Biomedical subjects
Publications and source records attributed to T H Watts.
As is the case with most proteins of the secretory pathway, the biogenesis of MHC class I and class II molecules occurs in association with molecular chaperones. Considerable progress has been made in identifying the chaperones involved and recent studies on two of these, calnexin and invariant chain, have shown that they influence multiple processes including protein stability, folding, assembly and intercellular retention.
The region of invariant chain encompassing residues 81-104 is critical for association with MHC class II molecules. This segment of invariant chain, termed CLIP for Class II-associated invariant chain Peptides, has been shown to inhibit antigenic peptide binding and T cell stimulation. Polymorphism affects the ability of CLIP to inhibit antigenic peptide binding, suggesting that CLIP may occupy the MHC II antigen binding site directly. However, CLIP may also mediate inhibition by binding to an alternate site causing an allosteric change to prevent antigenic peptide binding. The relationship between the apparent dissociation constant in the presence of a competitor (Kapp) and the competitor concentration can be examined to determine the nature of competition between two ligands. In competitive binding experiments between CLIP and antigenic peptide we find a linear dependence of Kapp on competitor concentration. These data are consistent with CLIP and antigenic peptide competing for the same site on the MHC class II molecule, thus arguing against an allosteric mechanism of CLIP inhibition. Mildly acidic conditions are thought to promote peptide loading in the endosome compartment by facilitating CLIP dissociation and enhancing antigenic peptide association. We have compared the effect of acidic pH on the equilibrium binding of murine CLIP and antigenic peptide to MHC class II molecules. Like antigenic peptide, CLIP binding can be greatly enhanced at mildly acidic pH, suggesting that a passive competitive mechanism for CLIP removal may not be sufficient to achieve loading of antigenic peptide in the endosome.
In this study we analysed the binding of the peptide HEL46-61 to purified Ak molecules which have been altered by site-directed mutagenesis at polymorphic positions to include amino acids from the Ad alpha-chain. We find that changes in the floor of the peptide binding groove, at positions 11, 14 and 28, abolish T cell recognition without changing peptide binding affinity. We further show that amino acid changes at these positions in the Ad molecule result in a conformationally altered molecule as evidenced by loss of binding of the Ad alpha specific monoclonal antibody K24. Thus the T cell receptor is highly sensitive to subtle changes in MHC II structure induced at sites that are unlikely to be involved in direct T cell contact. This has important implications with respect to allorecognition. The binding studies reported here were performed both at pH 7, to reflect binding of peptides at the cell surface, and at pH 5.5, to mimic binding in an intracellular acidic compartment. Binding to wild-type Ak was increased 2-3-fold at pH 5.5, whereas binding to some MHC II mutants was increased by greater than 20-fold at pH 5.5 relative to pH 7. These results show that the apparent peptide binding specificity for the mutants differs at pH 7 and 5.5, and suggest that caution should be used in defining the MHC-restriction of peptide epitopes at neutral pH.
M12 B lymphomas expressing transfected Ak molecules with truncated cytoplasmic domains have a defect in Ag presentation to some autoreactive T cell hybrids. This defect in Ag presentation is corrected by pretreatment of the B cells with agents that elevate intracellular cAMP. Here we show that dibutyryl-cAMP treatment of M12 B lymphomas leads to cell surface expression of the costimulatory molecule B7. Furthermore, CTLA4Ig, a ligand for B7, inhibits activation of an accessory signal-dependent T hybrid. B7 is also inducible in M12 B lymphomas upon MHC-restricted interaction with T cells that can be activated by the APC, but not by T cells that fail to respond to truncated MHC-bearing M12 cells. Activation of the unresponsive T hybrids with immobilized anti-CD3 confers on them the ability to induce B7 in the APC. Direct engagement by immobilized antibodies of MHC class II on M12 B lymphomas did not induce B7 expression. Taken together, these results imply that during T-B interaction, initial T cell activation events lead to the ability of the T cell to induce costimulatory activity in the B cell, which in turn further activates the T cell. Activated T cell supernatants induced a small amount of B7 but were not nearly as effective as cAMP or as coincubation of T and B cells. These results suggest a role for T-B contact or localized cytokine secretion in the induction of B7 during T-B interaction.
Major histocompatibility complex (MHC) class II molecules are critical restricting elements in the generation of thymus-dependent immune responses. Recent studies indicate that in addition to providing a composite epitope for recognition by T-cell antigen receptors, MHC class II molecules function in signal transduction through interaction with other cellular proteins. Mutational analyses indicate that structural information necessary for these functions is compartmentalized in different aspects of the molecular complex. Here, William Wade and colleagues review the structural basis of this MHC class II function as defined in the I-A alpha and -beta chains.
MHC class II molecules bind peptide antigens and present them to T cells. Recent evidence suggests that peptide--MHC class II interaction is regulated by pH. Both peptide binding to and dissociation from MHC class II molecules is enhanced at acidic pH. In this report we use circular dichroism to investigate the possibility that the modulation of peptide association with MHC class II molecules at acidic pH is associated with changes in MHC class II structure. Our results show that a change in the structure of the MHC class II protein A(d) occurs between pH 4 and 5, close to the pH optimum for peptide binding. More drastic changes in A(d) structure occur at lower pH levels, where peptide dissociation is enhanced. The structural changes at pH 4-5 are fully reversible upon neutralization, while changes at pH 3 are not. The pH stability of purified E(d) molecules is somewhat different, with changes observed only at pH less than 4. This may reflect a differential occupancy of the purified molecules with endogenous peptides.
In addition to their role as peptide binding proteins, MHC class II proteins can also function as signal transducing molecules. Recent work using B cells expressing genetically engineered truncated MHC class II molecules has suggested that signaling through the cytoplasmic domains of these proteins plays an important role in the generation of signals required for the activation of some T cell hybrids. Treatment of truncated Ia-expressing B cells with cAMP-elevating agents corrects the deficiency in Ag presentation by these cells. We report that the MHC class II-mediated signal appears to act by a mechanism that increases the efficiency of Ag presentation by B cells thereby lowering the amount of specific Ag required for T cell activation. We further show that the induction of the cAMP-induced signal in B cells is inhibited by cycloheximide and cytochalasin A, implicating protein synthesis as well as cytoskeletal rearrangements in Ag presentation to accessory signal- dependent hybrids. In contrast, these agents do not block Ag presentation to a T cell hybrid previously shown not to require the cAMP-induced signal for activation. The signal-dependent T hybrid is additionally dependent on LFA-1-ICAM-1 interaction for activation, whereas the signal-independent hybrid is not. These observations suggest the existence of two types of T cell hybrid with respect to their requirements for activation: those that require only the recognition of MHC class II-peptide complexes without accessory signals, as shown by their ability to respond to purified Ia on planar membranes, and those that, in addition to recognition of MHC II/Ag, require LFA-1-ICAM-1 interaction and the delivery of additional signal(s) induced in the B cell via signal transduction through MHC class II molecules.
MHC proteins are polymorphic cell surface glycoproteins involved in the binding of peptide Ag and their presentation to T lymphocytes. The polymorphic amino acids of MHC proteins are primarily located in the N-terminal domains and are thought to influence T cell recognition both by influencing the binding of peptide Ag and by direct contact with the T cell receptor. In order to determine the relative importance of individual polymorphic amino acids in Ag presentation, a number of groups have taken the approach of interchanging polymorphic amino acids between different alleles of MHC protein in an attempt to define which of the polymorphisms influence peptide binding and which influence T cell recognition by direct contact with the TCR. The peptide OVA323-339 has been previously shown to bind to the MHC class II protein Ad and to have a much lower affinity for Ak, whereas the peptide hen egg lysozyme 46-61 binds well to Ak and poorly to Ad. In the present report, we have analyzed the ability of purified wild-type MHC class II proteins as well as the ability of three different hybrid molecules between Ad and Ak to bind and present these peptides. We find that the alpha-chain of the MHC class II protein plays a critical role in the binding of HEL46-61 and confers the specificity for binding OVA323-339, regardless of which beta-chain is present. We also find that the beta-chain region 65-67 does not control the specificity of peptide binding to the MHC protein, but is important in T cell responses to preformed MHC-peptide complexes, suggesting a role for this region in contacting the TCR.
The peptide antigen OVA323-339 forms a highly stable complex with the I-Ad molecule which when reconstituted into lipid bilayers can stimulate the release of interleukin 2 (IL-2) by specific T cell hybrids. To probe the accessibility of various regions of the peptide in the I-Ad binding site, six analogues of OVA323-339, each with a unique site for fluorescein labeling, were synthesized. OVA peptides modified with fluorescein at the N terminus position 323 and at the epsilon-amino group of lysines at positions 328, 329, 330, 331 and 336 all bind to the I-Ad molecule. These substitutions include four amino acids previously identified as representing a structural motif common to MHC binding peptides. The fluorescein hapten on each analogue was fully accessible to quenching by anti-fluorescein antibody after binding of the peptide to the MHC class II protein, indicating that these regions of the peptide are exposed in the MHC binding site. These data suggest that the MHC class II peptide binding site is remarkably permissive with respect to tolerating bulky substitutions in the peptide antigen. The data further suggest that either the central region (328-331) of the peptide OVA323-339 is oriented in MHC class II binding site such that all of its side chains are exposed, or that the binding of the peptide is conformationally flexible allowing reorientation of the bulky substituent to the outside of the binding site in each case studied.
The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.
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.
The stability of a specific complex between the peptide Ag representing residue 323-339 of OVA and the MHC class II protein, I-Ad, in a lipid bilayer was investigated as a function of pH and temperature. The complex is much more stable in a lipid bilayer than previously reported for detergent micelles. Measureable dissociation was detectable only after several hours at a pH below 5. The results show that a purified preparation of MHC class II molecules can sequentially bind, release, and rebind peptide, indicating that, in principle, MHC class II molecules could be used more than once for peptide binding. However, the time and pH required for peptide-MHC dissociation suggests that, in an Ag presenting cell, either a prolonged residence in an acidic compartment or other factors will be required for regeneration of the peptide binding site.
B cell transfectants expressing MHC class II (Ia) molecules with truncated cytoplasmic domains are defective in both antigen presentation and in anti-Ia induced intracellular signaling. In this report we show that the Ag presentation defect in a truncated-Ia expressing transfectant can be overcome by providing the second messenger for the Ia-mediated signaling event. Preincubation with dibutyryl-cAMP restored the ability of the truncated Ia expressing-transfectant to stimulate IL-2 release by otherwise nonresponsive T hybrids. This provides direct functional evidence that signaling via the cytoplasmic domains of MHC class II proteins leads to the generation of accessory signals in the B cell that are important in T cell activation. The dibutyryl-cAMP induced signal must be on the same cell as the restricting element, does not bypass the requirement for occupancy of the T cell receptor with its normal ligand, and is lost upon fixation of the cells. Thus T cell-B cell interaction involves a two way communication in which both cells sense and respond to the formation of the antigen/MHC/TCR complex.
Planar membrane-bound complexes between a fluorescent peptide, FITC-OVA(323-339), and the class II MHC Ag, I-Ad, were analyzed by fluorescence microscopy and in biological assays to determine the optimum distance between peptide-Ia complexes required for maximum activation of IL-2 production by the Th cell hybridoma DO-11.10. Optimum responses were obtained when the average distance between peptide-Ia complexes was of the order of 200 A. This implies that T cell activation by Ag-MHC requires cross-linking of the TCR via closely packed Ag-MHC complexes. The same dose response curve to the preformed complexes was obtained whether one used a fixed concentration of Ia and varied the peptide concentration or a fixed concentration of peptide and varied the Ia concentration. In both cases there was a linear relationship between the number of peptide-Ia complexes and the response of the T cells. The association between Ia and peptide in vitro is an inefficient process, requiring prolonged incubation and a large excess of peptide over Ia. Once formed, however, the complex is extremely stable with no detectable dissociation at neutral pH after days at 4 degrees C. With several different preparations of Ia it was found that only about 10 to 20% of the purified Ia is capable of forming the long-lived complex with peptide.
The expression and function of surface immunoglobulin (sIg) receptors was analyzed using antigen-specific monoclonal B cells and T cells. When B cells were incubated with antigen (hapten carrier), hapten-specific sIg receptor expression was greatly reduced. The specific antigen-presentation activity of the B cells was also markedly reduced. Following the removal of antigen, expression of the sIg receptors was restored to original levels within 3-4 hr. Specific antigen-presentation activity of these B cells was concomitantly restored. Reduction of sIg receptor expression and specific antigen presentation were inhibited by the addition of competing free hapten, suggesting that crosslinking of sIg receptors is required for the observed changes in sIg receptor expression and that specific antigen presentation is hapten specific. Using oligopeptide-specific Th cells, we could show the requirement for processing of antigen in the interaction between hapten-specific B cells and carrier-specific T cells. These results indicated that the expression of sIg receptors for a specific antigen and the ability to present the antigen are interrelated.
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Membranes isolated from antigen-pulsed, antigen-presenting cells were extensively dialyzed and shown to retain the ability to present antigen to an I-Ad-restricted, antigen-specific T cell, 3DO-54.8. This ability to retain antigen on the membrane was duplicated in lysates of antigen-presenting cells as well as with pure I-A molecules in phospholipid vesicles. Measurement of the concentration of surface-associated fluorescent peptide on planar membranes prepared from antigen-pulsed phospholipid vesicles showed that about 1 peptide molecule was retained per 100 I-A molecules. We calculate that about 1000 I-A-associated peptide molecules are sufficient to stimulate the response of a single 3DO-54.8 cell. The association of fluorescent peptide with the surface depended on the presence of I-A and was blocked by unlabeled ovalbumin peptide or by a digest of hen egg lysozyme, added at the time of the pulse, but not after pulsing.