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Clonal analysis of the Mls system. A reappraisal of polymorphism and allelism among Mlsa, Mlsc, and Mlsd

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Abstract

Only two sets of antigenic determinants are recognized by T lymphocytes at uniquely high precursor frequencies: those encoded by the MHC and those encoded by Mls. The structural as well as functional characteristics of MHC products have been extensively analyzed. In contrast, little information concerning the nature of Mls genes or their products is available. Although it was originally described (5, 6) that the Mls locus on chromosome 1 is composed of four alleles that encode polymorphic cell surface structures, the issues of polymorphism and allelism in the Mls system have been controversial for some time. In the present study, T cell clones were generated by continuous stimulation of B10.BR (H-2k, Mlsb) T cells by CBA/J (H-2k, Mlsd) stimulators and they were used to analyze the relationship of putative Mlsa, Mlsc, and Mlsd determinants. All clones proliferated in response to determinants expressed by CBA/J stimulators. In addition, each of these clones exhibited a second reactivity to either AKR/J (H-2k, Mlsa) or C3H/HeJ (H-2k, Mlsc) stimulators. No clone responded to both AKR/J and C3H/HeJ. These second specificities were defined to be for Mlsa or Mlsc determinants, respectively, by the response patterns of clones and unprimed T cells to stimulators derived from congenic strains, recombinant inbred (RI) strains, and backcross mice. Moreover, a segregation analysis of the (CBA/J X B10.BR)F1 X B10.BR backcross indicated that the Mlsa-like and Mlsc-like determinants expressed on CBA/J (Mlsd) cells are in fact encoded by nonallelic, unlinked genes. These findings suggest a new concept of the polymorphism and genetics of the Mls system. It is proposed that two distinct and nonallelic gene products express, respectively, the noncrossreacting Mlsa and Mlsc determinants, and that the Mlsd phenotype does not represent an independent genotype but rather reflects the concurrent expression of Mlsa and Mlsc. The Mls system, therefore, consists of at least two systems that are distinct both genetically and antigenically, and that may be of different biologic or physiologic significance as well.

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Article
Festenstein originally described the Mls locus as a single dominant autosomal gene with four alleles which mapped in the 13th linkage group of chromosome 1. We subsequently presented evidence which indicated that the mixed leukocyte reaction stimulatory products of DBA/2 and CBA/J were controlled by two independently segregating Mls loci. Recently, Mls d of CBA/J was shown to be composed of Mls a of AKR and Mls c of C3H. In the present report, classic segregation data is presented which indicates that Mls c of C3H is controlled by three independently segregating loci. As defined by stimulatory patterns of numerous cell lines, we postulate the following: either one of the loci is shared with BALB.K, CE, C58, and partially with MA/MyJ, one is shared with CBA/H and CBA/J, and one is shared with BALB.K, CBA/J, and partially with CE; or the groups of shared determinants are controlled by different alleles of unique loci (or locus). In any event, Mls c appears to be composed of at least three independently segregating loci; the number of alleles/locus is being investigated. In addition, C3H was stimulated by BALB.K (both were recently postulated to be Mls c ); this epitope was shared with CBA/J, CBA/H, AKR/Cum, Ma/MyJ, and C58/J.
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Article
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Article
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Article
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Article
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Article
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Article
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Article
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Article
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Article
Recently a series of endogenous and exogenous superantigens have been described which have one common feature, namely, they lead to in vivo deletion and in vitro stimulation of T cells expressing particular T cell receptor V beta genes. The Mls antigens represent the prototypes of these molecules. We have mapped Mls-1 to the endogenous mammary tumor virus (MMTV) Mtv-7, while other SAG have also been associated with various MMTV. The open reading frame gene of the MMTV encodes the SAG. Thus, the new terminology MMTV sag has been proposed for this gene. Transfection experiments suggest that the expression of MMTV sag is tightly controlled, probably by a negative acting factor encoded within the open reading frame. Furthermore, a pronounced IL-4 effect is seen in the functional detection of the transfected Mtv-7 sag. Since this lymphokine does not influence the mRNA level of the endogenous or transfected MMTV genes, it is likely that it exerts its effect by increasing transcription of MHC class II genes, whose products are required for functional detection of Mls. We have identified one mouse strain, MA/MyJ, which has an Mls-1 phenotype but does not contain Mtv-7. The SAG activity of this strain was mapped to a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. Sequence analyses revealed that the predicted amino acid sequences of the Mtv-7 and the Mtv-43 sag genes are very similar. This is particularly striking in the C-terminus, where all other MMTV sag sequences differ 100%. Thus, this region of the molecule seems to control the V beta specificity of SAG molecules. It is likely that the SAG expression provides an advantage for the infectious MMTV, probably by facilitating its transmission by T cells from the site of primary residence in the gut to its final destination, the mammary glands.
Article
The Mls gene products, which have long been known for their potent T-cell stimulatory function, have recently come of age through two pivotal discoveries, revealing that they act as superantigens and originate from retroviruses. In addition, recent experiments suggest that two retroviruses, the murine B-type mammary tumor virus and the human lentivirus HIV, make use of the T-cell stimulatory capacity of a virally encoded superantigen for facilitating viral replication.
Article
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Article
Staphylococcal enterotoxins and a group of related proteins made by Streptococci cause food poisoning and shock in man and animals. These proteins share an ability to bind to human and mouse major histocompatibility complex proteins. The complex ligand so formed has specificity for a particular part of T cell receptors, V(β), and by engaging V(β) can stimulate many T cells. It is likely that some or all of the pathological effects of these toxins are caused by their ability to activate quickly so many T cells. It is also possible that encounters with such toxins have caused mice, at least, to evolve mechanisms for varying their T cell V(β) repertoires, such that they are less susceptible to attack by the toxins.
Article
The mechanisms responsible for major histocompatibility complex (MHC)-linked unresponsiveness are still poorly understood. Here we examine the cellular events that follow when B10. A mice are immunized with cow insulin, an antigen to which they make no apparent immunologic response. Despite the fact that there is no detectable antibody or T cell proliferative response to cow insulin, we have been able to clone out responding T cells after priming and restimulating in vitro with this "nonimmunogenic" antigen. These cells are L3T4+, and co-recognize specific antigen and class II MHC gene products. The data demonstrate that "nonresponder" mice to cow insulin have both the capacity to present antigen and T cells capable of recognizing that antigen. The diversity within this population was investigated by analyzing various parameters of cellular activation. These include fine specificity of both antigen and MHC recognition, as well as recognition of allogeneic MHC and M1s determinants. In addition, the antigen-presenting cell requirements were studied. The results demonstrate that this population comprise a surprisingly heterogeneous group in terms of its repertoire of receptors.
Article
Just as the development of B cell hybridization techniques and the availability of B cell monospecific products (monoclonal antibody) have revolutionized basic immunological approaches, recently established techniques for T cell cloning have provided another powerful tool. Experiments utilizing T cell clones have provided information relevant to T cell specificity, function, and the biochemistry of the T cell receptor which had been difficult to obtain with polyclonal T cell populations. In this communication, we describe a “clonological analysis”, i.e. the use of cloned T cells for genetic analysis of the strongly stimulatory, yet serologically undefined, T cell stimulatory determinants of the Mls (minor lymphocyte stimulatory locus) system.
Article
Two distinct suppressor systems have been described that are capable of down-regulating in vivo generation of cytotoxic T cells directed toward haptenaltered self-antigens. One system, induced by hapten, involves three T cells that others have shown to function sequentially to suppress DTH. The initiator of this cascade is a T cell that is readily induced in spleens of mice injected intravenously with syngenic membrane-coupled hapten. This Ts, when triggered by the same syngeneic membrane-coupled hapten that induced it, elaborates a factor. The other two Ts arise in lymph nodes and spleens of mice painted epidermally with hapten. One of the two Ts in this set is readily armed by the factor of the first Ts. The factor confers its specificity and genetic restriction upon the accepting Ts. The latter, when properly triggered, makes a factor that is taken up by its companion Ts, which actually suppresses by way of a nonspecific factor. Whereas this Ts cascade is operative at the efferent limb of DTH, it mediates suppression only at the afferent phase of the CTL response. A distinctly different suppressor system is induced by minor locus (Mls) antigen. When Mlsd lymphoid cells are injected intravenously into Mlsc-possessing mice, an Lyt-1+ T-suppressor cell is generated that can be found in the spleen as well as among peritoneal exudate cells. This Ts interacts with macrophages to accomplish nonspecific suppression of the CTL response that is detectable both in vivo as well as in vitro. A Ts soluble product has been found to be effective to suppress CTL generation in vitro only when macrophages are present in culture. The macrophage that accomplishes suppression is I-A-. Although the afferent limb of the CTL response is down-regulated by this suppressor system, our in vitro culturing system is so structured as to make the helper T cell inactive. Thus, the mechanism of suppression must be oriented to the other early participants in the response, namely, precursor CTL, helper and differentiation factors, and/or the antigen-presenting cell.
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Superantigens are bacterial, viral, or retroviral proteins which can activate specifically a large proportion of T cells. In contrast with classical peptide antigen recognition, superantigens do not require processing to small peptides but act as complete or partially processed proteins. They can bind to major histocompatibility complex class II molecules and stimulate T cells expressing particular T cell receptor V beta chains. The other polymorphic parts of the T cell receptor, which are crucial for classical antigen recognition, are not important for this interaction. When this strategy is used a large proportion of the host immune system can be activated shortly after infection. The activated cells have a wide variety of antigen specificities. The ability to stimulate polyclonal B (IgG) as well as T cell responses raises possibilities of a role for superantigens in the induction of autoimmune diseases. Superantigens have been a great tool in the hands of immunologists in unravelling some of the basic mechanisms of tolerance and immunity.
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A high proportion of T cell clones derived from bulk cultures selected to M1s a,d determinants were found to have joint specificity for allo-H-2 determinants, and vice versa. Significantly, the patterns of H-2 alloreactivity shown by clones selected to M1sa,b determinants appeared to be random. The possible implications of these findings are discussed.
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Negative and positive selection procedures were used to establish whether the strong proliferative response of T cells to M1sa determinants is H-2 restricted. After negative selection of H-2 determinants in vivo, it was shown that T cells give high primary mixed lymphocyte reactions in vitro to M1sa determinants presented on H-2-incompatible stimulator cells. Other studies demonstrated that (a) negative selection of T cells to M1sa determinants on H-2-incompatible cells removed T cells with specificity for M1sa-bearing H-2-compatible cells, and (b) T cells primed in vitro or in vivo to M1sa determinants on H-2-compatible cells gave high secondary responses to M1sa determinants presented either on H-2-compatible or H-2-incompatible stimulator cells. From these data we conclude that T cells recognize M1sa determinants per se rather than an association of M1sa plus self or allo-H-2 determinants.
Article
A high proportion of T cell clones derived from bulk cultures selected to M1s a,d determinants were found to have joint specificity for allo-H-2 determinants, and vice versa. Significantly, the patterns of H-2 alloreactivity shown by clones selected to M1sa,b determinants appeared to be random. The possible implications of these findings are discussed.
Article
Linkage between theMls locus and the chromosome 1 markersDip-1 andald was detected using two sets of recombinant inbred strains. Linkage betweenMls andDip-1 was confirmed in the fifth and sixth backcross generations of an incipient congenic strain. The AKXL data indicate that the gene order isDip-1-ald-Mls. The recombination frequency betweenald andMls is estimated to be 0.07 ±0.05, based on the AKXL data. The recombination frequency betweenDip-1 andMls is estimated to be 0.18 ±0.04, based on all the available data.
Article
A sensitive mixed leukocyte microculture (micro MLC) system has been developed in which both proliferative and cytolytic responses can be detected from small numbers of responding alloantigen-reactive T lymphocytes. Cytolysis and proliferation in micro MLC were assessed by conventional 51Cr release and 3H-thymidine uptake assays, respectively. Optimal sensitivity of the system was dependent on several parameters, the most important of which was the presence of supernatant fluid derived from secondary MLC (2° MLC SN). Under optimal conditions, formal limiting dilution analysis could be achieved since excellent discrimination between positive and negative microcultures was obtained for both the cytolytic and proliferative assays. The system was therefore used to obtain simultaneously minimal estimates of the frequency of cytolytic T lymphocyte precursors (CTL-P) and of the precursors of proliferating T lymphocytes (PTL-P). The results of a series of experiments involving C57BL/6 spleen cells responding against DBA/2 alloantigens indicated that the minimal frequency of CTL-P was approximately 2-fold lower than the frequency of PTL-P in the same responding cell population (average of 1:570 and 1:290, respectively). Since analysis of individual micro MLC further demonstrated that essentially all CTL-P were contained within the PTL-P population, the simplest interpretation of these findings would be that approximately 50% of alloantigen-reactive T cells in this strain combination have the capacity to give rise to specific CTL. Some applications of this model system are briefly discussed.
Article
We have investigated primary and secondary responses of mouse splenic T cells to strong mixed lymphocyte stimulating antigens controlled by the Mls locus using MHC-identical mixtures of cells. Our studies show that strong primary Mls-locus specific responses involve recognition of self I-A antigens, since BUdR and light suicide or F1 into parent radiation bone-marrow chimeras both demonstrate a preference of unprimed F1 T cells to respond to Mls-locus antigens associated with one parent's MHC antigens. Furthermore, conventional anti-I-A antisera and monoclonal anti-I-A antibody both inhibit Mls-locus responses in an MHC-specific manner. Finally, as is typical of T cells responding to I-A antigens or to nominal antigens associated with self I-A, Mls-locus responses are mediated by Lyt-1+, 2 cells. One striking finding in these studies was the very high frequency of cells capable of responding to Mls-locus antigens, the highest being 1/300 splenic T cells. This plus evidence for recruitment during primary Mls-locus responses may account for reports of a lack of I-A restriction in secondary anti-Mls locus responses to strong Mls-locus antigens, a finding with which we concur. The possibility that these secondary responses between noncongenic strains of mice may be directed at other genetic loci is also discussed. These experiments leave open the question of the biological role of the Mls-locus and of the very large number of T cells reactive to it.
Article
Virtually all T cell responses involve recognition of MHC gene products, with the possible exception of responses to Mls locus antigens. Reactions to Mls locus antigens, however, are normally tested between MHC identical strains of mice, so responses to Mls locus antigens may also involve recognition of self MHC antigens, probably encoded in the I region. Alternatively, the Mls locus might represent I region gene duplications translocated to another chromosome. The present experiments were designed to test these ideas. The authors report that T cell responses to Mls locus antigens involve recognition of the products of two gene clusters. One gene cluster is clearly self MHC genes, probably within the I region, while the second is almost certainly the genes of the Mls locus.
Article
A sensitive mixed leukocyte microculture (micro MLC) system has been developed in which both proliferative and cytolytic responses can be detected from small numbers of responding alloantigen-reactive T lymphocytes. Cytolysis and proliferation in micro MLC were assessed by conventional 51Cr release and 3H-thymidine uptake assays, respectively. Optimal sensitivity of the system was dependent on several parameters, the most important of which was the presence of supernatant fluid derived from secondary MLC (2° MLC SN). Under optimal conditions, formal limiting dilution analysis could be achieved since excellent discrimination between positive and negative microcultures was obtained for both the cytolytic and proliferative assays. The system was therefore used to obtain simultaneously minimal estimates of the frequency of cytolytic precursors (CTL-P) and of the precursors of proliferating T lymphocytes (PTL-P). The results of a series of experiments involving C57BL/6 spleen cells responding against DBA/2 alloantigens indicated that the minimal frequency of CTL-P was approximately 2-fold lower than the frequency of PTL-P in the same responding cell population (average of 1:570 and 1:290, respectively). Since analysis of individual micro MLC further demonstrated that essentially all CTL-P were contained within the PTL-P population, the simplest interpretation of these findings would be that approximately 50% of alloantigen-reactive T cells in this strain combination have the capacity to give rise to specific CTL. Some applications of this model system are briefly discussed.
Article
CBA and AKR mice are identical at the major histocompatibility locus (H-2) but differ at the strong, non-H-2, mixed leukocyte culture (MLC)-stimulating M-locus. Adoptive secondary in vivo antibody responses using immune spleen cells from mice of these strains demonstrate two interesting findings. One is a non-specific augmentation of the anti-hapten antibody response of CBA B cells by AKR T cells. This allogeneic effect is unidirectional in the same sense as the MLC between cells from these two strains. The second is that, despite an ongoing allogeneic effect, M-locus-incompatible T and B cells show strong specific cooperation. Thus, it seems unlikely that the failure of H-2-incompatible T and B cells to collaborate specifically is due to inhibitory allogenic reactions.
Article
Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.
Article
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.
Article
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)
Article
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.
Article
Cloned, protein antigen-specific, Ia-restricted T cell lines frequently (approximately 20%) also respond strongly to stimulator cells from strains expressing stimulatory alleles at the chromosome 1-encoded Mls-locus. Furthermore, such responses are blocked by monoclonal antibodies specific for Ia antigens expressed by the stimulator rather than the responder cells. However, such responses show no specificity for polymorphic determinants on Ia molecules, although in such responses, as in primary and secondary T cell responses to stimulating Mls-locus alleles, I-E molecules appear to play a central role. These results, combined with the unique immunobiology of the primary T cell proliferative response to Mls-locus-disparate stimulator cells, suggest to us that this response involves the interaction of the receptor on T cells for antigen:self Ia with a relatively nonpolymorphic region of Ia glycoproteins. This hypothesis is supported by the observation that a monoclonal antibody to the T cell receptor will inhibit both responses, although the response to Mls-locus-disparate stimulators appears to be more sensitive to these antibodies. We propose that the interaction of the T cell receptor with Ia is stabilized by a cell interaction molecule encoded or regulated by the Mls-locus gene product permitting the T cell receptor:Ia glycoprotein interaction to lead to T cell activation.
Article
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.
Article
To investigate whether small B cells can stimulate mixed lymphocyte reactions (MLR), highly purified populations of large vs. small B cell fractions were tested for their capacity to evoke MLR across Mls vs. H-2 barriers. Large B cell fractions stimulated high MLR to Mls and H-2 determinants, irrespective of whether the stimulators were exposed to irradiation or pretreated with mitomycin C. In accord with the findings of others, irradiated small B cell fractions proved to be very poor stimulators of MLR. Significantly, however, mitomycin C-treated small B cell fractions elicited high MLR, particularly to Mls determinants. The finding that small B cell fractions treated with irradiation are poor stimulators of T cells correlates with the known radiosensitivity of B cells. In this respect, the widely held view that small B cells do not have antigen-presenting cell (APC) function rests largely on studies with irradiated B cells. The present finding that T cells respond well to small B cells treated with mitomycin C, however, indicates that small B cell fractions do have APC function. Whether the APC function of small B cells reflects a response to resting B cells per se rather than to cells undergoing activation in vitro, however, remains to be ascertained.
Article
The Mls locus was originally defined to have four alleles; all controlled products that were detectable in MLR except b, which was described as being null. More recent evidence led other investigators to postulate that the Mls locus is nonpolymorphic, being composed of only the b null allele and a singly expressed allele previously ascribed to be the a and d alleles. Our results indicate that Mlsa and Mlsd control products that are antigenically distinct and, therefore, the products cannot be controlled by the same allele. In addition, the product of Mlsb was easily detectable by Mlsa and Mlsd responding cells and cannot be considered null. Alternative explanations are considered for these conflicting results.
Article
The mixed leukocyte culture (MLC) test is an in vitro model of the recognition phase of the homograft response. For the most part, activation in MLC is dependent on differences of the major histocompatibility complex (MHC). Our present studies in the mouse suggest that activation is primarily associated with differences of genetic regions of the MHC other than those which control the serologically defined (H-2) antigens. These differences do not lead to cytotoxic or agglutinating antibody formation despite extensive immunization; we have called these differences lymphocyte-defined (LD) differences. The strongest stimulation in MLC is associated with differences of the Ir region. It is possible that the Ir product is the T cell receptor and that it is this same molecule which can act as the stimulatory agent in MLC. Other possibilities are discussed.
Article
Mixed lymphocyte reactions occur when mouse spleen cell populations depleted of thymus-derived (T) lymphocytes are cultured with allogeneic target cells inactivated by mitomycin C or X irradiation, and when F(1) hybrid responder cells are cultured with inactivated parental target cells. These responses might be interpreted as indicating that T lymphocytes are not required for responsiveness and that F(1) lymphocytes recognize parental alloantigens. Data reported here indicate that the more likely explanation for these surprising results is that inactivated target cells recognize the "responding" cells and this recognition leads to the response observed.
Article
To examine the relationship of T cell specificity for Mls vs H-2 determinants, BALB/c (H-2d,Mlsb)(d,b) T cells were stimulated repeatedly in vitro with H-2-compatible, Mls-incompatible DBA/2(d,a) stimulators. This line of T cells gave strong mixed-lymphocyte reactions to the priming Mlsa determinants but, in addition, gave appreciable responses to various foreign H-2 determinants. When this T cell line was subsequently stimulated over a period of 2 mo with Mlsa-negative cells of a particular foreign H-2 haplotype, e.g., H-2k, the cells gave high responses to H-2k determinants but only very low responses to third-party H-2 determinants. Significantly, the cells retained high reactivity for Mlsa determinants. In other experiments, BALB/c T cells positively selected to Mlsa,d-negative H-2-incompatible stimulator cells retained high reactivity for Mlsa determinants. The implications of these findings are discussed.
Article
Gene products of the murine Mls locus elicit high primary mixed lymphocyte reactions (MLRs). Although this locus is considered to have four alleles, only two of these alleles, i.e., Mlsa and Mlsd, give strong responses in primary MLRs. This paper presents a series of experiments to show that Mlsa and Mlsd are in fact antigenically indistinguishable. Strong Mls determinants thus appear to be nonpolymorphic.
Article
To test whether M1s determinants, like other non-MHC or nominal antigens, are recognized by T-cells in association with H-2 determinants, the in vitro proliferative responses of T-cell lines and clones were studied. Lines and clones were prepared by soft agar cloning (B10.BR x BALB/c)F1 (H-2k/H-2d, M1sb/M1sb) T-cells responding in a primary MLR to AKD2F1 (H-2k/H-2d, M1sa/M1sa) stimulator cells. All the T-cell clones obtained could respond equally well in a proliferative assay to the Mlsa determinant in association with the H-2 haplotype of either parent, i. e., DBA/2 (H-2d, M1sa), and AKR (H-2k, M1sa) both stimulated equally well. When the T-cell lines and clones were screened against stimulators from recombinant inbred (RI) strains, it became apparent that strains exhibiting the H-2b, M1sa genotype stimulated poorly or not at all. This shows that the T-cell response to M1sa involves MHC recognition, and raises the possibility that the response to M1sa can involve recognition of H-2 specificities shared between the H-2k and H-2d haplotypes.
Article
The injection of anti-IgD antibody into mice has been shown to increase the expression of Ia antigens on splenic B cells. These antigens are the most potent lymphocyte-activating determinants (LAD) that trigger proliferation in an H-2-defined mixed lymphocyte reaction (MLR) and may play a role in the recognition of minor lymphocyte-stimulating (MIs) determinants. Therefore, we wished to investigate a possible correlation between apparent quantitative alterations in B cell Ia expression after anti-IgD activation with changes in the functional capacity to present allogeneic major histocompatibility complex (MHC) and MIs antigens to responsive T cells. We observed that the capacity of splenocytes removed 24 hr after the in vivo injection of anti-IgD to stimulate T cell proliferation across an H-2 barrier was most frequently enhanced two- to fourfold when a suboptimal concentration of stimulator cells was used or an early time point in the MLR was examined. In contrast, the capacity of splenocytes to stimulate across an MIsa, d difference after exposure to heterologous or hybridoma anti-IgD antibody often was increased 10-fold or more. Optimal MLR stimulatory capacity was induced by injection of 100 to 200 micrograms of heterologous anti-IgD. Augmented MIs stimulatory capacity of spleen cells peaked 24 hr after such treatment and continued to decline from this value at day 3 and at day 7 after injection. In contrast, the H-2 stimulatory capacity increased 1 day after injection of anti-IgD and remained at that elevated level 3 and 7 days after injection. The spleen cells from B cell-defective (CBA/N x DBA/2)F1 male mice were unaffected in their capacity to stimulate across an MIsa barrier after in vivo anti-IgD treatment; however, spleen cells from phenotypically normal (DBA/2 x CBA/N)F1 male mice after exposure to anti-IgD did evidence a considerably enhanced ability to stimulate in an MIsa-defined MLR. Because anti-IgD antibodies presumably have their major initial effect on surface IgD-bearing B cells, these studies suggest that anti-immunoglobulin-activated B cells may have a role (direct or indirect via interaction with accessory cells) in the presentation of allogeneic MHC and MIs antigens to responsive T cells.
Article
In the mouse, potent primary in vitro proliferation of T cells can be induced by allelic variants of cell-surface glycoproteins, Ia antigens, the genes for which are located in the I region of the major histocompatibility complex (MHC) on chromosome 17. The only other potent primary proliferative response known is induced by mixing MHC-identical lymphocytes from strains that differ at the locus termed Mls (ref. 1) (for mixed lymphocyte stimulating), which has been mapped to chromosome 1. While it is relatively easy to raise antibodies against Ia antigens, and thus determine both their chemical nature and their role in T-cell stimulation, the nature of the product of the Mls locus has remained obscure. It has been proposed that the Mls locus product is a minor antigen recognized in association with self-Ia antigens, a translocated Ia-like element, or a mitogenic molecule found on the surface of antigen presenting cells (APC). Here, we demonstrate that APCs from mice carrying stimulatory Mls locus alleles present antigen more efficiently to cloned, antigen-specific, Ia-restricted T cells than APCs from mice carrying nonstimulating Mls locus alleles. We propose that the Mls locus does not encode a unique cell-surface antigen at all; we suggest instead that the T-cell proliferative response induced by Mls-locus disparate cells reflects recognition of self-Ia molecules on APCs. If our interpretation is correct, it provides further evidence both for the quantitative nature of self tolerance and for the existence of a distinct recognition site for self-Ia molecules on antigen-specific T lymphocytes.
Article
This report demonstrates the expression of strong MIs locus MIsd) recognition by a cloned line of H-2-restricted influenza virus-specific CTL. This clone of F1 (H-2b/d; MIsb) origin was found to specifically proliferate in response to uninfected cells of CBA/J (H-2k, MIsd) origin but not to uninfected B10.BR or CBA/CaJ cells (H-2k, MIsb). In addition, proliferation by this cTL line was observed in response to histocompatible cells expressing cross-reactive MIsa determinants (DBA/2, NZB; H-2d, MIsa). This recognition was observed only at the level of CTL proliferation. The CTL line exhibited no cytotoxic activity for target cells of these MIs types. These observations are contrasted with the response of another cloned H-2-restricted influenza-specific CTL line that simultaneously exhibits alloreactivity for H-2k. The significance of these results for T lymphocyte recognition is discussed.
Article
Three alloreactive T cell clones are described which reveal a new lymphocyte-stimulating determinant (Lsd) controlled by a gene on chromosome 1 of the mouse but different from and possibly centromeric to Mls. Two clones were originally isolated as anti-H-2b-specific and showed and retained cross-reactivity to Lsd (clone OD3, BALB/c anti-C57BL/6; clone KB37, B10.BR anti-C57BL/10). The third clone (BD7, C57BL/6 anti-CBA/J) was probably originally directed against Lsd. So far, Lsd is only characterized by stimulation of T cell proliferation, and its physiological function is not yet known.
Article
We have used an immunofluorescence inhibition assay to identify 2 BALB/c plasmacytomas, TEPC-1017 and TEPC-1033, that secrete large quantitites of IgD. Both TEPC-1017 and TEPC-1033 myeloma proteins bound to anti-kappa as well as hybridoma and heterologous anti-delta antibodies, but not to anti-mu, gamma, alpha, or lambda antibodies. Both myeloma proteins were purified by (NH4)2SO4 precipitation, ion exchange chromatography, gel filtration, and Staphylococcus aureus Protein A absorption. These IgD kappa myeloma proteins were used to prepare affinity purified rabbit antibodies to delta-chain and the TEPC-1017 and TEPC-1033 idiotypes. Native TEPC-1017 and TEPC-1033 both had mobilities between those of mouse IgA kappa dimers and trimers when analyzed by polyacrylamide gradient gel electrophoresis. Both IgD myeloma proteins broke down under mild reducing conditions into subunits with electrophoretic mobilities slightly slower than those of an IgA kappa monomer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced TEPC-1017 and TEPC-1033 demonstrated kappa-chains and heavy chains that co-migrated with alpha chain. These data suggested that secreted IgD contains 2 delta 2 kappa 2 subunits that are linked by an easily reducible disulfide bond. The kappa-chains of IgD secreted by TEPC-1017 and TEPC-1033 have apparent m.w. of approximately 63,000 daltons, whereas the apparent m.w. of intracytoplasmic delta-chain, intracytoplasmic delta-chain synthesized in the presence of tunicamycin, and the cellfree translation product of TEPC-1017 delta-chain mRNA are 54,000, 43,000, and 44,000 daltons, respectively. This is compatible with the interpretation that the delta-chain peptide has a leader sequence and is N-glycosylated during or shortly after peptide synthesis and is glycosylated further shortly before IgD secretion.
Quantitative studies on the mixed lymphocyte reaction . III . Kinetics of the response
  • I Wilson
  • J L Blyth
  • P C Nowell
I. Wilson, D. B., J. L. Blyth, and P. C. Nowell. 1968. Quantitative studies on the mixed lymphocyte reaction. III. Kinetics of the response. J. Exp. Med. 128 :1157.
The minor lymphocyte stimulating Mlsd determinants : a composite of Mlsa and MW antigens
  • J J Ryan
  • R Abe
  • R J Hodes
  • J J Mond
  • F D Finkelman
  • J N Woody
Ryan, J. J., R. Abe, R. J. Hodes, J. J. Mond, F. D. Finkelman, and J. N. Woody. 1986. The minor lymphocyte stimulating Mlsd determinants : a composite of Mlsa and MW antigens. Int. Congr. Immunol., 6th, Toronto. 153. (Abstr.)