of spleen cells as shown in several inbred mouse strains, F1 animals, and with the use of several different Ab and T cell clones. In contrast, Langerhans cell-dependent APC function of epidermal cells remained completely unaltered. Even multiple injections of high doses of Ab never caused any inhibition of Langerhans cell function. Experiments with anti-I-Ak or anti-I-Ad Ab in an (H-2k X H-2d)F1 animal showed abrogation of APC function of spleen cells, but again not of Langerhans cells. Thus in vivo anti-I-A Ab administration appears to differentially affect Ia antigen expression and APC function from spleen and epidermis: Ia antigens are modulated from spleen cells but not from epidermis, and APC function disappears in the spleen but not in the epidermis. The abrogation of splenic but not of Langerhans cell APC function with anti-I-A Ab will facilitate the dissection of the relative contributions of Langerhans cells as compared with other APC in the generation of cutaneous immune responses.     

Comparison and partial characterization of guinea pig Ia alpha- and beta-chain oligosaccharides

The structures of the N-linked oligosaccharides of mature guinea pig Ia molecules were partially characterized by serial lectin affinity analysis. Those Ia antigens that are thought to be allelic products (Ia.3,5 and Ia.4,5) were found to bear identical oligosaccharides, whereas differences in glycopeptide distribution were found for Ia antigens known to be products of separate I subregions (Ia.2 and Ia.4,5). The two predominant oligosaccharides present on alpha-chains from all three Ia molecules were of the high mannnose type and the triantennary or tetraantennary complex type. Two structurally distinct beta-chains were isolated from Ia.3,5 and Ia.4,5 molecules; beta 1 bore primarily triantennary or tetraantennary complex oligosaccharides, and beta 2 had predominantly biantennary complex-type carbohydrate chains. The composition and distribution of the oligosaccharide moieties of guinea pig Ia molecules indicate that there are structural features shared among guinea pig, murine, and human Ia antigens.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

Murine class II (Ia) molecules associate with human invariant chain

Polymorphic class II (Ia) major histocompatibility complex (MHC) gene products associate intracytoplasmically with a third nonpolymorphic class II molecule, the invariant chain (Ii), which is encoded by gene(s) unlinked to the MHC. Although the role of the Ii chain in the expression of cell surface Ia molecules is unclear, it has been suggested that the Ii chain helps in the assembly and intracellular transport of class II antigens. In this study, we demonstrate that the murine polymorphic class II antigens of an interspecies mouse-human hybrid, which has segregated the murine invariant chain gene, associates with the human invariant chain gene intracytoplasmically. The murine Ia antigens are expressed on the cell surface and can function as restriction elements in antigen presentation to T cells. The biochemical analysis demonstrates that the regions of the Ii gene that are critical to its interaction with Ia molecules are conserved between species.     

Differential effects of monoclonal antibodies anti-L3T4 and anti-LFA1 on the antigen-induced proliferation of T-helper-cell clones: correlation between their susceptibility to inhibition and their affinity for antigen

Recognition by specific T helper (TH) cells of antigen presented by antigen-presenting cells (APC) involves, in addition to the antigen-specific receptor, non-antigen-specific molecules such as L3T4 and LFA1. In the present study, we analyzed the relationship between the avidity for antigen presented by APC of three TH cell lines and the participation of L3T4 and LFA1 cell surface antigens. We found a correlation between the avidity of TH cells for the complex GAT/Ia on APC measured by two independent assays and the participation of the cell-adhesion molecules L3T4 as measured by the ability of corresponding monoclonal antibody (MAb) to block the antigen-induced proliferation of TH cells. In contrast to the situation found with cytolytic T-lymphocyte (CTL) clones, we also found a differential inhibiting effect of anti-LFA1 MAb on the GAT-specific proliferation of the three TH clones. The results indicate a direct correlation between the inhibitory effects of anti-LFA1 and anti-L3T4 MAb and the affinity of TH cells for the complex formed by antigen and Ia.     

The syngeneic mixed leukocyte reaction: the genetic requirements for the recognition of self resemble the requirements for the recognition of antigen in association with self

We have used cells from inbred strain 2 and strain 13 guinea pigs in order to define further the role of Ia antigens in the syngeneic mixed leukocyte reaction (MLR). The guinea pig syngeneic MLR resembled the autologous MLR in man in that it demonstrated both memory and specificity. The Ia antigens appeared to be the proliferative stimuli in that the primary stimulator cell was an Ia-positive adherent peritoneal exudate cell (PEC) and the reaction could be specifically inhibited by anti-Ia sera directed to the stimulator cell. We also demonstrated the existence of two (2 x 13)F1 T cell populations that were capable of reacting to one or the other parental PEC in the absence of any known exogenous antigen. These results suggest that the syngeneic MLR may represent T cell activation mediated through a receptor for self Ia.     

Limited trypsin cleavage distinguishes MHC and thymus-leukemia antigens.

A simple technique is presented for the identification of particular cell membrane antigens. The method employs labeled membrane antigens that are isolated immunospecifically and subjected to limited trypsin digestion followed by polyacrylamide gel electrophoresis in detergent. A large "core" peptide is produced by proteolysis of murine thymus-leukemia antigens (TLA) and from antigens of the major histocompatibility complex (MHC). The tryptic cores from H-2K and H-2D are regularly distinguishable from the thymus-leukemia antigens (TLA) by gel electrophoresis in one dimension. This chemical distinction is particularly important in the analysis of antigen mixtures isolated with antisera specific for beta 2 microglobulin. These techniques have been used to identify thymus-restricted beta 2 microglobulin-associated antigens on cell membranes from mouse, man, guinea pig, and monkey. In appropriate inbred mouse strains, these are the TLA and it is proposed that in the three other species examined they may be analogues, although not necessarily homologues, of TLA. The broad species distribution of these thymus-restricted cell membrane antigens suggests that they are involved in the differentiation of thymus-dependent lymphocytes (T cells).     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

Antigenicity of carcinogen and viral induced sarcomas in inbred and random bred guinea pigs.

A tumor specific transplantation antigen (TSTA) has been detected in a methylcholanthrene (MCA) induced guinea pig tumor. It was possible to induce resistance to rechallenge with the tumor by immunization with irradiated cells in CFA. In contrast, the same technique failed to detect TSTA in two viral (Kirsten strain mouse sarcoma virus, Ki-MSV) induced guinea pig tumors; these results are similar to observations made with mouse Ki-MSV-induced tumors. Transplantation studies with these tumors in both inbred and random-bred guinea pigs showed a complexity of growth and rejection patterns. The B alloantigen, a major serologically defined antigen of the guinea pig histocompatibility complex, seemed to play a central role in acting as a guniea pig transplantation antigen. In all cases studied, the absence of B antigens in the recipient led to tumor rejection and anti-B antibody protection.     

In vivo treatment with monoclonal anti-I-A antibodies: disappearance of splenic antigen-presenting cell function concomitant with modulation of splenic cell surface I-A and I-E antigens

A single injection of anti-I-Ak antibody (AB) into H-2k mice resulted in abrogation of splenic antigen-presenting cell (APC) function for protein antigen-primed T cells or alloantigen-specific T cells. Spleen cells from anti-I-A-treated mice are not inhibitory in cell mixing experiments when using cloned antigen-specific T cells as indicator cells, thus excluding a role for suppressor cells in the observed defect. Also, nonspecific toxic effects and carry-over of blocking Ab were excluded as causes for the defect. Experiments with anti-I-Ak Ab in (H-2b X H-2k)F1 mice showed abrogation of APC function for T cells specific for both parental I-A haplotypes. In homozygous H-2k mice, anti-I-Ak treatment not only abrogated APC function for I-Ak-restricted cloned T cells but also for I-AekE alpha k-restricted cloned T cells. FACS analysis of spleen cells from anti-I-Ak-treated (H-2b X H-2k)F1 mice revealed the disappearance of all Ia antigens (both I-A and I-E determined), whereas the number of IgM-bearing cells was unaffected. The reappearance of APC function with time after injection was correlated with the reappearance of I-A and I-E antigen expression. In vitro incubation of spleen cells from anti-I-A-treated mice led to the reappearance of Ia antigen expression and APC function within 8 hr. Thus, it appears that B cells (as determined by FACS analysis) and APC (as determined by functional analysis) behave similarly in response to in vivo anti-I-A Ab treatment. We interpret these findings as suggesting that in vivo anti-I-A treatment temporarily reduces the expression of Ia molecules through co-modulation on all Ia-bearing spleen cells, thereby rendering them incompetent as APC. Such modulation of Ia molecules does not occur when spleen cells are incubated in vitro with anti-I-A antibodies. These results imply that a primary defect purely at the level of APC in anti-I-A-treated mice may be responsible for the observed T cell nonresponsiveness when such mice are subsequently primed with antigen.     

Guinea pig homologues of TL and QA-2 antigens.

The H-2, thymus-leukemia (TL), and Qa-2 antigens of mice are encoded by closely linked genes on murine chromosome 17, and have structural similiarity in that each antigen is borne on a approximately 44,000 dalton molecule associated with beta2 microglobulin (beta2mu). The extensive homology of major histocompatibility complex (MHC) products that exists for the mouse and guinea pig suggested that a similar homology might exist for products of genetic regions closely linked to the MHC. By taking advantage of the selective association of beta2mu with H-2, Qa-2, and TL antigens, and by using the technique of sequential immunoprecipitation, we demonstrated two previously undescribed guinea pig molecules reactive with anti-guinea pig beta2mu. The first molecule was composed of a 36,000 dalton glycoprotein associated with beta2mu and was found on guinea pig thymocytes, but not lymphocytes. The second molecule was composed of a 40,000 dalton glycoprotein associated with beta2mu, and was found on both guinea pig thymocytes and lymphocytes. By structure, chemical composition, association with beta2mu, and tissue distribution, the first molecule is an attractive candidate for the guinea pig homologue of TL antigen, whereas the second fits the criteria for the guinea pig homologue of Qa-2 antigen.     

Nature of T lymphocyte recognition of macrophage-associated antigens. IV. Inhibition of peptide antigen presentation by brief treatment of macrophages with anti-Ia serum

To examine the role of macrophage la antigens in T-lymphocyte stimulation, guinea pig macrophages were briefly treated with anti-Ia serum before or after antigen pulsing with the peptide antigen human fibrinopeptide B (hFPB). To assess their antigen-specific stimulatory capacity, the variously treated macrophages were added to culture with hFPB-immune guinea pig T cells and stimulation was determined by the incorporation of [³H]thymidine. Macrophages treated with anti-Ia serum before antigen pulsing stimulated T-cell responses equivalent to those observed with antigen-pulsed macrophages treated with normal serum. By contrast, brief anti-Ia treatment of macrophages immediately following a 2-hr antigen pulse reduced subsequent T-cell responses by 45 to 70%. Similar treatment of macrophages pulsed with antigen for only 1 hr produced only modest inhibition of T-cell responses. However, if macrophages pulsed for 1 hr with antigen were incubated several hours before brief anti-Ia serum treatment, the subsequent T-cell responses were reduced by 40 to 60%. This inhibition was specific for antiserum directed against Ia antigens of the guinea pig MHC, since brief macrophage treatment with antiserum directed against B.1 antigens, the guinea pig equivalent of murine H-2K and H-2D antigens, produced no inhibition of their T-cell stimulatory capacity. These results are discussed with respect to the formation of the immunogen presented by macrophages for T-cell recognition.     

The fine specificity of antigen and Ia determinant recognition by T cell hybridoma clones specific for pigeon cytochrome c

The activation of proliferative T lymphocytes normally involves the simultaneous recognition of a particular foreign antigen and a particular Ia molecule on the surface of antigen-presenting cells, the phenomenon of major histocompatibility complex (MHC) restriction. An analysis of T cell clones specific for pigeon cytochrome c, from B10.A and B10.S(9R) strains of mice, revealed the unusual finding that several of the clones could respond to antigen in association with Ia molecules from either strain. Using these cross-reactive clones, we performed experiments which demonstrated that both the Ia molecule and the T cell receptor contribute to the specificity of antigen recognition; however, MHC-linked low responsiveness to tuna cytochrome c (an immune response gene defect) could not be attributed solely to the efficacy with which the Ia molecules associated with the antigen. These results imply that antigen and Ia molecules are not recognized independently, but must interact at least during the process of T cell activation.     

Expression of ia antigens by murine keratinizing epithelial langerhans cells

Immunofluorescent and immunoelectron-microscopic staining methods were utilized to investigate the localization of Ia antigens in murine keratinizing epithelia. Approximately 3-5% of epidermal cells were shown to be Ia positive. Only dendritic Langerhans cells in the interfollicular epidermis and outer root sheaths were found to express Ia antigens. These Ia determinants were shown to be controlled by both theI- A andI- EC subregions of theH-2 complex. The results were confirmed by identifying positively stained cells containing Langerhans cell granules at the ultrastructural level. No staining was noted on the surface of keratinocytes, melanocytes, or immigrant lymphocytes. The results presented are in close agreement with those previously reported for Ia-bearing Langerhans cells in human and guinea pig epidermis.     

Assessment of roles for calreticulin in the cross-presentation of soluble and bead-associated antigens

Antigen cross-presentation involves the uptake and processing of exogenously derived antigens and their assembly with major histocompatibility complex (MHC) class I molecules. Antigen presenting cells (APC) load peptides derived from the exogenous antigens onto MHC class I molecules for presentation to CD8 T cells. Calreticulin has been suggested to mediate and enhance antigen cross-presentation of soluble and cell-derived antigens. In this study, we examined roles for calreticulin in cross-presentation of ovalbumin using a number of models. Our findings indicate that calreticulin does not enhance in vitro cross-presentation of an ovalbumin-derived peptide, or of fused or bead-associated ovalbumin. Additionally, in vivo, calreticulin fusion or co-conjugation does not enhance the efficiency of CD8 T cell activation by soluble or bead-associated ovalbumin either in wild type mice or in mice lacking Toll-like receptor 4 (TLR4). Furthermore, we detect no significant differences in cross-presentation efficiencies of glycosylated vs. non-glycosylated forms of ovalbumin. Together, these results point to the redundancies in pathways for uptake of soluble and bead-associated antigens.     

Alloreactive T cells from individual soft agar colonies specific for guinea pig Ia antigens. II. Cellular and molecular requirements for optimal proliferative responses

We have investigated the cellular and molecular requirement for optimal proliferative responses of several alloreactive T cell lines that were derived from individual soft agar colonies and were specific for guinea pig Ia antigens. Optimal proliferation of several colonies was observed in cultures containing purified allogeneic macrophages and growth factor(s) present in supernatant fluids of Con A-activated T cells (Con A-S). Significant proliferative responses of these alloreactive T cell colonies were also routinely detected in cultures only supplemented with unfractionated irradiated allogeneic peritoneal exudate cell (PEC). The T cell component of the stimulator cell population was crucial for these responses by producing necessary growth factor(s) endogenously in the culture. Thus, 2 signals, allogeneic Ia antigens and growth factor(s), were required for optimal proliferative responses of these alloreactive T cell colonies. Furthermore, macrophage-associated Ia antigen was more efficient than B cell-associated Ia for these responses. The requirement for allogeneic Ia antigen was not absolute, since the colonies could easily be expanded when the cultures were supplemented with irradiated syngeneic PEC and the T cell mitogens, Con A or PHA. The effect of the mitogen was mediated via the T cells in the irradiated PEC, since removal of the T cells from these PEC markedly reduced the responses. Thus, it is likely that a nonspecific signal(s) presumably from T cells can promote proliferation of alloreactive T cell colonies in the absence of allogeneic Ia antigen. These results suggest 2 mechanisms of activation of these alloreactive T cells.     

Structural identity of the TL antigen homologues derived from strain 2 and strain 13 guinea pigs

Strain 2 and strain 13 guinea pig thymocytes have been shown to bear a molecule that by several criteria appears to be a homologue of the murine TL antigen. The existence of a TL polymorphism in the mouse system as evidenced by TL- strains and various TL phenotypes in TL+ strains prompted a study to determine if a similar polymorphism could be demonstrated in the guinea pig system. By using two-dimensional gel electrophoresis, the thymocytes of a third inbred strain, DHCBA, were shown to bear a TL antigen, and the TL antigens of strains 2 and DHCBA were shown to give identical patterns of spots. A biochemical comparison of the strain 2 and strain 13 TL antigen heavy chains by tryptic and chymotryptic peptide mapping demonstrated that these molecules have identical peptides. Thus, no polymorphism could be demonstrated within the guinea pig TL system for the three inbred strains studied. Comparative tryptic peptide mapping of the guinea pig TL and class I B.1+S antigens demonstrated 43% homology, significantly higher than that reported for murine H-2 and TL antigens. These results provide suggestive evidence that the gene duplication giving rise to the genes determining the class I and TL antigens may have occurred more recently in the guinea pig than in the mouse.     

Factors affecting the presentation of exogenous hepatitis B virus core antigen

Hepatitis B virus core antigen (HBcAg) plays a critical role in terminating acute Hepatitis B virus infection and may be used as a potential vaccine candidate. The cell surface major histocompatibility complex (MHC) class 1 molecules are thought to be involved in the presentation of HBcAg. Surface MHC class 1 HLA A2 heavy chain (HC) and trimeric molecules were characterized on transfected Hela cells used as antigen presenting cells (APC) for the presentation of HBcAg. The results show that antibodies against HC HLA A2 and trimeric HLA-A2 molecules resulted in increased activation of HBcAg 18-27 minimal peptide specific cytotoxic T lymphocytes (CTLs), while the addition of exogenous beta2-microglobulin decreased the activation of HBcAg specific CTLs. Further, specific CD8+ T cells were activated only when Hela cells as APCs were primed with HBcAg (peptide, soluble or embedded on virosomes) at pH 6.5.