Genetic analysis of the presentation of minor lymphocyte-stimulating determinants. II. Differing non-MHC control of super-stimulatory and more poorly stimulatory Mls phenotypes

Analysis of the capacity of splenocytes from non-prototypic Mlsa or Mlsc mouse strains to stimulate allogeneic H-2k-compatible T cells in a primary Mls-defined MLR provided interesting examples of exceptions to the usually stated characterization of Mlsa and Mlsc determinants as highly stimulatory of weakly stimulatory, respectively. Across the Mlsa barrier, MA/My stimulator cells had a significantly reduced capacity to elicit responder proliferation in comparison with prototypic AKR/J or less well studied C58/J, CE/J, or RF/J splenocytes. Across the Mlsc barrier, a gradient of stimulatory ability was observed with RF/J splenocytes being virtually nonstimulatory, prototypic C3H/HeJ splenocytes having an intermediate capacity, and CE/J and C58/J being highly stimulatory presenters of this non-MHC specificity. The differing capacity of each of these H-2k stimulator cells to elicit unprimed responder cell proliferation across an Mlsa or Mlsc difference correlated with the T cell growth factor activity that was secreted into the MLR supernatants. The super stimulatory form of Mlsc was expressed in an autosomal dominant fashion by (Mlsc poorly stimulatory x Mlsc super-stimulatory)F1 animals, (BALB.K x C58/J)F1 or (RF/J x CE/J)F1. The segregation of Mlsc stimulatory ability among first backcross and F2 animals derived from the former F1 was compatible with a single non-MHC gene controlling the expression and presentation of the super-stimulatory form of Mlsc. The regulatory nature of this gene was indicated by the observation that F1 animals generated from the Mlsc nonprototypic and poorly stimulatory BALB/c parental strain were self-tolerant to the super-stimulatory form of Mlsc. The existence of an Mls specificity other than a and c was suggested by positive non-MHC MLR responses in certain responder/stimulator cell combinations of Mls prototypic and nonprototypic mouse strains.     

The Mlsd-defined primary mixed lymphocyte reaction: a composite response to Mlsa and Mlsc determinants

Considerable disagreement exists among immunologists regarding the polymorphic nature of the murine Mls system. An estimate of the capacity of a given putative Mls allelic gene product expressed on a stimulator population to elicit proliferation of H-2-compatible Mls-disparate unprimed T cells may vary widely among different groups of investigators. This laboratory has shown previously that preactivation of B lymphocytes in a splenocyte stimulator population by exposure to goat anti-mouse IgD (GaMD) before irradiation dramatically enhanced the in vitro presentation not only of the strongly stimulatory (and highly cross-reactive) Mlsa and Mlsd, but also the more poorly stimulatory Mlsc specificity. Therefore, by the use of GaMD-treated splenocytes that optimally present the various Mls non-H-2 stimulatory epitopes, we attempted in this study to obtain a clearer understanding of Mls polymorphism by re-examining the conflicting claims associated with the mixed lymphocyte reaction (MLR) stimulatory capacity of different Mls specificities. Among H-2k responder cells of the Mls null, Mlsa, Mlsb, or Mlsd genotypes, only T cells from Mlsd-bearing CBA/J mice did not respond to Mlsc determinants present on GaMD-treated C3H/HeJ stimulator cells. Crossing CBA/J with an Mlsc-responsive mouse strain yielded an F1 animal in which nonresponsiveness to Mlsc was dominant. Although Mlsa (AKR/J) and Mlsc (C3H/HeJ) parental T cells both proliferated vigorously to Mlsd (CBA/J) stimulator cells, the Mlsa/c (AKR X C3H)F1 T cells responded poorly to GaMD-treated Mlsd stimulator cells. In addition, Mlsd (CBA/J) T cells were nonresponsive to Mlsa (AKR/J), Mlsc (C3H/HeJ), and Mlsa/c (AKR X C3H)F1 GaMD-treated stimulator cells. Because Mlsa (AKR/J) and Mlsc (C3H/HeJ) specificities are mutually stimulatory, at least limited polymorphism must exist in the Mls system. However, because Mlsa/c (AKR X C3H) and Mlsd (CBA/J) specificities are mutually nonstimulatory, T cell proliferation in an Mlsd-defined primary MLR is most likely due to a composite response to Mlsa and Mlsc epitopes present on CBA/J stimulator cells.     

T cell recognition of Mls. T cell clones demonstrate polymorphism between Mlsa, Mlsc, and Mlsd

The determinants encoded by the minor lymphocyte stimulating locus (Mls) are defined as determinants that induce strong T cell proliferative responses in primary mixed lymphocyte reactions. Although the Mls locus was originally described as having four alleles, a, b, c, and d, a number of recent observations have led several investigators to challenge the idea that Mls is truly a polymorphic system. To better define this system of determinants recognized at high frequency by T cells, the present studies were undertaken to evaluate the polymorphism of Mls products. In the present study, the in vitro proliferative responses of Mlsa- and Mlsc-specific T cell clones were employed to analyze Mls products. The identification of determinants recognized by Mlsa- and Mlsc-reactive clones was established by the pattern of responses to stimulators derived from congenic strains, recombinant inbred strains, and backcross mice. T cell clones and unprimed T cells gave concordant responses that confirmed the Mlsa or Mlsc specificity of the cloned populations. With the use of these two sets of Mls-specific T cell clones, the existence or absence of polymorphism of Mls-encoded gene products was examined. It was found that Mlsa-specific cloned T cells responded to Mlsa but not Mlsc stimulators, whereas Mlsc-specific clones responded to Mlsc but not Mlsa. This reciprocal pattern of specificity indicates that the Mls system as currently defined is therefore truly polymorphic. In addition, it was observed that both Mlsa- and Mlsc-specific clones were stimulated by Mlsd stimulators. In particular, the possibility that Mlsa and Mlsc are not alleles but products of different loci and that Mlsd strains are those that express both Mlsa and Mlsc is considered.     

Characterization of a new minor lymphocyte stimulatory system. I. Cluster of self antigens recognized by "I-E-reactive" V beta s, V beta 5, V beta 11, and V beta 12 T cell receptors for antigen.

In the mouse, two sets of V beta gene products have been shown to be associated with T cell recognition of endogenous self Ag. One of these is the set of V beta associated with T cell reactivities to stimulatory Mls gene products, Mlsa (V beta 6, V beta 8.1, V beta 9) or Mlsc (V beta 3); another is the set of V beta, such as V beta 5, V beta 11, V beta 12, or V beta 17a, which were originally found to be related to I-E recognition. Although the Mls system has been well characterized, little is known about the nature of the ligands for the second set of V beta. In this work, we describe the evidence that the natural ligand or ligands of V beta 5, V beta 11, and V beta 12 may be novel Mls determinants that are recognized by naive T cells at a high precursor frequency and function as the ligand for clonal deletion of self-reactive T cells by negative selection. However, surprisingly, unlike the conventional Mls system, in which all V beta associated with Mlsa recognition or Mlsc recognition are uniformly deleted in those animals expressing the relevant Mls type, expression of these three V beta segregates independently among strains. Based on these observations, the nature of T cell recognition for this new Mls gene product(s) is discussed.     

Altered expression of self-reactive T cell receptor V beta regions in autoimmune mice.

Intrathymic tolerance results in elimination of T cells bearing self-reactive TCR V beta regions in mice expressing certain combinations of I-E and minor lymphocyte stimulatory (Mls) phenotypes. To determine if autoimmune strains of mice have a defect in intrathymic deletion of self-reactive TCR V beta regions, expression of V beta 3, V beta 6, V beta 8.1, and V beta 11 were examined in lpr/lpr and +/+ strains of mice; MRL/MpJ(H-2K, I-E+, Mlsb,), C57BL/6J(H-2b, I-E-, Mlsb,), C3H/HeJ(H-2k, I-E+, Mlsc), AKR/J(H-2k, I-E+, Mlsa); and in autoimmune NZB/N(H-2d, I-E+, Mlsa) and BXSB(H-2b, I-E-, Mlsb) mice. The results suggest that, during intrathymic development, self-reactive T cells are deleted in autoimmune strains of mice as found in normal control strains of mice. However, the TCR V beta repertoire is skewed in autoimmune strains compared to normal strains of mice. For example, MRL-lpr/lpr mice, but not other lpr/lpr strains, had increased expression of V beta 6 relative to expression in control MRL(-)+/+ mice, which is associated with collagen-induced arthritis. These data are consistent with a model of normal affinity for negative selection of self-reactive T cells in the thymus of autoimmune strains of mice followed by expansion of autoreactive T cell clones in the peripheral lymphoid organs. The peripheral lymphoid organs of lpr/lpr mice contain an expanded population of abnormal CD4-, CD8-, 6B2+ T cells. Elimination of self-reactive peripheral T cells suggests that these abnormal cells are derived from a CD4+ subpopulation in the thymus. Flow cytometry analysis of peripheral lymph node T cells from MRL-lpr/lpr mice reveal three populations of CD4+ T cells expressing low, intermediate and high intensity of B220 (6B2). This supports the hypothesis that in lpr/lpr mice, self-reactive CD4+ T cells are eliminated in the thymus, and that these cells lose expression of CD4 and acquire expression of 6B2 in the periphery.     

Activation requirements of cloned inducer T cells. III. Need for two stimulator cells in the response of a cloned line to Mls determinants

To gain insight into the nature of Mls determinants, we examined the stimulator cells responsible for the activation of inducer T cell clones by Mls determinants. Two types of clones responding to Mls determinants were identified. One type responded to purified B cells, but not to splenic adherent cells (SAC), from mice bearing Mls stimulatory determinants. The other type of Mls-reactive T cell clone, including the representative clone Ly1-N5, demonstrated a vigorous response to unfractionated spleen cells, but showed little or no response to B cells alone or to SAC alone from mice bearing the Mlsa or Mlsd stimulatory determinant. The response of these clones to Mls determinants required stimulation by two cell types. The failure of clone Ly1-N5 to respond to Mlsa-bearing B cells was reversed by the addition of SAC taken from mice bearing the Mlsa allele. In addition, SAC from mice bearing the nonstimulatory Mlsb allele could synergize with B cells from Mlsa-bearing animals. B cells were required to provide the Mlsa determinant, because the combination of Mlsa-bearing SAC and Mlsb-bearing B cells did not activate the clone. The response of clone Ly1-N5 to Mls is restricted by Ia determinants (shared by H-2b, H-2d, and H-2k haplotypes but not by the H-2q haplotype). The permissive H-2 alleles can be present either on the stimulator B cell or on the SAC. The optimal response of the clone was obtained by using B cells bearing Mlsa and the permissive Ia epitopes. However, a significant response of the clone to B cells bearing Mlsa but an inappropriate Ia (Iaq) was also seen in the presence of SAC bearing the nonstimulatory Mlsb allele but the permissive Ia epitopes.     

Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens.

In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.     

Nonresponsiveness to Mlsd in F1 hybrid mice carrying Mlsa and Mlsc genes

Neither the biological function nor a basic understanding of the enigmatic chromosome 1-encoded Mls locus of the mouse has yet been uncovered despite extensive investigations. The present report is a continuation of our genetic analyses of the Mls locus in an attempt to better define the system. Data presented here indicate that in contrast to cells of mice expressing either the Mlsa or Mlsc allele which respond in mixed leukocyte reactions to cells expressing the Mlsd allelic products, cells from (Mlsa X Mlsc)F1-hybrid mice do not. In addition, the nonresponder phenotype appears to segregate as a single autosomal genetic system in backcross animals. These findings fail to support two recently advanced hypotheses: first, that the Mls locus is nonpolymorphic, or second, that the Mls locus controls differential expression of Ia antigenic determinants. Although the mechanism by which a (responder X responder) converts to a nonresponder remains unknown, three models involving gene complementation are discussed.     

T cell responses to Mls determinants are restricted by cross-reactive MHC determinants

The studies presented here investigated the relationship between T cell recognition of MHC-encoded products and non-MHC-linked Mls determinants. The first aspect addressed whether Mls-reactive T cells recognize Mls-encoded products alone or in association with MHC-encoded determinants. Initial studies used Mlsa-specific T cell clones that were generated by repeated stimulation of C57BL/6 or B10.A(5R) spleen cells with DBA/2 lymphoid cells. These clones recognized Mlsa on cells expressing MHC products of the H-2b, H-2d, and H-2k haplotypes, but not the H-2q haplotype. Thus, these cloned T cells were found to recognize Mlsa products in association with public but demonstrably polymorphic H-2 determinants. The question of whether T cell clones that were specific for self-H-2 determinants (autoreactive) or soluble antigen plus syngeneic H-2 (antigen-specific) could also be stimulated by Mlsa determinants was also addressed. A substantial proportion of the antigen-specific or autoreactive T cell clones tested were stimulated by Mlsa determinants. Furthermore, stimulation of these clones by Mlsa was H-2 restricted. The pattern of H-2-restricted recognition of Mlsa by these clones was not distinguishable from that observed in the Mlsa-specific T cell clones, nor was it influenced by the primary specificity or H-2 restriction pattern of a given clone. Although these findings provide a means of explaining the observation that Mls-reactive T cells exist at extremely high precursor frequencies, they also raise questions regarding the nature of the receptor structures which are used by a single T cell in the recognition of two or more apparently distinct stimuli.     

Effect of Mlsa on antigen presentation to class II-restricted T cells

The nature of the gene products encoded or regulated by the minor lymphocyte-stimulating (Mls) loci remains enigmatic despite extensive experimental evaluation. This work tested the hypothesis that the Mlsa genotype, when compared to the Mlsb genotype, facilitates Ag presentation to class II-restricted T cells. Titrated numbers of H-2-identical, Mls-disparate APC were used to stimulate proliferation of autoreactive, alloreactive, or Ag-specific class II-restricted T cell clones or lines. Apparent preferential presentation by Mlsa vs Mlsb APC obtained from H-2-identical strains was seen infrequently, and when observed, analysis with the use of APC from recombinant inbred lines revealed that preferential presentation did not correlate with the Mls genotype of the APC. These studies show that the Mlsa genotype does not influence overall Ag presentation to class II-restricted T cells.     

Isolation and characterization of an I-A-restricted T cell clone with dual specificity for poly(Glu60Ala30Tyr10) (GAT) and Mlsa,dl

We have isolated a BALB/c (H-2d, Mlsb) T cell clone (JTL-G12) specific for the synthetic polypeptide antigen poly(Glu60Ala30Tyr10) (GAT) in the context of self I-A determinants and for Mlsa,d antigens in the absence of GAT. JTL-G12 proliferation in response to GAT was mapped to the Kd, I-Ad subregions by using inbred H-2 congenic and recombinant strains. In addition, monoclonal antibody directed against I-Ad but not Kd or I-As determinants blocked JTL-G12 proliferation in response to GAT presented by syngeneic splenocytes, indicating I-A restriction. The Mls cross-reactivity of this clone was verified by using a panel of inbred strains bearing the Mlsa,b,c,d alleles and by using BXD recombinant inbred strains bearing the Mlsa allele or the Mlsb allele. All of the Mlsa BXD strains of the H-2d or H-2b haplotypes stimulated JTL-G12 in the absence of GAT, whereas all of the Mlsb BXD strains were nonstimulatory. This response pattern is in complete accordance with recognition of the Mlsa determinant encoded by Mls or closely linked loci on chromosome 1. JTL-G12 proliferation in response to GAT/I-Ad and Mlsa,d determinants could be blocked with a monoclonal antibody (GK1.5) directed against L3T4, a structure involved in class II major histocompatibility complex antigen recognition. These results suggest that antigen/class II responsiveness, Mls reactivity, and expression of L3T4 can be properties of a single T cell population.     

The immunobiology of the T cell response to Mls-locus-disparate stimulator cells. I. Unidirectionality, new strain combinations, and the role of Ia antigens

The primary mixed lymphocyte reaction of T cells to Mls-locus-disparate stimulator cells differs from that to non-self Ia antigens in several respects. In the present experiments, the unidirectional nature of this response is shown in several strain combinations, including the newly detected Mlsa and Mlsa-like alleles expressed by strains PL/J, RF/J, and SM/J. All of these strains stimulate MHC-identical T cells strongly. In addition, they stimulate a variety of cloned T cell lines specific for Mlsa,d, which can thus be shown to respond to Mlsa,d stimulators of the H-2b,d,k,u, and v haplotypes. Although these results suggest that primary T cell responses to Mlsa,d are unlikely to be MHC restricted, these primary responses are readily inhibited by monoclonal antibodies specific for the I-A and especially the I-E products borne by the stimulator cells, as well as by monoclonal antibodies specific for L3T4a on the responding T cells. This effect of anti-Ia antibodies is not overcome by exogenous interleukin 1. Thus, I-A and especially I-E molecules are centrally involved in the unidirectional primary T cell response to the potently stimulating Mlsa and Mlsd alleles expressed by cells of several different MHC haplotypes.     

Structural analysis of the mouse T-cell receptor Tcra V2 subfamily

Cosmid clones containing T-cell receptor Tcra V2 subfamily gene segments have been isolated from a BALB/c cosmid library and subjected to DNA sequence analysis. The V gene segments in the Tcra V2 subfamily differ from each other by 3%–7% at the nucleotide level and 5%–16% at the amino acid level. T-cell receptor Tcra V2 gene segment polymorphisms have been identified in the B10.PL and PL/J mouse strains with a Tcra V2 subfamily-specific probe. These V gene segment polymorphisms may cause the differential Tcra V gene usage in induced experimental allergic encephalomyelitis between B10.PL and PL/J mice.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession numbers U04312 and U04622-U04626     

Regulation of the in vitro presentation of minor lymphocyte stimulating determinants by major histocompatibility complex-encoded immune response genes

Activation of murine B lymphocytes in a splenocyte stimulator population with affinity-purified goat anti-mouse IgD (G alpha M delta) antibody was previously shown by this laboratory to enhance the presentation of strongly stimulatory major histocompatibility complex (MHC) and minor lymphocyte-stimulating (Mlsa,d) determinants in a primary mixed lymphocyte reaction. In the present study, the G alpha M delta treatment of murine splenocytes was employed to enhance the detection of the weakly stimulatory non-MHC Mlsc determinant in order to study the role the MHC might play as a restricting element for the recognition of these minor antigens in a primary mixed lymphocyte reaction. Indeed, enhanced T cell proliferation to Mlsc determinants presented on G alpha M delta-treated splenocytes was observed when the responder and activated H-2-compatible stimulator cell shared certain MHC haplotypes. High responsiveness was associated with the H-2a,k,j,p haplotypes, intermediate responsiveness was associated with the H-2f,g haplotypes and low responsiveness was associated with the H-2b,s haplotypes. (Low X high responder)F1 T cells preferentially responded to the Mlsc determinants presented on G alpha M delta-treated stimulator cells of the F1 or parental high responder H-2 haplotype. When mitomycin C instead of irradiation was used to inactivate normal (non-IgD-treated) splenocytes, a similar preferential response of T cells to Mlsc determinants presented on stimulator cells of a high responder H-2 haplotype was also observed. The inability of G alpha M delta-treated splenocytes of the low responder haplotype to elicit substantial levels of T cell proliferation across an Mlsc difference could not be attributed to the failure of these stimulator cells to become activated by the anti-Ig antibody. In addition, co-culture experiments could not identify the poor T cell response to Mlsc determinants presented on certain MHC haplotypes as being caused by the induction of nonspecific suppressor cells. Presentation of Mlsc determinants caused by transgene product complementation was detectable in F1 mice derived by crossing one parent that had the Mlsc non-MHC genes and a poorly permissive H-2 haplotype with a parent that expressed a permissive H-2 haplotype but lacked the Mlsc non-MHC genes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The expression of Mls c determinants on Mls a,Mls b,and Mls x prototypic strains

In the mouse sytem, specific determinants other than major histocompatibility complex (MHC) gene products are capable of inducing strong primary proliferative responses in naive T cells. These determinants are encoded by at least two gene loci designated as minor lymphocyte stimulatory (Mls) loci. In order to elucidate the biological role of the Mls system, an effort has been initiated to clarify the fundamental immunogenetic characteristics of the Mls system. In this report, we describe the unexpected finding that Mls ^c determinants are expressed on splenocytes of strains including those which have been used as prototypic examples of three other Mls types: Mls ^a (DBA/2, DBA/1), Mls ^b , (BALB/c), and Mls ^x (PL/J). The expression of Mls ^c by these strains was demonstrated both by the response patterns of unprimed T cells from MHC-identical inbred or F₁ hybrid strains and by the responses of a panel of Mls-specific T-cell clones. The experimental results reported here also suggest that the expression of Mls determinants may be influenced by multiple other genes, including MHC-linked genes.Abbreviations used in this paper MHC major histocompatibility complex - MLR mixed lymphocyte reaction - Mls minor lymphocyte stimulating locus antigen - MMC mitomycin C - NNT nylon wool nonadherent T cells     

Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes

V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.     

Genetic and nongenetic control of the immune response of mice to a synthetic glycolipid, stearylisomaltotetraose

Evidence for genetic control of antibody response to stearylisomaltotetraose (ST-IM4), a chemically defined synthetic glycolipid, was studied in various mouse strains. Anti-glycolipid antibodies were induced by repeated injections of ST-IM4 in complete Freund's adjuvant. C57BL and CBA/J mice were found to be good responders, while A/J, SJL/J, and AKR/J mice were poor responders. Responsiveness is independent of the H-2 genotype since AKR/J and CBA/J mice share the same H-2 locus. In addition, B10.A and B10.PL mice developed antibody levels similar to those of C57BL. The immunoglobulin heavy-chain locus (IgCH) was also found not to control antibody levels to ST-IM4 since C57BL and SJL/J mice share the same IgCH allotype. In C58/J mice, wide variation in response was observed among individual mice. Study of the response to ST-IM4 in 12 breeder pairs from different family lines of the C58/J colony also indicated that the regulation of the immune response to ST-IM4 is apparently more complex than can be explained by single gene control. C58 mice, unlike many other strains, had very low preimmune titers.     

Deficiency of the murine fifth complement component (C5). A 2-base pair gene deletion in a 5'-exon

To ascertain the molecular mechanism that causes murine C5 deficiency, genomic and cDNA libraries were constructed from mouse liver DNA and mRNA employing the congenic strains B10.D2/nSnJ and B10.D2/oSnJ that are sufficient and deficient for C5, respectively. Genomic fragments were isolated which correspond to PvuII and HindIII restriction fragment length polymorphisms associated with C5 deficiency. Sequence analyses demonstrated that each of these polymorphisms resulted from single base pair substitutions and that neither substitution would probably cause or contribute to the C5 deficiency. Sequence analyses of C5 sufficient and deficient cDNAs revealed a 2 base-pair deletion in the deficient cDNAs. The "TA" deletion was located near the 5' end of the cDNA. This deletion shifts the reading frame of the C5 mRNA so that the termination codon UGA is present 4 base pairs downstream from the deletion. Genomic DNA was amplified and sequenced corresponding to the area surrounding the 2-base pair deletion. Six C5-deficient strains, A/HeJ, AKR/J, DBA/2J, NZB/B1NJ, SWR/J, and B10.D2/oSnJ, and four C5-sufficient strains, Balb/CJ, C57Bl/6J, DBA/1J, and B10.D2/nSnJ, were analyzed. The sequencing data revealed that the 2 base pairs were deleted from the C5 gene of each deficient mouse tested but not from the C5 gene of any sufficient mouse. These data demonstrate that: 1) there is an identical 2-base pair deletion in an exon of the C5 gene in several different C5-deficient mouse strains; 2) the mRNA transcribed from the C5D gene retains this deletion; and 3) this mutation should result in C5 protein deficiency.     

Characterization of the Mlsf system. II. Identification of mouse mammary tumor virus proviruses involved in the clonal deletion of self-Mlsf-reactive T cells.

The genetic linkage of loci encoding stimulatory Mlsa and Mlsc determinants with proviruses of mouse mammary tumour viruses (MMTV) has been shown. We previously have reported that the ligand(s) for V beta 5, V beta 11, and V beta 12 behaves as a novel minor lymphocyte-stimulating (Mls) determinant(s), Mlsf, to induce the strong proliferation of unprimed T cells, and that this ligand(s) also functions as a self-Ag for the clonal deletion of self-reactive T cells. In the accompanying paper (Part I), a unique polymorphism characteristic of the Mlsf gene product is presented. In order to determine the genetic basis for this novel Mls system, we examined the progeny of multiple genetic crosses to identify the MMTV proviral loci involved in the clonal deletion of self-Mlsf-reactive T cells. Results from these investigations indicated that at least three known MMTV proviruses, Mtv-8, Mtv-9, and Mtv-11 are involved in the expression of Mlsf gene products. Presence of Mtv-9 results in the complete deletion of V beta 5, V beta 11, and V beta 12; Mtv-8 is associated with the complete deletion of V beta 12, but only a partial deletion of V beta 11 (primarily CD4-positive T cell subset) with little or no deletion of V beta 5; and Mtv-11 induces the complete deletion of V beta 11 and V beta 12, but no deletion of V beta 5. Given the significant sequence homology in the C-terminal portion of the open reading frame (ORF) region among these three MMTV and the almost equivalent effect of these three MMTV provirus upon the V beta 12 repertoire, their apparent hierarchic effect upon the V beta 5 and V beta 11 repertoires suggests that affinity differences in recognition of the same determinant by different TCR V beta may play a significant role in the clonal deletion of self-reactive T cells.