Fine-mapping quantitative trait loci affecting murine external ear tissue regeneration in the LG/J by SM/J advanced intercross line.

External ear hole closure in LG/J mice represents a model of regenerative response. It is accompanied by the formation of a blastema-like structure and the re-growth of multiple tissues, including cartilage. The ability to regenerate tissue is heritable. An F34 advanced intercross line of mice (Wustl:LG,SM-G34) was generated to identify genomic loci involved in ear hole closure over a 30-day healing period. We mapped 19 quantitative trait loci (QTL) for ear hole closure. Individual gene effects are relatively small (0.08 mm), and most loci have co-dominant effects with phenotypically intermediate heterozygotes. QTL support regions were limited to a median size of 2 Mb containing a median of 19 genes. Positional candidate genes were evaluated using differential transcript expression between LG/J and SM/J healing tissue, function analysis and bioinformatic analysis of single-nucleotide polymorphisms in and around positional candidate genes of interest. Analysis of the set of 34 positional candidate genes and those displaying expression differences revealed over-representation of genes involved in cell cycle regulation/DNA damage, cell migration and adhesion, developmentally related genes and metabolism. This indicates that the healing phenotype in LG/J mice involves multiple physiological mechanisms.

Healing quantitative trait loci in a combined cross analysis using related mouse strain crosses.

Inbred mouse strains MRL and LG share the ability to fully heal ear hole punches with the full range of appropriate tissues without scarring. They also share a common ancestry, MRL being formed from a multi-strain cross with two final backcrosses to LG before being inbred by brother-sister mating. Many gene-mapping studies for healing ability have been performed using these two strains, resulting in the location of about 20 quantitative trait loci (QTLs). Here, we combine two of these crosses (N = 638), MRL/lpr × C57BL/6NTac and LG/J × SM/J, in a single combined cross analysis to increase the mapping power, decrease QTL support intervals, separate multiple QTLs and establish allelic states at individual QTL. The combined cross analysis located 11 QTLs, 6 affecting only one cross (5 LG × SM and 1 MRL × B6) and 5 affecting both crosses, approximately the number of common QTLs expected given strain SNP similarity. Amongst the five QTLs mapped in both crosses, three had significantly different genetic effects, additive in one cross and over or underdominant in the other. It is possible that allelic states at these three loci are different in SM and B6 because they lead to differences in dominance interactions with the LG and MRL alleles. QTL support intervals are 40% smaller in the combined cross analysis than in either of the single crosses. Combined cross analysis was successful in enhancing the interpretation of earlier QTL results for these strains.

Lack of immunodominance in the T cell response to herpes simplex virus glycoprotein D after administration of infectious virus.

Glycoprotein D (gD) of HSV has been shown to be a potent immunogen. To analyze the T cell antigenic determinants on gD, a series of 28 overlapping 20-mer peptides that span the extracellular portion of gD-1 were examined for their ability to stimulate T cells from rgD-1 or infectious HSV-1-primed H-2d mice in vitro. rgD-1-primed cells responded exclusively to peptide 241-260, the immunodominant determinant of gD in H-2d mice. In contrast, infectious HSV-primed T cells were shown to respond to 17 (and up to 22) of 28 synthetic gD peptides. These results indicate an extensive diversity in the T cell repertoire to gD in H-2d mice with T cells directed to a broad array of peptide determinants being recruited during the acute phase of an HSV infection.

Protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotype on rat T cell receptors specific for myelin basic protein.

Immunizing Lewis rats with guinea pig myelin basic protein (MBP) yielded an encephalitogen specific, Ia-restricted, rat-mouse T cell hybridoma 5.10, which was used to establish a clonotypic mAb (10.18) that binds to and precipitates the rat TCR. By two-dimensional gel electrophoresis, the rat TCR was shown to consist of two disulfide-linked peptide chains with mol wt of 48,000 and 39,000. 10.18 binds the majority of cells in MBP-specific T cell lines that are capable of transferring experimental allergic encephalomyelitis (EAE) to Lewis rat recipients, but does not bind to either a purified protein derivative of tuberculin-specific cell line or an OVA-specific line. Furthermore, soluble 10.18 can block antigen-specific stimulation of hybridoma 5.10 but cannot control hybridomas, while immobilized 10.18 stimulates 5.10, but cannot control the hybrids. Though 10.18+ cells are very rare in normal rats, increase of 10.18+ cells is observed in MBP-primed paralyzed rats. Finally, when 10.18 is injected into MBP-primed Lewis rats, EAE is abrogated. We have thus characterized EAE as a "mono-idiotypic" autoimmune disease.

Differences in the repertoire of the Lewis rat T cell response to self and non-self myelin basic proteins.

We have examined the fine specificity of a panel of cloned T cell hybridomas generated from Lewis rats immunized with guinea pig (GP) or Lewis rat myelin basic protein (MBP) to determine the autoimmune T cell repertoire that develops in experimental allergic encephalomyelitis (EAE). This analysis has demonstrated that GP MBP, which was approximately 10-fold more potent for EAE induction than the autologous rat MBP, produced a population in which almost one-fourth of the members responded to GP-unique determinants and displayed no crossreactivity on the self antigen. The remaining majority of GP MBP-induced clones were specific for the 68-88 encephalitogenic determinant and could be subdivided into three groups based on their varying responses to the 68-88 peptide and rat and rabbit MBPs. Surprisingly, one of these groups showed equal reactivity to GP and rat MBPs. In contrast, the clonotype composition of the T cell population induced by rat MBP was quite different. One-half of these clones comprised a single group responding to the 68-88 determinant, reacting equally with GP and rat MBP. All of these responded to the same range of antigen concentrations as their GP-induced counterparts. The remaining half of that population contained a collection of clones that was nearly as encephalitogenic as the 68-88 population after propagation as a short-term T cell line. These clones were specific for at least three distinct antigenic determinants, all displaying extensive cross-species reactivity, and required as little or less rat MBP for maximal stimulation as did the 68-88-reactive clones. We therefore conclude that the T cell repertoire for MBP does include clones with reactivity to both 68-88 and non-68-88 determinants of GP and rat MBPs, and that both MBPs appear to be equally capable of stimulating these clones in vitro. However, the differences in the clonotype composition of the populations induced by immunization with these two antigens suggest that rat and GP MBPs are subject to different immunoregulatory constraints in the animal and may account for the difference in the encephalitogenic potential of these two antigens.

Sheep red blood cell-specific helper activity in rat thoracic duct lymphocyte populations positively selected for reactivity to specific strong histocompatibility alloantigens.

These studies show that positively selected T-cell populations, having enriched reactivity in the mixed lymphocyte interaction and the graft-versus-host reaction to strong alloantigens of a chosen major histocompatibility complex haplotype, also possess helper activity which is quantitatively normal in the generation of primary antibody responses to sheep red blood cells in vitro. Such positively selected populations give a linear dose plaque-forming cells response curve indistinguishable from that seen with normal unselected T-cell populations. These findings imply that T cells reactive to histocompatibility antigens also react to conventional antigens, and the possibility is raised that they may do so by some recognition mechanism involving multiple specificities.

Collaboration of allogeneic T and B lymphocytes in the primary antibody response to sheep erythrocytes in vitro.

This study provides a direct quantitative comparison of the helper effects of allogeneic and syngeneic rat T cells in the production of direct SRBC plaque-forming cell (PFC) responses by B cells in culture. In syngeneic T-B combinations, log-log plots of the number of PFC generated after 5.5 days in culture vs. the number of T cells employed as helpers showed a linear response between 10(4) and 2.5 times 10(5) T cells added. Allogeneic T-B combinations, in which the T cells possess the capacity for reactivity to major alloantigens of the B-cell donor, showed a different dose/response relationship in which PFC responses were decreased at high T/B ratios and augmented at low T/B rations. In this system responses were detected with as few as 10(3) allogeneic T cells. Use of negatively selected allogeneic T populations, specifically depleted of mixed lymphocyte interaction (MLI) and graft-vs-host reactivity for B-cell alloantigens, as helpers gave dose/response curves quantitatively identical to responses with syngeneic T-B combinations and also with F1 T-cell parental B-cell combinations. These data indicate that rat T and B cells need not share a major histocompatibility complex haplotype in order to collaborate effectively in a primary direct PFC response to SRBC in culture. In addition, the PFC response required the combinaed presence of T and B cells as well as antigen in the cultures, a finding consistent with the two signal model of B-cell activation. Finally, the dose/response data obtained suggest the possibility that although SRBC antigen is required in the cultures helper activity with low numbers of normal allogeneic T cells may not depend on T cells having specificity for this antigen.

Overlapping T cell antigenic sites on a synthetic peptide fragment from herpes simplex virus glycoprotein D, the degenerate MHC restriction elicited, and functional evidence for antigen-Ia interaction.

Analysis of the B10.A T cell response to synthetic peptides representing the NH2-terminal 23 amino acids from the HSV glycoprotein D sequence revealed two antigenic determinants for T cells: one localized between residues 1-16 and the other between residues 8-23. The 1-16 site, which is helical, was recognized in the context of the Ia molecule, whereas the 8-23 site, which is nonhelical, was recognized in the context of the I-E molecule. The I-E-restricted response was found to be highly MHC degenerate in that T cell hybridomas specific for the 8-23 peptide responded to antigen on APCs derived from B10.A, B10.A(5R), and B10.A(9R) mice and showed differences in antigenic fine specificity with APCs of different haplotypes. These data support the idea of antigen-Ia interaction.

Idiotype-anti-idiotype regulation. I. Immunization with a levan-binding myeloma protein leads to the appearance of auto-anti-(anti-idiotype) antibodies and to the activation of silent clones.

BALB/c mice immunized multiple times with ABPC48 (A48 or Ab1), a BALB/c bacterial levan (BL)-binding myeloma proteins, produce anti-Ab1 antibodies (Ab2). Immunization with only tow doses of AB1 often leads to the production of anti (antiA48) (Ab3) as does immunization with hemocyanin conjugates of Ab2. Finally, immunization with hemocyanin conjugates of Abf3 leads to the production of anti- (anti-[anti-A48]) (Ab4). Normal BALB/c mice immunized with BL produce an anti BL antibody response containing no detectable Ab1 idiotype (Id)-bearing molecules. Mice producing Ab3 express substantial amounts of Ab1 Id in their anti-BL response whereas mice producing Ab2 and Ab4 show a generalized inhibition in their anti BL response. These results indicate that states of immunity within an idiotypic chain may have marked effect on antibody responses to the antigen (i.e., BL) which is the putative initiator of the chain. Strikingly, the chain itself has an interesting feature. That is, Ab3 and Ab1 share a cross-reactive Id in that both are bound by Ab4 and Ab2. We propose a model of Id-anti-Id systems to explain this unexpected result. This is based on the concept of regulatory idiotopes on Ab1 molecules which initiate Ab2 (anti-idiotope) responses. In contrast, Ab2 molecules generally fail to initiate anti-Ab2 Id responses eigher because any individual idiotope is present at very low concentration or because Ab2 molecules tend to lack regulatory idiotopes. Thus, Ab2 molecules immunize syngeneic animals because they interact with cells bearing Ab1 like regulatory idiotopes. Thus, Ab3 will share regulatory idiotopes with Ab1. Ab4 and Ab2 will share the ability to bind the Ab1-like regulatory idiotope.

Major histocompatibility complex-controlled, antigen-presenting cell-expressed specificity of T cell antigen recognition. Identification of a site of interaction and its relationship to Ir genes.

In previous work (5,6), we have reported studies on a T lymphocyte hybridoma clone and the peritoneal exudate T cells (PETLES) from B10.A(5R) mice primed with the cytochrome c carboxyl terminal peptide (residues 81-103) of the tobacco horn worm moth (Manducca sextus). As expected, since B10.A(5R) is a low responder to pigeon fragment 81-104, it was found that the B10.A(5R) lymphocytes were unable to respond to the pigeon cytochrome c 81-104 fragment presented on syngeneic B10.A(5R) antigen-presenting cells (APC). However, these same T lymphocytes did respond to the pigeon fragment when presented on B10.A APC. Thus, some structural difference between the pigeon and moth peptides had prevented B10.A(5R) APC from effectively presenting the pigeon fragment to moth-primed B10.A(5R) lymphocytes. This structural difference was found to be the deletion of an alanine at position -103 (Ala103) from the pigeon sequence in the moth peptide. Two additional T cell specificities were created by changing residue-99. These T cell populations from the B10.A(5R) showed an identical dependence on the Ala103 deletion when B10.A and B10.A(5R) APC were compared. The relationship of APC-expressed antigen specificity and MHC-linked immune responsiveness differences was also examined. The B10.A(5R) was found to be a high responder to each of three peptides that lack Ala103 but not to the Ala103-containing analogues. B10.A mice, in contrast, respond to both types of peptides. Utilizing allogeneic antigen-presentation to B10.A PETLES by pulsed APC, it was shown that the poor response of the B10.A(5R) to the Ala103-containing peptides was, in two of three cases, not associated with any differences in T cell repertoires but due to two different APC capabilities of B10.A and B10.A(5R). The exception apparently represents a case of T cell repertoire polymorphism between B10.A and B10.A(5R) that can also affect immune responsiveness.

A synthetic peptide induces long-term protection from lethal infection with herpes simplex virus 2.

Immunization against viral pathogens is generally directed toward the induction of virus neutralizing antibody (VNA) and the maintenance of the potential for a second-set (IgG) response. Indeed, an elevated level of specific antibody is considered a reliable clinical indicator that a state of immunity exists in the host. However, in the case of herpes simplex virus (HSV), the presence of circulating VNA does not necessarily correlate with protection. Thus, it has been found that secondary infections occur in individuals even with high neutralizing titers to HSV, suggesting that antibody to the virus may be useless or even deleterious. In consideration of these facts, we were interested in inducing a T cell response to HSV. We had already shown that synthetic peptides corresponding to the NH3-terminal region of the glycoprotein D (gD) molecule of HSV could induce a strong T cell response when injected into mice, but did not, by themselves, confer protection. In this report, we examined the ability of peptides, covalently coupled to palmitic acid and incorporated into liposomes, to induce virus-specific T cell responses that confer protection against a lethal challenge of HSV-2. We have demonstrated that long-term protective immunity is achieved with a single immunization in the absence of neutralizing antibody when antigen is presented in this form. Furthermore, T cells but not serum from such immune mice can adoptively transfer this protection.

Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V alpha and V beta chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different.

Prospects for specific immune intervention in T cell-mediated autoimmune disease via anti-idiotypic regulation depend on the degree of diversity of the responder cell antigen receptor repertoire. A highly heterogenous response against self epitopes offers little chance for such regulation. We report here that the Lewis rat autoimmune disease experimental allergic encephalomyelitis, generally considered to be a model of human multiple sclerosis, is caused by T cells that use a limited set of TCR V genes. We have cloned the rat TCR alpha and beta chain cDNAs from the Lewis rat x mouse T cell hybridoma 510, which retains the rat specificity for the encephalitogenic determinant of myelin basic protein (MBP). Using Northern blot analysis of T cell RNA with the cloned V region probes, we have found a specific, and near perfect, correlation between expression of TCR message hybridizing to the V alpha 510 and VB510 probes and specificity for the encephalitogenic determinant of MBP in both T cell hybridomas and encephalitogenic T cell clones. This restricted V gene usage provides a basis for observed idiotypic regulation of auto-reactive T cells, and possible therapy for autoimmune disease. A curious and unexplained observation is that the Lewis rat V alpha/V beta combination that dominates the encephalitogenic response to the 68-88 peptide of MBP is precisely the same V alpha/V beta combination used by the B10.PL mouse response to the encephalitogenic response to the 1-9 peptide of MBP.

Contribution of antigen-presenting cell major histocompatibility complex gene products to the specificity of antigen-induced T cell activation.

Previous studies from our laboratory showed that B 10.A mice are high responders to pigeon cytochrome c fragment 81-104, whereas'B 10.A(5R) mice are low responders. In the present studies, the C-terminal cyanogen bromide cleavage fragment and homologous synthetic peptides of tobacco horn worm moth cytochrome c were shown to be immunogenic in both B10.A and B10.A(5R) mice. These strains, however, showed different patterns of cross-reactivity when immune lymph node T cells were stimulated with cytochrome c fragments from other species. To examine the two patterns of responsiveness at a clonal level, cytochrome c fragment-specific T cell hybridomas were made and found to secrete interleukin 2 in response to antigen. The patterns of cross- reactivity of these B 10.A and B 10.A(5R) clones were similar to that seen in the whole lymph node population. Surprisingly, when these clones were tested for major histocompatibility complex (MHC)-restricted antigen recognition, they were all found to respond to antigen with both B10.A and B10.A(5R) antigen-presenting cells (APC). Furthermore, the cross-reactivity pattern appeared to be largely determined by the genotype of the APC, not the genotype of the T cell clone. That is, a given T cell clone displayed a different fine specificity when assayed with B10.A or B10.A(5R) APC. This observation indicates that the APC MHC gene product and antigen interact during the stimulation of the T cell response and that as a consequence the specificity of antigen-induced T cell activation is influenced by these MHC gene products. (During the preparation of this manuscript it has come to our attention that results similar to our own, concerning the fine specificity of cytotoxic T cell clones, have been obtained by Dr. T. R. Hunig and Dr. M. J. Bevan, Massachusetts Institute of Technology, Boston, MA. T. R. Hunig and M. J. Bevan. 1981. Specificity of T-cell clones illustrates altered self hypothesis. Nature. 294:460.)

T cell determinants of myelin basic protein include a unique encephalitogenic I-E-restricted epitope for Lewis rats.

The major encephalitogenic epitope for Lewis rats is the 72-89 sequence of guinea pig basic protein (GP-BP) or rat basic protein (Rt-BP). T cells responsive to this epitope are I-A restricted and preferentially express the V alpha 2:V beta 8 gene combination in their TCR. In this work, we describe for the first time the delayed appearance of T cells specific for additional discrete determinant of BP, the nonencephalitogenic 55-68 sequence of GP-BP restricted by I-A, and the encephalitogenic 87-99 sequence of Rt-BP restricted by I-E. The TCR V alpha 2:V beta 8 gene combination was expressed by both encephalitogenic GP-BP S72-89 and Rt-BP S87-99 T cell specificities but not by GP-BP 44-68-specific T cells. This is the first demonstration of I-E-restricted encephalitogenic T cells in Lewis rats and supports the conclusion that the I-E class II locus is involved in autoimmune diseases.

Spallanzani's mouse: a model of restoration and regeneration.

The ability to regenerate is thought to be a lost phenotype in mammals, though there are certainly sporadic examples of mammalian regeneration. Our laboratory has identified a strain of mouse, the MRL mouse, which has a unique capacity to heal complex tissue in an epimorphic fashion, i.e., to restore a damaged limb or organ to its normal structure and function. Initial studies using through-and-through ear punches showed rapid full closure of the ear holes with cartilage growth, new hair follicles, and normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to the scarring usually seen in mammals. Since the ear hole closure phenotype is a quantitative trait, this has been used to show-through extensive breeding and backcrossing--that the trait is heritable. Such analysis reveals that there is a complex genetic basis for this trait with multiple loci. One of the major phenotypes of the MRL mouse is a potent remodeling response with the absence or a reduced level of scarring. MRL healing is associated with the upregulation of the metalloproteinases MMP-2 and MMP-9 and the downregulation of their inhibitors TIMP-2 and TIMP-3, both present in inflammatory cells such as neutrophils and macrophages. This model has more recently been extended to the heart. In this case, a cryoinjury to the right ventricle leads to near complete scarless healing in the MRL mouse whereas scarring is seen in the control mouse. In the MRL heart, bromodeoxyuridine uptake by cardiomyocytes filling the wound site can be seen 60 days after injury. This does not occur in the control mouse. Function in the MRL heart, as measured by echocardiography, returns to normal.

Observations, legends, and conjectures concerning restricted T-cell receptor usage and autoimmune disease.

It has become clear over the past few years that a variety of experimental autoimmune conditions are mediated by T cells bearing a highly restricted subset of antigen receptors. This restricted TcR usage raises important questions concerning not only the recognition of autoantigens, but also the pathogenic mechanisms underlying many models of autoimmunity. Furthermore, the extension of these findings in certain cases to human disease has raised the possibility of specific therapeutic immune intervention. In this review, we examine the available data on restricted T-cell receptor usage in autoimmune disorders and explore the interpretations and the theoretical and practical implications of these findings.

An alternative view of T-cell receptor-MHC interaction: T-cell receptor binds transversally to the alpha-helices of the MHC molecule.

We have attempted to elucidate the relative orientation of the T-cell receptor (TCR) to the major histocompatibility complex (MHC)-antigen complex during antigen recognition, using the T-cell response to B10.A (I-Ek) and B10.A(5R) (I-Eb) mice to the 1-23(H) peptide derived from glycoprotein D of the herpes simplex virus. The 1-23(H)-specific T-cells derived from both B10.A and B10.A(5R) mice use the same set of V alpha genes and a different array of V beta genes. The CDR1s of these TCR beta chains share residues at particular positions. The CDR2s of the TCR beta chains have a negative charge, which correlates with I-Eb reactivity and with the positively charged polymorphic residues residing at the C-terminal end of the alpha-helix of the I-Eb beta chain of the class II molecule. Taken together, the data suggest that the TCR beta chain interacts with both the alpha and beta chains of the MHC class II molecule, as does the TCR alpha chain.

The interplay of T cell responses to viral and autoimmune epitopes.

The examination of the immune T cell response to herpes simplex virus (HSV) antigen glycoprotein D (gD) in an ongoing infection has revealed a uniquely broad range of antigenic determinants seen. This has been shown in the murine T cell response to gD determinants where over 60% of the overlapping peptides are recognized as opposed to 1 of 30 peptides seen when gD was injected in Freund's adjuvant. This has also been seen in the response to local autoantigens when the HSV infection is produced by corneal scarification. Furthermore, analysis of the response to the autoantigen, Golli myelin basic protein (MBP), present in the developing thymus is explored.

Use of a solid-phase 3H-radioimmunoassay for the measurement of immunoglobulin produced in short-term cultures of antibody-secreting cells.

A slid-phase radioimmunoassay (RIA) for assaying immunoglobulin produced from antibody-secreting myeloma, hybridoma and immune spleen cells is described. Specific antibody is detected by culturing antibody-producing cells on antigen-coated flexible polyvinylchloride microtiter wells, washing away the cells, and measuring the bound specific antibody with tritiated affinity-purified anti-isotype reagents. Antibody produced from 10(3) myeloma cells can be detected with as little as 4 h of incubation. With 24-48 h of incubation, antibody from as few as approximately 3-15 myeloma, hybridoma or immune spleen plaque-forming cells (PFC) can be detected. This culture-well RIA has certain distinct advantages over the hemolytic PFC assay and RIA assays in which antibody in culture supernatants is measured.

Cytotoxic effects of myelin basic protein-reactive T cell hybridoma cells on oligodendrocytes.

Cells of a rat/mouse T cell hybridoma reactive to the encephalitogenic peptide of myelin basic protein (MBP) was found to be cytotoxic to 51Cr-labelled rat oligodendrocytes (oligos) inducing 52 +/- 5% 51Cr release vs. 28 +/- 2% spontaneous 51Cr release from replicate oligos. The hybridoma cells were not toxic for rat astrocytes or concanavalin A-stimulated lymphoblasts. Hybridoma T cells reactive to an experimental allergic encephalomyelitis-irrelevant antigen (ovalbumin) were not cytotoxic to oligos. The cytotoxic reaction required cell-cell contact but did not require the in vitro presence of antigen-presenting cells MBP. The target antigen on the oligos is not yet defined. These studies suggest that MBP-reactive T cells can be directly cytotoxic to oligos in the absence of other cell populations.