Analysis of T hybridomas prepared from a T cell clone with three specificities. Recognition of self + X and allo-H-2 determinants segregates from recognition of Mlsa determinants

To see k information on T cell recognition of Mlsa determinants, hybridomas were prepared from a well-characterized F23.2+ (V beta 8.2+) T cell clone specific for three different ligands, i.e., 1) Mlsa determinants, 2) fowl gamma-globulin (F gamma G) plus self-H-2 (H-2d), and 3) allo-H-2, e.g., H-2p, determinants. Fusion of the clone to the BW5147 thymoma line produced a triple-reactive T hybridoma which generated two types of spontaneous variants. One type of variant (type I) lost Mlsa reactivity but retained reactivity to both F gamma G/H-2d and allo-H-2p. These variants, which were generated at high frequency, stained strongly with a mAb, A1.57, with idiotypic specificity for the TCR molecules of the parental clone. The second type of variant (type II) reacted to Mlsa determinants but showed no reactivity to F gamma G/H-2d or to allo-H-2p. These variants failed to express the A1.57 idiotypic determinants of the parent clone, but were F23.2+ (V beta 8.2+); nonequilibrium pH gradient electrophoresis analysis suggested that these hybrids expressed a mixed TCR heterodimer composed of the parental clone beta-chain and the BW5147 alpha-chain. Three aspects of the data are very difficult to accommodate with the view that Mlsa, F gamma G, and allo-H-2 determinants are all recognized via a common TCR molecule: 1) the independent (and frequent) segregation of Mlsa reactivity from F gamma G/H-2d and allo-H-2p reactivity, 2) the retention of Mlsa reactivity by the type II variants despite loss of the parental clone alpha-chain, and 3) the loss of Mlsa reactivity by the type I variants despite high expression of the A1.57+ TcR molecules derived from the parental clone. The data support a model in which Mlsa determinants are recognized by a separate T cell structure, which we envisage as a monomorphic accessory molecule unrelated to the TCR. Since the type II hybridoma variants invariably retained quantitatively normal TcR expression, the triggering phase of anti-Mlsa responses appears to be TCR dependent. The model we favor is that anti-Mlsa/Mlsa interaction increases TCR binding with Ia epitopes to above the threshold required for cell triggering. A key feature of this model is that Mlsa and Ia determinants are recognized as separate structures rather than as a complex.     

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.     

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.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. II. Significance of MHC antigens in anti-Mls tolerance

Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a popliteal lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of F1 hybrid mice. When thymus cells were obtained from 1-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/2)F1 (H-2d, Mlsb/a) [CDF1] recipients and of other hybrid recipients, heterozygous in Mlsa,c,d alleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not cause the response in MHC-heterozygous F1 hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDF1 bone marrow cells, complete tolerance to Mlsa-H-2d antigens of CDF1 cells was induced in the thymus, while responsiveness to Mlsa antigens in the context of H-2k and H-2b antigens, was not affected. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)F1 (H-2d/k, Mlsb/a) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mlsa-H-2k antigens as well as to Mlsa-H-2d antigens, which suggests the involvement of MHC, probably class II antigens of tolerance-inducing 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.     

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.     

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.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

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.     

Primary in vitro cytotoxic response of F1 T lymphocytes against parental antigens.

Spleen cells from adult female (AKR/J x BALB/(c)F1 mice can respond to mitomycin C-treated spleen from AKR/J mice and can generate effector CTL in a 5-day primary in vitro culture. The response is comparable in magnitude to the response to allogeneic H-2K or H-2D antigens. The response is T cell mediated and is directed to antigen(s) present only on the parental cells. The target cell must be homozygous at H-2Kk to be lysed and H-2Dk antigens do not serve as a target in this response. Spleen cells from (B10.BR x B10.D2) hybrids that have been stimulated with AKR/J lyse B10.Br as well as AKR/J target cells. Similar H-2k/d hybrid F1 anti-H-2k parent responses are seen in certain other strain combinations. A number of possible interpretations of these responses are discussed.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to Mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.     

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.     

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.     

Characterization of the in vitro murine T-cell proliferative responses to murine and human thyroglobulins in thyroiditis-susceptible and -resistant mice

The in vitro proliferative response to autoantigenic mouse thyroglobulin (MTg) of lymph node cells (LNC) from thyroiditis-susceptible (high-responder) CBA/J (H-2k) mice was further characterized. The relatively weak response was enhanced by adding irradiated spleen cells from normal syngeneic mice to cultures of responding LNC. Furthermore, the adjuvant used for immunization was found to influence the magnitude of the response. Results of experiments varying both the adjuvant and the route of immunization (footpad versus subcutaneous) demonstrated that marked proliferative response to MTg in vitro was not necessarily a predictor of the severity of disease. However, the capacity to proliferate in response to MTg correlated with disease susceptibility, as reported previously. The response to MTg was dependent on Thy-1+, Lyt-1+2- cells and was inhibited by monoclonal I-A antibodies. Thus, proliferation is mediated by T cells of the helper/amplifier phenotype recognizing the autoantigen in association with Ia molecules. The determinants on human thyroglobulin (HTg) and MTg stimulating the proliferative responses of LNC from thyroiditis-susceptible and thyroiditis-resistant (low-responder) BALB/c (H-2d) mice were found to differ. Cells from resistant mice proliferated only in response to foreign determinants on HTg and not to shared or mouse-specific epitopes of MTg, whereas susceptible mice had T cells reactive to shared determinants expressed on MTg and HTg as well as to foreign determinants on HTg.     

Comparison of antigen specificity, class II major histocompatibility complex restriction, and in vivo behavior of myelin basic protein-specific T cell lines and clones derived from (BALB/c x SJL/J) mice

Immunization with myelin basic protein (BP) causes experimental allergic encephalomyelitis (EAE) in certain strains of mice. SJL/J (H-2s) is the prototype sensitive strain. Although BALB/c (H-2d) is resistant to EAE through use of an identical immunization protocol, (BALB/c x SJL/J)F1 hybrid mice develop EAE after immunization with BP. T cell clones specific for BP have been isolated from a highly encephalitogenic line of (BALB/c x SJL/J)F1 hybrid T cells raised against bovine BP. The clones were examined for their H-2 restriction and specificity for heterologous forms of BP (mouse, rat, and bovine BP). The results revealed the clones cross-reacting with mouse (self) BP were almost always restricted to F1 hybrid class II major histocompatibility complex (MHC) elements. In contrast, mouse cross-reactive clones derived from a nonencephalitogenic (BALB/c x SJL/J) T cell line raised against rat BP were largely restricted to H-2d elements. These clones did not cross-react with bovine BP. Four additional lines were generated by carrying the original rat and bovine F1 T cell lines on parental antigen-presenting cells thus generating lines biased toward homozygous (SJL/J, H-2s, or BALB/c, H-2d) restriction elements. These "parentally restricted" T cell lines did not induce EAE when injected in vivo. These results suggest that in this F1 strain sensitivity to T cell-induced EAE is associated with epitopes on murine BP that associate with F1 class II MHC restricting elements. In contrast, nonencephalitogenic T cell lines contain a high proportion of murine cross-reactive clones restricted to H-2d, the haplotype of the classically resistant BALB/c mouse. This work illustrates the use of T cell lines and clones in a model system to further analyze the role of MHC restriction elements in autoimmune disease occurring in heterozygous individuals.     

Enhanced priming of multispecific,murine CD8+ T cell responses by DNA vaccines expressing stress protein-binding polytope peptides

A polytope DNA vaccine (pCI/pt10) was used that encodes within a 106-residue sequence 10-well characterized epitopes binding MHC class I molecules encoded by the K, D, or L locus (of H-2(d), H-2(b), and H-2(k) haplotype mice). The pCI/pt10 DNA vaccine efficiently primed all four K(b)/D(b)-restricted CD8(+) T cell responses in H-2(b) mice, but was deficient in stimulating most CD8(+) T cell responses in H-2(d) mice. Comparing CD8(+) T cell responses elicited with the pCI/pt10 DNA vaccine in L(d+) BALB/c and L(d-) BALB/c(dm2) (dm2) mice revealed that L(d)-restricted CD8(+) T cell responses down-regulated copriming of CD8(+) T cell responses to other epitopes regardless of their restriction or epitope specificity. Although the pt10 vaccine could thus efficiently co prime multispecific CD8(+) T cell responses, this priming was impaired by copriming L(d)-restricted CD8(+) T cell responses. When the pt10 sequence was fused to a 77-residue DnaJ-homologous, heat shock protein 73-binding domain (to generate a 183-residue cT(77)-pt10 fusion protein), expression and immunogenicity (for CD8(+) T cells) of the chimeric Ag were greatly enhanced. Furthermore, priming of multispecific CD8(+) T cell responses was readily elicited even under conditions in which the suppressive, L(d)-dependent immunodominance operated. The expression of polytope vaccines as chimeric peptides that endogenously capture stress proteins during in situ production thus facilitates copriming of CD8(+) T cell populations with a diverse repertoire.     

T cell allotypic determinants encoded by genes linked to the immunoglobulin heavy chain locus. I. Establishment of monoclonal antibodies against allotypic determinants

Monoclonal alloantibodies for T cell allotypic determinants were obtained by hybridizing SP-2 tumor cells with BALB/c (H-2d, Igh-1a) spleen cells, which had been repeatedly immunized with Con A-stimulated CB-20 (H-2d, Igh-1b) spleen cells. It was found that these monoclonal anti-CB-20 antibodies detect the new allotypic determinants (distinct from the B cell Igh-C region determinant) expressed only on the augmenting or suppressor T cells. Genetic analysis of these antigenic determinants revealed these antibodies react with the gene products located on the telomeric side chromosome of the Igh variable region gene (Igh-V) cluster. These antibodies when given in vivo caused a modification of antibody production. The antibody activity was absorbed by Con A-stimulated B10.BR (H-2b, Igh-1b), C57BL/6 (H-2b, Igh-1b), CWB (H-2b, Igh-1b), CB-20 (H-2d, Igh-1b), and BAB-14 (H-2d, Igh-1b) spleen cells, but not by Con A-stimulated C3H.SW (H-2b, Igh-1j), BALB/c (H-2d, Igh-1a), A/Sn (H-2a, Igh-1e), and C.AL-20 (H-2d, Igh-1d) spleen cells. In addition, in vivo these monoclonal antibodies modified anti-SRBC antibody production only in Igh-1b allotype-bearing mice. One monoclonal antibody reacted with 4-hydroxy-3-nitrophenyl acetyl- (NP) hapten-specific augmenting T cells, and the other two batches of monoclonal antibodies reacted with NP-specific suppressor T cells of NP-mediated cutaneous responses. A mapping study with these recombinants limits the gene coding for the T cell-specific determinants to a gene within the variable region to the telomeric side of NP-VH and to the centromeric side of prealbumin. This segment is inclusive of all immunoglobulin genes, the region Owen named IgT-C, and a histocompatibility gene (H-Ig).     

Immunogenicity of B chain in insulin responder and nonresponder mice.

The potential immunogenicity of insulin B chain in beef insulin low-responder H-2k,a and high-responder H-2b,d mice was examined using lymph node proliferation assays. Oxidized B chain was immunogenic in H-2k,a, but not H-2b,d, mice. The T cell population recognized a determinant in OX-B chain associated with I-Ak. These cells did not respond to intact insulin, suggesting that the B chain determinant was not available to I-Ak during immunologic processing of insulin. Responses were observed in H-2k and H-2d, but not H-2b, after immunization with reduced and carboxyamidomethylated-insulin which contains equimolar A chain and B chain. These responses were I-A-restricted and heterogeneous, with reactivity to A chain and B chain determinants. In each case, little or no cross-reactivity was observed between RCAM-insulin and intact insulin. Furthermore, T cell populations induced in H-2k mice selectively recognized OX-B chain or RCAM-B chain, which differ in chemical modification of the thiols of Cys B7 and Cys B19. Similarly, RCAM-BINS-immune T cells from H-2d did not react to OX-B chain. These results indicate that derivatization of the cysteine thiols, through disulfide bonds, oxidation, or carboxyamidomethylation, radically affects T cell recognition of insulin B chain.     

Murine IgA binding factors (IgA-BF) suppressing IgA production: characterization and target specificity of IgA-BF

Chemical and functional properties of IgA binding factor(s) (IgA-BF) from both murine Con A-activated spleen cells and Fc gamma R+, Fc alpha R+ T hybridoma cells (T2D4) were studied. IgA-BF produced from the cells after preculture with IgA were purified with IgA-Sepharose. Purified IgA-BF inhibited the binding of IgA to Fc alpha R+ L5178Y T lymphoma cells, and class-specifically suppressed in vitro IgA synthesis of the pokeweed mitogen (PWM)-stimulated murine spleen cells. Both IgA-specific suppressive activity and IgA binding activity of the factor(s) were co-fractionated between BSA and OVA in gel filtration analysis. SDS-PAGE analysis of IgA-BF biosynthetically labeled with [35S]methionine showed a specific band on 56,000. Suppressive activity of IgA-BF was absorbed with lentil-lectin-Sepharose and was eluted with 0.2 M alpha-methyl-D-mannoside. The suppressive activity obtained from T2D4 cells (H-2k) and BALB/c Con A blasts (H-2d) was absorbed with the corresponding anti-H-2 and anti-I-A column and recovered in the acid-eluate. The activity was not absorbed with the unrelated anti-H-2 column. Despite the presence of MHC products, IgA-BF from both cell sources equally suppressed IgA-specific responses of BALB/c (H-2d), C3H/He (H-2k), and C57BL/10 (H-2b) spleen cells. They also suppressed IgA production as well as IgA synthesis of PWM-stimulated culture of human peripheral blood lymphocytes without affecting IgM and IgG responses. Suppression of murine and human IgA responses both in mouse and human were mediated by the molecules having the same Ia products, suggesting that there is no MHC, as well as species restriction, for the interaction between IgA-BF and their target cells. IgA-specific suppressive activity was absorbed with human B blastoid cells bearing surface IgA (Dakiki) but not with those bearing surface IgG (CESS) or murine and human T cell line cells (BW5147, L5178Y, HPB-ALL, and MOLT4), indicating that IgA-BF interact with B cells bearing IgA to suppress their differentiation.     

Antigen-specific T cell-mediated suppression. II. In vitro induction by I-J-coded L-glutamic acid50-L-tyrosine50 (GT)-specific T cell suppressor factor (GT-T8F) of suppressor T cells (T82) bearing distinct I-J determinants.

The induction of new suppressor T cells (Ts2) by suppressive extracts (TsF) from L-glutamic acid50L-tyrosine50 (GT) nonresponder mice was examined. Incubation of normal spleen cells with allogeneic GT-TsF for 2 days in vitro led to the generation of Ts2 cells able to suppress subsequent responses to the immunogen GT-methylated bovine serum albumin (GT-MBSA) in vivo. This induction occurred efficiently when TsF donor and target cells differed at all of H-2, including the I-J subregion. B10.BR (H-2k) GT-TsF, adsorbed on, then acid eluted from GT-Sepharose and anti-I-Jk [B10.A (3R) anti-B10.A (5R)]-Sepharose in a sequential fashion could induce BALB/c (H-2d) spleen cells to become Ts2 only if nanogram quantities of GT were added to the purified GT-TsF. This indicates a requirement for a molecule or molecular complex possessing both I-J determinants and antigen (GT)-binding specificity, together with GT itself, for Ts2 induction. The induced Ts2 are I-J+, since their function can be eliminated by treatment with anti-I-Jk plus C. These I-J determinants are coded for by the precursor of the Ts2 and do not represent passively adsorbed, I-J coded TsF, since anti-Ijk antiserum [(3R X DBA/2)F1 anti-5R] which cannot recognize the BALB/c (I-Jd) TsF used for induction still eliminates the activity of induced A/J (I-Jk) Ts2. These data provide further evidence for and information about the minimum of two T cells involved in antigen-specific suppressor T cell systems.