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P Marrack

Publications and source records attributed to P Marrack.

At least 73 records · Page 4Linked to original sources
PubMed1994

Binding of a soluble alpha beta T-cell receptor to superantigen/major histocompatibility complex ligands.

The genes for the alpha and beta chains of a murine T-cell receptor were truncated just prior to the portions encoding the transmembrane regions and introduced into baculovirus by recombination. Insect cells infected with the virus secreted a soluble form of the receptor that could be purified to homogeneity. This soluble receptor reacted with a set of six monoclonal antibodies originally raised to different epitopes on the natural transmembrane-region-containing receptor and bound with appropriate specificity to a cell surface complex of the human major histocompatibility complex class II molecule DR1 with the bacterial superantigen staphylococcal enterotoxin B.

Amino Acid Sequence↗
PubMed1994

Monoclonal antibodies defining functional sites on the toxin superantigen staphylococcal enterotoxin B.

Four monoclonal antibodies (mAbs) were produced binding to four nonoverlapping epitopes on the superantigen staphylococcal enterotoxin B (SEB). The mAbs were tested for their ability to detect SEB bound to major histocompatibility complex (MHC) class II, to inhibit SEB binding to MHC class II, to inhibit SEB stimulation of T cell hybridomas, to bind to various nonfunctional mutants of SEB, and to capture and present SEB and its mutants to T cells in the absence of MHC class II. We concluded that two mAbs, B344 and B327, bound to epitopes not required for superantigen function, one mAb, 2B33, blocked an MHC interaction site on SEB, and the fourth mAb, B87, blocked the T cell recognition site on SEB. Moreover, two mAbs (B344 and 2B33) were capable of presenting SEB, although much less efficiently than APC, to CD4- but not CD4+ T cell hybridomas. The results confirm the functional domains on SEB originally defined by mutation and show that MHC class II is not always an essential component of the superantigen ligand.

Animals↗
PubMed1994

Production of soluble MHC class II proteins with covalently bound single peptides.

The alpha beta T-cell receptors (TCRs) react with complex ligands composed of peptides bound to major histocompatibility complex (MHC) proteins. In the absence of foreign antigens the peptides bound to MHC molecules come from the proteins of the host itself. Interactions between TCRs and these self-peptide-MHC ligands work positively to drive T-cell development in the thymus and negatively to delete or inactivate T cells with potential self-reactivity. On the cell surface, MHC proteins are associated with many different self peptides, making it impossible to know which self peptide was involved in positive or negative interactions with a particular T cell. These studies as well as in vitro studies on TCR-peptide-MHC interactions would be aided by a means of producing MHC molecules containing a single peptide. We have tackled this problem for MHC class II proteins by genetically attaching the peptide by a flexible peptide linker to the amino terminus of the class II beta-chain. Here we report that a secreted, soluble form of this covalent peptide-MHC complex can be expressed in insect cells. The peptide is engaged by the peptide-binding groove of the secreted MHC molecule and this complex is recognized by T cells bearing receptors specific for that combination.

Amino Acid Sequence↗
PubMed1994

Genetic analysis of low V beta 3 expression in humans.

While studying the T cell receptor (TCR) repertoire of normal individuals, we found that more than 20% of adults have low levels of circulating V beta 3.1+ T cells in both CD4 and CD8 populations. A similar frequency was found in fetal cord blood samples, suggesting that in most cases, the V beta 3.1low phenotype is inherited. In support of this conclusion, children expressing low levels were only found in families where one of the parents expressed this phenotype. In two large families, genetic studies showed that low expression was a recessive trait and dependent on inheritance of particular TCR VB gene complexes. Family members with the low phenotype, however, expressed VB3.1 genes with normal sequences and expressed normal levels of receptor per cell. Results from these families suggest that up to 50% of normal individuals may carry a VB3.1 allele that is defective in its ability to rearrange effectively. In another large family, low expression in one individual was shown not to be determined by genes within the TCR VB gene or major histocompatibility complexes, suggesting a different mechanism for low V beta 3.1+ T cells. Overall, our results describe novel mechanisms that result in low levels of V beta 3.1+ T cells in a relatively large subset of the normal human population.

Adult↗
PubMed1994

Stimulation with specific antigen can block superantigen-mediated deletion of T cells in vivo.

The T-cell response to pigeon cytochrome c peptide, residues 88-104 (pcytC), in B10.BR mice is mediated largely by cells bearing both V beta 3 and V alpha 11 variable regions of the T-cell antigen receptor. These cells are, therefore, reactive with the superantigen staphylococcal enterotoxin A (SEA). Recent reports have shown that in vivo exposure to superantigen can lead to deletion of superantigen-reactive T cells from the pool of mature T cells in the periphery. Here we show that upon cotreatment of animals with both SEA and pcytC, bulk deletion of the population of SEA-reactive cells is maintained, while the subpopulation of SEA-reactive T cells that also responds to pcytC is not deleted but instead proliferates in response to pcytC. These results are discussed with regard to mechanisms regulating the balance between T-cell tolerance and T-cell activation in vivo.

Amino Acid Sequence↗
PubMed1994

Identification of two V beta 7-specific viral superantigens.

The commonly used strains of laboratory mice have mouse mammary tumor viruses (MTV) integrated at various locations in their DNA. The number and position of these integrants varies from one strain of mouse to another. It has recently been shown that the genomes of many of the MTV code for superantigens. The predicted amino acid sequences of these superantigens and their specificity for TCR V beta differs for each MTV integrant. This study contains the predicted amino acid sequence and V beta specificity of two MTV superantigens that had not previously been analyzed. The results show that both of these MTV superantigens are specific for TCR that bear V beta 7, but unlike the MTV7 superantigen not for receptors bearing V beta 6 or V beta 8.1. The data also support the conclusion of previous studies that the COOH-terminal sequence of these proteins is a major factor in controlling their V beta reactivity.

Amino Acid Sequence↗
PubMed1994

Processing and major histocompatibility complex binding of the MTV7 superantigen.

Mouse mammary tumor viruses produce superantigens (vSAGs) which interact with class II major histocompatibility complex (MHC) proteins and stimulate T cells. vSAGs are synthesized as Type II membrane proteins, but at least one of these proteins (vSAG7) is found on the cell surface in a proteolytically processed form. Monoclonal antibodies (MAbs) were used to characterize vSAG7 and its binding to class II molecules. vSAG7 is synthesized in the endoplasmic reticulum (ER) as a 45 kd glycoprotein containing N-asparagine-linked oligomannosyl carbohydrates. vSAG7 transits the golgi complex, where it is modified by the addition of complex-type glycans and proteolysed at three positions. After proteolysis, the amino and carboxyl termini remain noncovalently associated. The ER, golgi, and surface forms of vSAG7 are stably bound to class II, but one of the proteolysed forms comprises the majority of the class II-bound material.

Amino Acid Sequence↗
PubMed1993

Unexpected expansions of CD8-bearing cells in old mice.

As mice age, spontaneous changes occur in the receptor repertoire of their T cells. The receptor repertoire of CD4+ T cells does not change with age. By contrast, however, the percentage of alpha beta+, CD8+ T cells bearing particular V elements varies considerably between individual aged mice, although it is remarkably consistent among individual young animals within a given strain. Changes of receptor V element use among CD8+ T cells in individual mice are unpredictable. However, when a large number of mice of the same strain is analyzed, strain-specific trends in V element skewing are found. Old C3H.SW and B10.BR mice have mono- or oligoclonal expansions of CD8+ T cells. These expansions of peripheral CD8+ T cells with age are probably due to deregulation of proliferation of individual CD8+ T cells after recognition of viral or environmental Ag, accompanied, perhaps, by partial transformation of particular T cell clones. Another phenomenon documented herein is the fact that the CD4/CD8 ratio drops steadily as a function of age. Shifts in CD4/CD8 ratio were not due to increased numbers of CD8+ T cells in spleen and lymph nodes, rather the CD4+ T cells disappeared from aging mice faster than CD8+ T cells.

Aging↗
PubMed1993

Comparison of peptides bound to spleen and thymus class II.

In the past we and others have suggested that positive selection of developing thymocytes may depend upon interaction between the alpha beta receptors on these cells and major histocompatibility complex (MHC) proteins bound to peptides found uniquely in the selecting tissue, thymus cortical epithelium. To test this hypothesis, peptides were isolated from MHC class II proteins of spleen, thymus cortical plus medullary epithelium, or thymus cortical epithelium alone. The results showed that the major peptides bound to class II on thymus cortical epithelium were also associated with spleen class II. Some peptides could only be detected in isolates from spleen, probably because of differences in the distribution or uptake of the donor proteins between spleen and thymus. Thus, although we found some tissue-specific distribution of self-peptides, our data suggest that there are no fundamental differences among these tissues in the occupancy of class II MHC by self-peptides. These results limit hypotheses which depend on a specialized mechanism of peptide generation and/or MHC class II loading to account for the positive selection of T cells on thymic cortical epithelium.

Amino Acid Sequence↗
PubMed1993

Fibroblasts can induce thymocyte positive selection in vivo.

During development in the thymus, thymocytes bearing alpha beta T-cell receptors are selected to mature if the receptors they bear are able to interact in some way with major histocompatibility complex (MHC) proteins expressed on thymic stromal cells. It has been shown that thymus cortical epithelial cells are usually the cells presenting the MHC molecules involved in this process of so-called positive selection. Here we tested the ability of fibroblasts to mediate positive selection in vivo. Fibroblasts transfected with the genes for the MHC I-Ab proteins were injected intrathymically into irradiated H-2k animals reconstituted with H-2bxk F1 fetal liver cells. Eight weeks later, the recipient mice were immunized and shown to contain peptide-specific I-Ab-restricted T cells. This demonstrates the ability of I-Ab-transfected fibroblasts to participate in positive selection. Thus a cell type that is not specialized to process and present antigens in the context of MHC class II molecules can mediate positive selection when transfected with an appropriate MHC molecule. The data also support the idea that the ability to mediate positive selection may not be limited to thymic cortical epithelium.

Animals↗
PubMed1993

Antigen recognition properties of mutant V beta 3+ T cell receptors are consistent with an immunoglobulin-like structure for the receptor.

We examined the effect of mutations in the V beta portion of a pigeon cytochrome c (cyto c)-specific V beta 3+/V alpha 11+ T cell receptor on its ability to recognize cyto c/IEk and various superantigens. The results were consistent with an immunoglobulin-like structure for the receptor V beta domain and with separate interaction sites on V beta for conventional antigen and superantigens. An amino acid predicted to lie in CDR1 was critical for cyto c/IEk but not superantigen recognition, while several amino acids predicted to lie in the hypervariable region 4 loop were critical for superantigen but not cyto c/IEk recognition.

Amino Acid Sequence↗
PubMed1993

Superantigens and their potential role in human disease.

In the past few years, there has been a virtual explosion of information on the viral and bacterial molecules now known as superantigens. Some structures have been defined and the mechanism by which they interact with MHC class II and the V beta region of the T cell receptor is being clarified. Data are accumulating regarding the importance of virally encoded superantigens in infectivity, viral replication, and the life cycle of the virus. In the case of MMTV, evidence also suggests that superantigens encoded by a provirus may be maintained by the host to protect against future exogenous MMTV infection. Experiments in animals have also begun to elucidate the dramatic and variable effects of superantigens on responding T cells and other immune processes. Finally, the role of superantigens in certain human diseases such as toxic shock syndrome, some autoimmune diseases like Kawasaki syndrome, and perhaps some immunodeficiency disease such as that secondary to HIV infection is being addressed and mechanisms are being defined. Still, numerous important questions remain. For example, it is not clear how superantigens with such different structures, for example, SEB, TSST-1, and MMTV vSAG, can interact with MHC and a similar region of the TCR in such basically similar ways. It remains to be determined whether there are human equivalents of the endogenous murine MMTV superantigens. The functional role of bacterial superantigens also remains to be explained. Serious infection and serious consequences from toxin-producing bacteria are relatively rare events, and it is questionable whether such events are involved in the selection pressure to maintain production of a functional superantigen. Hypotheses to explain these molecules, which can differ greatly in structure, include T cell stimulation-mediated suppression of host responses or enhancement of environments for bacterial growth and replication, but substantiating data for these ideas are mostly absent. It also seems likely that only the tip of the iceberg has been uncovered in terms of the role of superantigens in human disease. Unlike toxic shock syndrome, other associations, especially with viral superantigens, may be quite subtle and defined only after considerable effort. The definition of these molecules and mechanisms of disease may result in new therapeutic strategies. Finally, it is apparent that superantigens have dramatic effects on the immune system. One wonders whether these molecules or modifications of them can be used as specific modulators of the immune system to treat disease.

Amino Acid Sequence↗
PubMed1993

The bacterial and mouse mammary tumor virus superantigens; two different families of proteins with the same functions.

In conclusion, the bacterial toxins are completely unlike the MTV superantigens in primary sequence and structure. The former are soluble globular proteins which do not have to be proteolytically cleaved before they act. The latter are synthesized as type II membrane proteins and may be clipped before they reach the cell surface and act to stimulate T cells. Table III summarizes the similarities and differences between the two sets of superantigens. The most notable quality of these molecules is that both sets of families have developed strategies whereby they bind to Class II and engage V beta. As far as the microorganisms which produce them are concerned, these two properties appear to be essential since they are absolutely conserved over proteins of a number of different structures. Several questions can now be addressed as follows. a. Why do all known superantigens bind to Class II? For the microorganism which produces them, the function of superantigens appears to be T-cell and perhaps directly or indirectly B-cell and macrophage stimulation. Activation of virgin T cells requires engagement with antigen plus MHC on professional antigen-presenting cells. Unlike other cell surface proteins, for example Class I, most Class II in animals is expressed on such cells. Therefore it is likely that superantigens have evolved to engage Class II because presentation to T cells by Class II-bearing cells offers the superantigen the best chance of activating its target T cells. b. Why do superantigens engage TCR V beta and not V alpha or CD3? It is possible that superantigens bind to the V beta portion of the TCR rather than V alpha because the latter does not have a consistently well exposed face for engagement. The fact that it is perhaps relatively easier to produce anti-V beta rather than anti-V alpha antibodies supports this idea. We have shown that N-glycosylation of V beta can interfere with recognition by vSAGs (Pullen et al. 1991), perhaps glycosylation of V alpha tends to conceal otherwise available sites. As far as C beta, C alpha or CD3 engagement is concerned, this may be just too dangerous for MTVs. The role of MTVs SAgs in the life history of the virus seems to be to stimulate T cells in the suckling recipient and thereby create a pool of activated lymphocytes in which the virus may survive until the mouse gives birth and transmits the virus to her own progeny (Hainaut et al. 1990, Golovkina et al. 1992).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Lack of complete correlation between emetic and T-cell-stimulatory activities of staphylococcal enterotoxins.

This study examined the emetic activity of several staphylococcal enterotoxin type A and B (SEA and SEB, respectively) mutants that had either one or two amino acid residue substitutions. New sea gene mutations were constructed by site-directed mutagenesis; gene products were obtained with glycine residues at position 25, 47, 48, 81, 85, or 86 of mature SEA. Culture supernatants from Staphylococcus aureus RN4220, or derivatives containing either sea or a sea mutation, were analyzed for the ability to stimulate proliferation of murine splenocytes, as determined by incorporation of [3H]thymidine. Culture supernatants containing SEA-N25G (a SEA mutant with a substitution of glycine for the asparagine residue at position 25), SEA-F47G, or SEA-L48G did not stimulate T-cell proliferation, unlike supernatants containing the other substitution mutants. Purified preparations of SEA-N25G had weak activity and those of SEA-F47G and SEA-L48G had essentially no activity in the T-cell proliferation assay. All mutants except SEA-V85G, which was degraded by monkey stomach lavage fluid in vitro, were tested for emetic activity. SEA-C106A and two SEB mutants, SEB-D9N/N23D and SEB-F44S (previously referred to as BR-257 and BR-358, respectively), whose construction and altered immunological properties have been reported previously, were also tested in the emetic assay. Each mutant was initially administered intragastrically at doses of 75 to 100 micrograms per animal; if none of the animals responded, the dose was increased four-to fivefold. SEA-F47G, SEA-C106A, and SEB-D9N/N23D were the only mutants that did not induce vomiting at either dose tested; these three mutants had reduced immunological activity. However, there was not a perfect correlation between immunological and emetic activities; SEA-L48G and SEB-F44S retained emetic activity, although they had essentially no T-cell-stimulatory activity. These studies suggest that these two activities can be dissociated.

Amino Acid Sequence↗