The T cell response to sperm whale myoglobin in the H-2d haplotype has been shown to be largely focused on a limited region around glutamic acid 109 recognized in association with I-Ad. T cell clones 9.27 and 1.2 have been previously (4, 5) shown to reflect this specificity and MHC restriction. In this study we have used a panel of synthetic peptides from the region 102-118 of myoglobin to characterize the specificities of these representative clones. The segment from 106-118 was found to represent a consensus region for recognition by both clones. However, we saw significant differences between clones in the hierarchy of responsiveness to peptides within the panel. In as much as the peptide and the I-Ad molecule remain constant, these differences derive from differences in how each T cell receptor interacts with the antigen. This peptide segment is an amphipathic alpha helix in native myoglobin, meaning that one side is hydrophobic and the other hydrophilic. It is one of the prototype cases that led us to find that amphipathic helices constitute the majority of immunodominant sites recognized by helper T cells (1). It is likely that the peptide will refold into an amphipathic helix stabilized by the interface at the surface of the presenting cell. When such secondary conformation is considered, these data are consistent with a model of multiple T cell specificities arising from multiple views of a single antigen conformation at a single Ia-binding site and do not require postulation of multiple conformations or binding sites. Additionally, the finding of distinct specificities suggests that the immunodominance of this site depends not on the dominance of a single clone, but on the focusing of a polyclonal response on a single region of the molecule in association with I-Ad. The immunodominance of this particular region of the protein may thus depend on intrinsic features of the site, such as potential to form an amphipathic helix, as well as extrinsic factors such as binding properties of the I-A molecule.
The present study reports the surprising observation that IL-2-R+ cells can be detected in fresh, unstimulated, murine spleen T cells from unimmunized mice by flow cytometry using the monoclonal anti-receptor antibody 7D4. Also, unexpectedly, these cells were found exclusively in the L3T4+Lyt-2- population by two-color fluorescence, in contrast to receptor+ cells after stimulation, in which both L3T4+Lyt-2- and Lyt-2+L3T4- cells were found. The fraction of splenic T cells bearing IL-2-R reproducibly varies twofold under non-H-2-linked genetic control, with high expression in DBA/2 and BALB/c (approximately 6-7%) and low expression in B10.D2 and C57BL/6 (3%). This correlates quantitatively with a greater responsiveness of the DBA/2 and BALB/c splenic T cells to high doses of IL-2, compared with B10.D2 T cells; twice as many B10.D2 T cells as DBA/2 T cells were required to get the same response. Studies with 23 B X D RI strains revealed that the level of IL-2-R+ cells in unstimulated spleen cells was regulated by multiple genes, very likely including at least one gene on chromosome 7, near the HBB locus. The mapping makes novel use of nonparametric (Smirnov) statistics, which we suggest may be of general usefulness in similar analyses of RI strains.
In this study, we used monoclonal antibodies to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cell free IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [3H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL 2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To our knowledge, this is the first demonstration of release or secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen.
Although studies of the association of antigen with APC have been complicated by antigen-processing requirements, recent studies have suggested that immunologically relevant antigen should be present on the APC surface. Nevertheless, blocking of antigen presentation with antibody to the antigen has not been demonstrable in most systems. To study this problem we developed a system using avidin to block presentation of amino-terminal biotinylated synthetic peptide 132-146 of sperm whale myoglobin (B132) to a murine T cell clone specific for this site in association with I-Ed. greater than 95% specific inhibition was observed with doses of B132 equipotent to unmodified peptide. Specific blocking could be observed: (a) after pulsing APC with antigen, washing, and incubating for a chase period of 8-16 h before addition of avidin and T cells to assure adequate time for intracellular trafficking and maximal display of antigen on the cell surface, or (b) when monensin is present during the antigen pulse to inhibit such traffic. Therefore, the inhibition appeared to be occurring at the cell surface unless dissociation and reassociation were constantly occurring. To distinguish these, B10.GD APC (I-Ed-negative) were pulsed with antigen and cocultured with B10.D2 APC (I-Ed-positive). No detectable antigen presentation resulted. Thus, minimal dissociation and reassociation between antigen and APC occurs and, consequently, blocking by extracellular solution-phase binding of avidin to antigen is unlikely. Taken together, these data suggest that the blocking is occurring at the cell surface. Thus, under physiologic conditions, immunologically relevant antigen necessary for T cell activation appears to be present on the APC surface and is freely accessible to macromolecules the size of avidin. These findings hold specific implications for models of antigen presentation for T cell recognition.
A functional analysis of mutant class II molecules was conducted to identify regions important for antigen-specific T cell activation. Site-directed mutagenesis was used to construct a panel of mutant A beta k genes containing either single or multiple d allele substitutions in the beta 1 domain. The product of each of these genes was expressed with either the A alpha d or A alpha k polypeptide in the Ia-negative B cell lymphoma M12.C3. These mutant class II molecule-bearing cells were tested for their ability to present antigen to a panel of Ak-restricted T cell clones specific for various epitopes of myoglobin. Results from this analysis demonstrate that T helper clones recognized complex determinants interacting with multiple residues on the beta 1 domain and also requiring the haplotype-matched alpha 1 domain. This is in contrast to monoclonal antibodies that recognize a domain-specific, immunodominant region involving residues 40, 63, and 65-67. Every T helper clone was found to interact with a distinct pattern of residues, even among clones recognizing the same combination of peptide and major histocompatibility complex (MHC) molecule. The 3 for 1 residue substitution between k and d alleles at residues 65-67 was one of the most important, because it resulted in loss of ability to present antigen to 7 of 7 I-Ak-restricted T cell clones. These residues have been shown previously to comprise the immunodominant allo-specific serological determinants and to stimulate some alloreactive T cell clones. Substitution at residues 12 and 13 also abrogated antigen presentation to all the T cell clones, but this may be a consequence of a conformational change due to altered alpha beta chain pairing. Substitution at position 9, which is predicted to be located in the floor of the peptide-binding groove where it should not interact directly with the T cell receptor, enhanced presentation of the antigenic site 102-118 to some T cells and diminished it to others. This finding suggests a most interesting conclusion that the same antigenic site may bind in different conformations or orientations to the same MHC molecule, although an indirect effect on the conformation of the MHC molecule itself cannot be excluded. Substitutions at residues 85, 86 and 88 also abrogated the response of one T cell clone but not others specific for the same peptide with the same Ia molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
T cell activation is widely believed to depend on interleukin 1 (IL 1) provided by antigen (Ag)-presenting cells (APC). Because IL 1 is not a constitutive product of APC, we examined the features of its production during the interaction of murine T cell clones and APC. We observed that IL 1 was detectable in supernatants of most myoglobin-specific T cell clones grown with APC and Ag. Two of these T cell clones induced exceptionally high levels of IL 1 in their supernatants, and these same clones demonstrated the unusual restriction to I-Ek, which is a low responder type for sperm whale myoglobin. One of these clones was characterized additionally as to the mechanism of IL 1 induction. This clone rapidly stimulated IL 1 production in the APC population (detectable at 4 hr of co-culture) or in macrophages (M phi) or a M phi-like cell line. IL 1 induction was Ag dependent and H-2 restricted. Induction was radioresistant, both on the part of the T cell and of the IL 1 producer. The IL 1-induction process was attributable to a lymphokine produced by the T cell clone. This lymphokine was distinct from IFN-gamma, TNF and CSF-1 and may account for a principal mechanism of T----APC signalling. The induced IL 1 was the same in size, co-mitogenicity, and pyrogenicity as lipopolysaccharide-induced IL 1.
It has been assumed, without direct evidence, that T cell hybridomas and non-transformed T cell clones are both good models of normal Ag-specific T cells. To compare directly the difference in activation of cloned normal T cells and T hybridoma cells with the same TCR, cloned T hybridoma cells were obtained by fusing pre-established, myoglobin-specific, Iad-restricted T cell clones (14.5 and 9.27) with BW5147 cells. T cell clones were pre-activated with IL-2 as well as specific Ag before fusion. Cloned T hybridoma A3.4C6 was derived from Lys 140-specific and I-Ed-restricted clone 14.5. The other cloned T hybridoma, C7R14, was a fusion product of Glu 109-specific and I-Ad-restricted clone 9.27. Both T hybridomas showed the same Ag specificity and Ia restriction as the parental cloned T cells. However, C7R14 showed higher apparent affinity and broader cross-reactivity than 9.27. Clone 14.5, but not hybridoma A3.4C6, appeared to stimulate splenic cells to secrete cytokines inhibiting HT-2A cell proliferation. The most striking difference between the clones and hybridomas was that both clones, but neither of the matched hybridomas, were induced to synthesize IL-1 on stimulation with Ag. Finally, both cloned T cells and T hybridomas killed Ag-pulsed Iad-bearing B lymphoma target cells. This evidence suggests that killing function can be inherited from clones to hybridomas. However, the clones were much more efficient at killing than the hybridomas, and the hybridomas were more efficient at IL-2 production than the clones. Thus, matched pairs of clones and hybridomas differ in their capacity to mediate the two functions or may tend to be selected differently during cloning. Thus, although our results generally support the validity of T cell hybridomas as faithful models of the corresponding T cell clones, a number of subtle and not-so-subtle differences indicate that caution must be used in such an extrapolation.
The role of IgD in the immune response has remained elusive, although the predominance of IgD on the B cell surface and the paucity of IgD in serum have suggested a receptor function. In support of this hypothesis, it has recently been shown that receptors for IgD on helper T cells can be induced by exposure to IgD in vivo and in vitro. Such IgD receptor-positive T cells (i.e., T delta cells), detectable as RFC using IgD-coated SRBC, augment antibody responses. In this report, we demonstrate that cloned, antigen-specific T cells of helper phenotype show only very low percentages of IgD-RFC, if allowed to rest in vitro after antigen exposure in the absence of IL 2. Exposure to IgD or to IL 2 for 24 hr causes the IgD-specific RFC to increase as much as 25-fold to nearly 80%. Clones that have recently been stimulated with antigen, or T cell hybridomas prepared from such clones, exhibit 40 to 50% IgD-RFC before exposure and twofold higher levels after exposure to IgD. IL 2 also causes a dose-dependent induction of OgD-RFC in normal splenic T cells. Thus, antigen stimulation, IL 2 and IgD can all induce these receptors for IgD which presumably enable helper T cells to interact more effectively with IgD+ B cells.
'/%3e%3cuse xlink:href='%23a' transform='translate(0 -400)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -600)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -800)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -1000)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -1200)'/%3e%3cpath d='M0 0h1400v800H0z' style='fill:%23010066'/%3e%3cpath d='M576 100c-166.146 0-301 134.406-301 300s134.854 300 301 300c60.027 0 115.955-17.564 162.927-47.783-27.353 9.44-56.71 14.602-87.271 14.602-147.327 0-266.897-119.172-266.897-266.01 0-146.837 119.57-266.01 266.897-266.01 32.558 0 63.746 5.815 92.602 16.468C696.217 118.91 638.305 100 576 100z' style='fill:%23fc0'/%3e%3cpath d='m914.286 471.429-99.538-53.25 29.43 108.982-66.575-91.165-20.77 110.96-20.428-111.024-66.857 90.96L699.314 418l-99.701 52.943 74.065-85.192-112.8 4.441 103.694-44.62-103.555-44.94L673.8 305.42 600 220l99.538 53.25-29.43-108.982 66.575 91.165 20.77-110.96 20.428 111.023 66.857-90.959L814.97 273.43l99.702-52.944-74.065 85.193 112.8-4.441-103.694 44.62 103.555 44.94-112.785-4.79z' style='fill:%23fc0' transform='matrix(1.2738 0 0 1.2423 -89.443 -29.478)'/%3e%3c/svg%3e)
