Alpha-galactosyl antibody redistributes alpha-galactosyl at the surface of pig blood and endothelial cells.

The interaction of antibodies with cell surface antigens may induce redistribution of immune complexes, followed by antigen depletion, with increased resistance to injurious effect of antibody and complement (antigenic modulation). Human natural antibodies to Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha Gal) epitopes expressed at the surface of pig cells are a major obstacle to xenotransplantation. Recent studies have shown that these antibodies do not modulate alpha Gal, but the morphological consequences of the antigen-antibody interaction are unknown. Pig blood and endothelial cells, were exposed to baboon alpha-Gal antibodies, and studied by immunofluorescence and phase contrast microscopy, flow cytometry, and inhibition enzyme-linked immunosorbent assay. In cells studied at 4 degrees C or fixed, alpha Gal was diffusely expressed at the surface. After cross-linking at 37 degrees C, antigenic modulation did not occur, but granular redistribution of alpha Gal immune complexes was seen in all cell types. In other systems a similar redistribution is known to induce perturbation of the plasma membrane/cytoskeletal structure with changes in adhesive properties, gene regulation, and T cell activation, which could be important if pig xenografts will be made to survive for prolonged periods.

Interaction of baboon anti-alpha-galactosyl antibody with pig tissues.

As barriers to xenotransplantation are surmounted, such as suppression of hyperacute rejection allowing improved graft survival, it becomes important to define longer-term host-xenograft interactions. To this end we have prepared in baboons high titer anti-alpha-Galactosyl (alphaGal) and anti-porcine aortic endothelial cell antibodies, similar to human natural xenoantibodies and reactive with epitopes of thyroglobulin, laminin, and heparan sulfate proteoglycans. When injected into pigs with a protocol similar to that used in the rat to show the nephritogenic potential of heterologous anti-laminin and anti-heparan sulfate proteoglycan antibodies, baboon immunoglobulins bound first to renal vascular endothelium, and later to interstitial cells, especially fibroblasts and macrophages, and to antigens in basement membranes and extracellular matrix, where they colocalized with laminin- and heparan sulfate proteoglycan-antibodies, and with bound Griffonia simplicifolia B4. A similar binding was observed in other organs. The pigs did not develop an acute complement-dependent inflammation, but rather chronic lesions of the basement membranes and the extracellular matrix. Incubation of renal fibroblasts with baboon anti-alpha-Galactosyl antibodies resulted in increased synthesis of transforming growth factor-beta and collagen, suggesting a possible basis for the fibrotic response. The results demonstrate that in this experimental model a consequence of alphaGal antibody interaction with porcine tissues, is immunoreactivity with alphaGal on matrix molecules and interstitial cells, priming mechanisms leading to fibrosis resembling that in chronic allograft rejection. The possibility that similar lesions may develop in long-surviving pig xenografts is discussed.

Staphylococcal enterotoxin B-induced T-cell anergy is mediated by regulatory T cells.

Naive T cells mount a vigorous proliferative response to superantigen (SAg) stimulation in vivo. The proliferative response is followed by a partial deletion of responder T cells. Part of the deletion process has recently been attributed to the action of regulatory cytotoxic T cells that recognize major histocompatibility complex (MHC) class I-associated antigen receptor determinants on the target cell surface. Responder T cells that survived the SAg response were found to be incapable of generating a secondary proliferative response to a SAg challenge. We show here that this 'anergy' is enforced by CD8-positive regulatory suppressive T cells. These regulatory cells inhibit cell division of preactivated T cells but not the Sag response of naive T cells. Regulatory T cells are not generated in the presence of cyclosporin A and, once activated, become inactivated or deleted when restimulated in the presence of this immunosuppressive drug.

T cell vaccination induces T cell receptor Vbeta-specific Qa-1-restricted regulatory CD8(+) T cells.

Vaccination of mice with activated autoantigen-reactive CD4(+) T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8(+) regulatory T cells that are specific for pathogenic CD4(+) T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8(+) T cells emerge that preferentially lyse and regulate activated autologous CD4(+) T cells in a T cell receptor (TCR) Vbeta-specific manner. This TCR Vbeta-specific regulation is not observed in beta2-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vbeta8-specific Qa-1-restricted CD8(+) T cells are also induced by TCV with activated CD4(+) Vbeta8(+) T cells. These CD8(+) T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vbeta8. Further, CD8(+) T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4(+)Vbeta8(+) T cell clone specifically recognize other CD4(+) T cells and T cell tumors that express Vbeta8 and the syngeneic Qa-1(a) but not the allogeneic Qa-1(b) molecule. Thus, Vbeta-specific Qa-1-restricted CD8(+) T cells are induced by activated CD4(+) T cells. We suggest that these CD8(+) T cells may function to specifically regulate activated CD4(+) T cells during immune responses.

Human CD8+ T lymphocyte clones specific for T cell receptor V beta families expressed on autologous CD4+ T cells.

CD8+ T cells control immune responses, and recent studies suggest that this regulation is, in part, specifically directed towards TCR structures expressed by CD4+ cells. To develop a system to study the role of the TCR in regulatory interactions, we isolated clones of CD4+ cells expressing identified TCR V beta chains. These CD4+ clones were used to stimulate and expand autologous CD8+ cells, which kill the inducing CD4+ clone as well as independently isolated autologous CD4+ clones sharing the same TCR V beta as the inducing cell but not CD4+ T cells expressing different V beta TCRs. This V beta-specific cytotoxicity is dependent on the state of activation of the target cells and is not inhibited by an anti-class I monoclonal antibody, W6/32. We envision that V beta-specific CD8+ T cells of this type may regulate immune responses by direct interaction with antigen-activated CD4+ cells.

Murine CD8+ T cells that specifically delete autologous CD4+ T cells expressing V beta 8 TCR: a role of the Qa-1 molecule.

Interactions mediated by TCRs expressed on different T cell subsets may play a role in immunoregulation. To investigate this idea, we studied the regulation of superantigen-induced TCR V beta-restricted responses. We asked whether the in vivo regulation of CD4+ V beta 8+ T cells following SEB injection is controlled by CD8+ T cells. We found that in mice deficient in CD8+ T cells, the down-regulation of CD4+ V beta 8+ T cells below baseline is not observed. Moreover, following SEB administration, CD8+ T cells emerge that preferentially kill subpopulations of activated CD4+ V beta 8+ but not CD4+ V beta 8- T cells in vitro. This TCR V beta-specific cytotoxicity is dependent on beta 2-microglobulin and is inhibited by antisera specific for Qa-1 but not by antibody to MHC class Ia. These data suggest the idea that the specificity of immune regulation may involve CD8+ T cell recognition of TCR V beta determinants and Qa-1 molecules expressed on CD4+ T cells.

Characteristics of the cytosol-synthesized proteins generating class-I-bound peptides.

The proteins synthesized in the cytosol are several thousand, and the number of peptides potentially able to be bound by class I molecules they can generate is therefore huge. On the other hand, the actual number of peptide-class I complexes required for CTL activation is around 200. We focused on the peptides bound by B27 molecules and by the whole class I. By comparing our results with analogous data from other laboratories, we found that 31 peptides matched protein sequences in data bases; in four cases, two peptides are derived from the same protein. The finding of four pairs of identical samples in a sampling of 31 peptides from a pool of unknown magnitude suggests that this pool is quite small. We have estimated the size of this pool by combinatorial analysis and by computer simulation, and we have found a most probable distribution of about 100 to the number of self-proteins that can actually generate peptides bound by class I molecules.

Cellular mechanisms of exogenous peptide binding to HLA class II molecules in B cells.

We have investigated the ability of APC Class II molecules to bind and release exogenous peptides, two phenomena that are still poorly understood. In order to investigate the half-life of the complex of an exogenous peptide with DR molecules we have evaluated the uptake and release of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein (MA 17-29-Y) by a B-EBV cell line at different times and under different conditions. We have found that the kinetics of both binding and release of the peptide are very fast in living cells; using glutaraldehyde-fixed cells, the kinetics of the two phenomena are slow, closely resembling those observed with the same peptide and purified, immobilized DR molecules. As confirmed by the study of a specific T-cell clone activation, the Class II-MA 17-29-Y complexes are short-living ones, with an average half-life of 55 min, and the DR molecules that bind exogenous peptides continuously undergo peptidic exchange. These data, taken together, suggest that the APC are endowed with cellular mechanisms that increase the efficiency of both the loading and the unloading of Class II HLA with exogenous peptides. These mechanisms do not appear to require ATP or to involve newly synthesized Class II molecules, intracellular acidic compartments, or the microtubule-microfilament system. On the other hand, an undamaged cell membrane appears to be crucial for an efficient binding.

Do anergic T cells induce suppressor T lymphocytes through idiotypic interactions?

Suppression by T cells and T cell anergy have been implied, at different periods of immunological research, as the main agents of peripheral down regulation of the immune response. This article discusses the possibility that anergic T cells, with the participation of appropriate co-stimulatory molecules on their membranes, stimulate CD8 cells with an alpha/beta TCR specific for peptides of the TCR of the anergic cell itself processed and presented by class I MHC. The non-anergic (orthoergic) members of the same clone, if activated, process and present their TCR in the same way, but, lacking the co-stimulatory molecule, are unable to stimulate the anti-idiotype CD8 cells. On the other hand the orthoergic, but not the anergic, cells can be induced into death (possibly by apoptosis) by the specific CD8 lymphocytes or, alternatively, can be pushed into the anergic pool by the same CD8 suppressors, thus contributing to the generation of a TCR-restricted circuit in which suppression is dominant. This simple immunosuppressive circuit can adequately explain some recent experiments on the course of experimental allergic encephalomyelitis. It is to be stressed that many elements of the proposal are hypothetical. They are, however, open to experimental study.

Role of CD8+ T cells in murine experimental allergic encephalomyelitis.

The course of experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis, is affected by immunoregulatory T lymphocytes. When animals are immunized with encephalitogenic peptide of myelin basic protein and recover from the first episode of EAE, they become resistant to a second induction of this disease. Animals depleted of CD8+ T cells by antibody-mediated clearance were used to examine the role of CD8+ T cells in EAE. These cells were found to be major participants in the resistance to a second induction of EAE but were not essential for spontaneous recovery from the first episode of the disease.

The major rheumatoid factor cross-reactive idiotype is dominantly expressed by Staphylococcus aureus Cowan strain I-activated CD5+ control B cells.

Some seropositive (RF+) and seronegative (RF-) rheumatoid arthritis (RA) patients selectively express high concentrations of the major RF cross-reactive idiotype (RCRI) in their sera and generate high frequencies of RCRI+ pokeweed mitogen (PWM)-induced plasma cells from their peripheral blood mononuclear cells (PBM). To determine if normal individuals can express RCRI in vitro, B cells from controls were activated with Staphylococcus aureus Cowan strain I (SAC) bacteria to identify RCRI and RF production. In addition, we studied the relationship of RCRI expression with the subset of B cells bearing CD5. Control CD5+ B cells are responsible for RCRI expression following SAC activation. We also observed that RCRI is dominantly expressed by control SAC-induced B cells in frequencies comparable to that expressed by some RA and juvenile rheumatoid arthritis patients' PBM activated by PWM. Therefore, the frequency of RCRI+ B cells in control and arthritis patients' PBL may be similar, or the selection and/or regulation of RCRI+ B-cell expression in vitro and in vivo may be different in arthritis patients compared to normal individuals.

Endocytosis of the TCR/CD3 complex and the class-I major histocompatibility complex in a human T cell line.

We investigated the expression of the T cell receptor (TCR)/CD3 complex on a CD4-positive human T cell lymphoma cell line treated with phorbol myristate acetate (PMA) and/or CA2+ ionophore using fluorescence flow cytometry and fluorescence microscopic analysis. PMA induced a significant decrease in the expression of the CD3 complex on the cell membranes. Fluorescence microscopy confirmed that the down regulation is due to internalization of the antigens. Ca2+ ionophore treatment had no effect on the internalization of the CD3 complex. Double staining revealed that the vesicles containing the internalized CD3 complex and those containing intra-cytoplasmic class I major histocompatibility complex antigen had similar distribution in the PMA-stimulated cells, implying coexistence of these two antigens in a cytoplasmic perinuclear distribution.

Cellular mechanisms of artificial peptides binding to HLA.

On the basis of the consideration that cell-free models cannot precisely mimic the complexity of the intracellular environment, we used a system to investigate the mechanisms that enable antigen-presenting cells (APC) to bind exogenous peptides through their human leukocyte antigen (HLA) molecules. We evaluated the uptake of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein by B-EBV cell lines, under various conditions. The results can be summarized as follows: a) the kinetics of peptide binding and release are very fast in living, fully competent cells; b) the peptide-HLA complexes are short-living and the DR molecules continuously undergo peptidic exchange; c) using glutaraldehyde-fixed cells, the kinetics of the two phenomena are slow, closely resembling those observed with the same peptide and purified, immobilized DR molecules. The data suggest that in APC, cellular mechanisms are operative that increase the efficiency of both loading and unloading of Class II HLA with exogenous peptides. This is likely to be related to the recycling of Class II molecules to intracellular compartments, were binding takes place. The observation that the HLA-peptide complex is a dynamic structure, suggests the possibility of replacing natural peptides with synthetic ones at this level, in order to regulate the immune response.

Endocytosis and dissociation of class I MHC molecules labeled with fluorescent beta-2 microglobulin.

Membrane class I MHC molecules of Con-A activated and lymphoma murine cells have been labeled by exchange of the cell's beta 2m with soluble fl-beta 2m. It has previously been shown that this method of labeling is specific and does not affect the biologic properties of class I MHC Ag. With this labeling it has been possible to demonstrate the constitutive endocytosis of class I MHC by fluorescence microscopy and by measuring the resistance to quenching by crystal violet of the internalized fl-beta 2m molecules. We could also follow the kinetics of beta 2m dissociation from the class I molecules at different pH. At pH 5.5, that is the average pH of endosomes, there is considerable dissociation within 15 to 20 min, that is the average recycling half time of class I MHC containing endosomes in activated T cells. Inasmuch as the process is reversible it is likely that, in the recycling endosomes of T cells, class I MHC molecules undergo conformational changes with beta 2m going off and on and with consequent changes of the peptide binding site. This process might be involved in Ag presentation, but, because it is apparently limited to T cells, it would play a role in the presentation of the cell's own TCR in idiotypic interactions between T cells.

Class II MHC molecules are spontaneously internalized in acidic endosomes by activated B cells.

The antibody response to protein antigens requires specific cooperation between B and T cells. In order to deliver the helper signal, T cells must recognize, in the context of Class II MHC, processed antigen on the membrane of B cells. Processed antigen is in the form of peptides bound in a given site of the Class II MHC molecule; in order to address the question of where, in the B cell, the complex of Class II MHC and processed antigen is formed, we studied the subcellular localization of these two molecules. Since the formation of this complex is the crucial step in antigen processing and presentation, the answer to this question is central to the whole problem of the physiology of antigen handling by B cells. To collect information pertinent to the question, we have compared, in B cells, the intracellular traffic of Class II MHC and of monovalent and divalent anti-immunoglobulin antibodies used as protein ligands of the membrane immunoglobulins. We have done so by two-color immunofluorescence microscopy, and we have detected extensive confluence of Class II MHC molecules with the immunoglobulin ligand, both mono- and bi-valent, in the endosomes of LPS-activated murine B cells. Whereas the ligand clearly reaches the endosomes by internalization from the cell membrane, the Class II MHC molecules could reach the same location either by endocytosis from the membrane or through targeting to the endosomes of newly synthesized Class II MHC molecules. We have collected quantitative evidence for endocytosis of Class II MHC by following, with the fluorescence activated cell sorter, the quenching of the fluorescence of fluoresceinated Fab' anti Class II MHC in LPS-activated murine B cells; this quenching indicates the entry of the label into an acidic intracellular compartment. Together with the results of others, obtained with different methods, our observations support the concept that, at least in mature activated B cells, Class II MHC molecules reach the organelles where they meet processed protein antigens, mainly through the endocytic route. Since activated B cells endocytose their membrane Class II MHC, and not their membrane Class I, our results contribute to the understanding of how B cells present antigens, that have bound to their membrane immunoglobulins, to Class II-restricted helper T cells and not to Class I-restricted cytolytic T cells.

Differential endocytosis of IgM and IgD in murine B cell lines.

The internalization of surface immunoglobulin (Ig) by B lymphocytes is the first step in the antigen-presenting function performed by these cells. Mature B cells coexpress on their surface IgM and IgD. At this time, there is controversy over whether these two isotypes serve different functions in the antigen-presenting process. The results presented here show that the intracellular pattern of distribution of IgM and IgD after internalization is strikingly different in the B cell lines studied. These findings support the hypothesis that the role of the two Ig classes in the antigen-presenting function may be different.