RESUMO
Invariant chain (Ii) contributes in a number of distinct ways to the proper functioning of major histocompatibility complex (MHC) class II molecules. These include promoting effective association and folding of newly synthesized MHC class II alpha and beta subunits, increasing transit of assembled heterodimers out of the endoplasmic reticulum (ER), inhibiting class II peptide binding, and facilitating class II movement to or accumulation in endosomes/lysosomes. Although the cytoplasmic tail of Ii makes a key contribution to the endocytic localization of class II, the relationship between the structure of Ii and its other diverse functions remains unknown. We show here that two thirds of the lumenal segment of Ii can be eliminated without affecting its contributions to the secretory pathway events of class II folding, ER to Golgi transport, or inhibition of peptide binding. These same experiments reveal that a short (25 residue) contiguous internal segment of Ii (the CLIP region), frequently found associated with purified MHC class II molecules, is critical for all three functions. Together with other recent findings, these results raise the possibility that the contributions of Ii to the early postsynthetic behavior of class II may depend on its interaction with the class II binding site. This would be consistent with the intracellular behavior of unoccupied MHC class I and class II molecules as incompletely folded proteins and imply a related structural basis for the similar contributions of Ii to class II and of short peptides to class I assembly and transport.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/fisiologia , Fragmentos de Peptídeos/fisiologia , Dobramento de Proteína , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/metabolismo , Dados de Sequência MolecularRESUMO
The intracytoplasmic forms of class II (or Ia) major histocompatibility complex heterodimers are associated with a third glycoprotein, termed the invariant chain (Ii). This specific interaction has led to the view that Ii plays a necessary role in the assembly, intracellular transport, and/or membrane insertion of Ia molecules. To test this hypothesis directly, we have transfected complementary DNA clones that encode murine class II alpha and beta chains into cells that do not express any endogenous Ii messenger RNA (mRNA) (COS-7 and BALB/c 3T3 cells). After DNA-mediated gene transfer, significant cell surface expression of Ia was observed in transient expression assays using COS-7 cells and a stable expression system using BALB/c 3T3 cells. Furthermore, the total levels of class II alpha and beta mRNA were similar in Ii- cells (transfected BALB/c 3T3) and in Ii+ cells (B cell hybridoma) that expressed nearly identical amounts of surface Ia, suggesting that the efficiency of Ia expression was equivalent in the two cell types and, therefore, independent of Ii. These results indicate that the physiologic role for Ii is not simply to mediate membrane expression of Ia molecules, and that alternative hypotheses concerning the true function of this molecule need to be considered.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Antígenos de Superfície/análise , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/fisiologia , Células Cultivadas , DNA/análise , Antígenos de Histocompatibilidade Classe II/genética , Plasmídeos , RNA Mensageiro/análise , TransfecçãoRESUMO
T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4(+) cell population. Low concentrations of TCR ligand elicit only interferon-gamma (IFN-gamma) production. Increasing ligand recruits more cells into the IFN-gamma+ pool, increases IFN-gamma produced per cell, and also elicits IL-2, but only from cells already making IFN-gamma. Most cells producing only IFN-gamma show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-gamma synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-gamma/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-gamma and not IL-2, and the amount of IFN-gamma exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.
Assuntos
Interferon gama/biossíntese , Interleucina-2/biossíntese , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos CD/imunologia , Células Clonais , Técnicas de Cocultura , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Interleucina-3/biossíntese , Ligantes , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Células Th1/metabolismoRESUMO
Calnexin is a resident protein of the endoplasmic reticulum (ER) that associates with nascent protein chains. Among the newly synthesized integral membrane proteins known to bind to calnexin is invariant chain (Ii), and Ii release from calnexin coincides with proper assembly with major histocompatibility complex (MHC) class II heterodimers. Although calnexin association with several membrane glycoproteins depends on interactions involving N-linked glycans, we previously reported that a truncation mutant of mouse Ii (mIi1-107) lacking both N-glycosylation sites was highly effective in associating with MHC class II heterodimers and escorting these dimers through the secretory pathway. This could indicate that calnexin, despite binding to both Ii and class II, is not necessary for the proper interaction of these proteins, or that in contrast to most membrane glycoproteins, the N-linked glycans of Ii are not critical to its interaction with this chaperone. To examine this issue, we have directly explored the binding of calnexin to both Ii truncation mutants lacking the typical sites of N-glycosylation or Ii produced in cells treated with tunicamycin to prevent glycan addition. These experiments revealed that either method of eliminating N-linked carbohydrates on Ii also inhibited association with calnexin. A lumenally truncated form of Ii (mIi1-131) that still has N-linked carbohydrates showed a decreased affinity for calnexin compared with intact Ii, however, indicating that calnexin-Ii binding is not determined solely by the sugar moieties. All forms of Ii lacking N-linked sugars and showing defective association with calnexin also had enhanced rates of preendosomal degradation. Despite this effect on degradation rate, tunicamycin treatment did not inhibit the association of class II with glycan-free Ii. These data support the view that calnexin is not an absolute requirement for the proper assembly of class II-Ii nonamers, but rather acts primarily to retain Ii in the ER and to inhibit its degradation. These two properties of calnexin-Ii interaction may help ensure that sufficient intact Ii is available for efficient inactivation of the binding sites of newly synthesized class II molecules, while limiting the ability of excess free Ii to alter the transport properties of the early endocytic pathway.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/química , Sequência de Bases , Transporte Biológico , Calnexina , Células Cultivadas , Chlorocebus aethiops , Primers do DNA/química , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicosilação/efeitos dos fármacos , Glicosiltransferases/antagonistas & inibidores , Antígenos de Histocompatibilidade Classe II/química , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Tunicamicina/farmacologiaRESUMO
(Responder [R] X nonresponder [NR])F1 mice give indistinguishable primary in vitro plaque-forming cell (PFC) responses to either R or NR parental macrophages (Mphi) pulsed with the Ir-gene controlled antigen L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). However, such (R X NR)F1 mice, if primed to GAT, retained in vitro responsiveness to GAT-R-Mphi, but no longer responded to GAT-NR-Mphi. This suggested (a) a possible Mphi-related locus for Ir gene activity in this model, and (b) the occurrence of active suppression after priming with GAT leading to a selective loss of the usual primary responsiveness of (R X NR)F1 mice to GAT-NR-Mphi. This latter interpretation was tested in the current study. [Responder C57BL/6 (H-2b) X nonresponder DBA/1 (H-2q)]F1 mice were primed with 100 microgram GAT in pertussis adjuvant. 4-8 wk later, spleen cells from such mice were tested alone or mixed with normal unprimed F1 spleen cells for PFC responses to GAT-R-Mphi and GAT-NR-Mphi. The primed cells failed to respond to GAT-NR-Mphi, and moreover, actively suppressed the normal response of unprimed F1 cells to GAT-NR-Mphi. If the primed spleen cell donor had been treated with 5 mg/kg cyclophosphamide 3 days before priming or with 5-10 microliter/day of an antiserum to the I-Jb subregion [B10.A(5R) anti B10.A(3R)] during the first 4 days postpriming (both procedures known to inhibit suppressor T-cell activity), cells from such mice responded in secondary culture to both GAT-R-Mphi and also GAT-NR-MPhi. In addition, such spleen cells no longer were capable of suppressing normal F1 cells in response to GAT-NR-Mphi. Similar data were obtained using [CBA (H-2k) X DBA/1 (H-2q)]F1. Further, it was shown that (a) primary responsiveness to GAT-NR-Mphi was not an artifact of in vitro Mphi pulsing, because in vivo GAT-pulsed Mphi showed the same activity and (b) the secondary restriction for Mphi-antigen presentation was controlled by H-2 linked genes. These data suggest an important role for suppressor T cells in H-2 restricted secondary PFC responses, and also provide additional support for the hypothesis that Ir-gene controlled differences in Mphi antigen presentation are related to both suppressor cell generation and overall responsiveness in the GAT model.
Assuntos
Formação de Anticorpos , Genes MHC da Classe II , Memória Imunológica , Terapia de Imunossupressão , Macrófagos/imunologia , Linfócitos T/imunologia , Alanina , Animais , Formação de Anticorpos/efeitos dos fármacos , Ciclofosfamida/farmacologia , Glutamatos , Camundongos , Camundongos Endogâmicos , Peptídeos/imunologia , TirosinaRESUMO
The occurrence of distinct genetic defects affecting the generation of T cell-derived suppressor factor (TsF) or the suppressive activity of such TsF was investigated. For the synthetic polypeptide L-glutamic acid50-L-tyrosine50 (GT), it could be shown that the nonsuppressor strain A/J fails to produce suppressor T cells (Ts1) capable of GT-TsF generation upon challenge with GT. Conversely, B6, another nonsuppressor strain, produces GT-TsF active on other allogeneic strains such as A/J, but itself fails to be suppressed by this material. (B6A)F1 mice both make GT-TsF, and are suppressed by it. Further experiments revealed that the production of GT-TsF and the ability to be suppressed by GT-TsF are under the control of H-2-linked genes. Finally, the defect in GT-TsF activity in B6 mice was shown to be exquisitely antigen specific, in that this strain can be suppressed by a closely related TsF specific for L-glutamic acid60-L-alanine30-L-tyrosine10. It is suggested that H-2 (I) control of suppressor T cell (Ts) activity may reflect the involvement of I-A and I-C gene products in antigen presentation to Ts in analog with other T cell subsets, and that TsF function might also involve such presentation, in this case of the idiotypic structures of the TsF-combining site. Predictions deriving from this hypothesis are discussed, including the possibility that H-2 linked immune response genes regulate auto-anti-idiotypic responses in immune networks.
Assuntos
Antígenos H-2/genética , Tolerância Imunológica , Linfocinas/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Animais , Comunicação Celular , Genes MHC da Classe II , Ligação Genética , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos/imunologia , Peptídeos/imunologiaRESUMO
Feedback suppression of the primary humoral immune response to sheep erythrocytes (SRBC) in vitro was induced with cell-free supernate material derived from antigen-(SRBC) activated B (sIg+) cells. This soluble products bears Ig determinants and binds to the eliciting antigen (SRBC). The activity of this antibody in suppressing anti-SRBC plaque-forming cell responses is restricted to spleen cell cultures containing B cells sharing VH genes with the B cells producing the suppressive antibody. The anti-hapten (trinitrophenyl) response to derivatized SRBC is not affected by antigen-primed B cells or their products. These data are compatible with suppression being mediated by anti-antigen antibody, either (a) via blockade of different SRBC epitopes recognized by a limited set of B cell clones in each mouse strain, (b) via triggering of an anti-idiotypic response, either antibody or suppressor T cell in nature, restricted to activity in cultures containing B cells sharing VH structures with the original antibody, or (c) via interference by preformed antibody with T cell help directed at idiotype bearing B cells.
Assuntos
Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Tolerância Imunológica , Região Variável de Imunoglobulina , Linfócitos T Reguladores/imunologia , Animais , Células Cultivadas , Eritrócitos/imunologia , Retroalimentação , Alótipos de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas , Idiótipos de Imunoglobulinas , Camundongos , SolubilidadeRESUMO
Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II beta chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for li-dependent determinants. This demonstrates that related leucine-based trafficking signals in li and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.
Assuntos
Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Endopeptidases/fisiologia , Endossomos/metabolismo , Feminino , Leucina , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
CD4 is a membrane glycoprotein on T lymphocytes that binds to the same peptide:major histocompatibility complex (MHC) class II molecule recognized by the antigen-specific receptor (TCR), thereby stabilizing interactions between the TCR and peptide;MHC class II complexes and promoting the localization of the src family tyrosine kinase p56lck into the receptor complex. Previous studies identified a solvent-exposed loop on the class II beta 2 domain necessary for binding to CD4 and for eliciting CD4 coreceptor activity. Here, we demonstrate that a second surface-exposed segment of class II is also critical for CD4 function. This site is in the alpha 2 domain, positioned in single class II heterodimers in such a way that it cannot simultaneously interact with the same CD4 molecule as the beta 2 site. The ability of mutations at either site to diminish CD4 function therefore indicates that specifically organized CD4 and/or MHC class II oligomers play a critical role in coreceptor-dependent T cell activation.
Assuntos
Antígenos CD4/metabolismo , Antígenos HLA-D/metabolismo , Ativação Linfocitária/fisiologia , Modelos Moleculares , Conformação Proteica , Linfócitos T/imunologia , Animais , Sítios de Ligação , DNA Complementar/genética , Genes MHC da Classe II , Antígenos HLA-D/química , Antígenos HLA-D/genética , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , TransfecçãoRESUMO
Major histocompatibility complex (MHC) class I molecules generally present peptides derived from cytoplasmic proteins, but recent reports have suggested that macrophages (M phi) may be uniquely able to present exogenous antigens via these molecules, and that particle-associated antigens show a marked increase in the efficiency of such presentation. We confirm here that particle uptake by M phi permits exogenous ovaalbumin (OVA) to gain access to the endogenous class I processing pathway, an event that occurs rarely, if at all, in the absence of phagocytic stimuli. Presentation of soluble protein antigens by MHC class I molecules, however, is not limited to M phi, nor is direct coupling of antigen to the particle required. A variety of unconjugated particles promoted presentation of simultaneously offered soluble OVA to Kb-restricted T cells by both M phi and non-M phi antigen-presenting cells (APC), provided the latter could phagocytose the particles. Enhancement of presentation by phagocytic stimuli could not be explained by greater delivery of soluble antigen to endosomal compartments because such stimuli did not increase soluble tracer accumulation, nor did they improve presentation of OVA to an MHC class II-restricted T cell hybridoma. OVA presentation induced by cophagocytosis of particles and free antigen was nevertheless very inefficient in comparison to presentation of OVA peptide, and even modest responses required high concentrations of protein and particles. Furthermore, only a fraction of APC exposed to OVA and particles were lysed by anti-OVA cytotoxic T lymphocytes, despite virtually all cells showing OVA accumulation, particle uptake, and Kb expression. Titration experiments were most consistent with a model in which, by disrupting membrane integrity, phagocytic overload ("indigestion") allows escape of OVA into the cytosol of some APC, rather than with a model in which phagocytosis activates a novel antigen processing pathway that has evolved to permit class I loading of exogenous antigen. These data suggest caution in the development of vaccine strategies based on use of particle conjugates for elicitation of CD8+ T cell immunity, but, at the same time, may be relevant to understanding class I-restricted responses to some intracellular pathogens normally resident in membrane-bound vesicles.
Assuntos
Apresentação de Antígeno , Antígenos H-2/imunologia , Macrófagos/imunologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Fagocitose , Sequência de Aminoácidos , Animais , Citosol/metabolismo , Feminino , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Dados de Sequência Molecular , Ovalbumina/metabolismo , Fragmentos de Peptídeos/metabolismo , Fagossomos/metabolismoRESUMO
Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectiviely a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be medicated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-D surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.
Assuntos
Antígenos de Histocompatibilidade , Imunidade Celular , Nitrobenzenos/imunologia , Linfócitos T/imunologia , Trinitrobenzenos/imunologia , Animais , Reações Antígeno-Anticorpo , Reações Cruzadas , Testes Imunológicos de Citotoxicidade , Isoanticorpos , Camundongos , Camundongos Endogâmicos , Baço/imunologiaRESUMO
After appropriate in vivo or in vitro immunization, cytotoxic T lymphocytes (CTL) are generated which efficiently kill cells bearing particular membrane antigens in common with the immunizing cell (reviewed in reference 1). Such CTL have been most thoroughly studied in mice, employing alloimmunization with cells differing at the major histocompatibility locus, H-2. in such cases, the predominant cell surface antigens recognized by the CTL appear to be the molecules carrying the serologically defined H-2 specificities, coded for by the K and D regions of the H-2 complex (2). In other syngeneic models of cell-mediated specific cytolysis, involving lymphocyte chariomeningitis (LCM) virus- or ectromelia virus-infected cells or TNP-modified lymphoid cells, thymus-derived cells also constitute the main effector cell type. The CTL generated in these latter systems function most efficiently when virus-infected or TNP-modified target cells share identitites at the H-2K or H-2D loci with the effector CTL and stimulator cells (3-5). Another set of experimental systems in which CTL are generated and play a significant biological role is that of immunity to tumor-associated antigens (TAA) (6). The nature of the TAA which the CTL recognize is only beginning to be understood. Several recent reports indicated the existence of physiochemical and/or antigenic relationships between TAA and H-2 antigens (7,8). These relationships, together with the genetic restrictions cited above in the generation of CTL involving products of the H-2K or H-2D loci suggested the possibility that in certain tumor systems, the TAA which are able to most effectively stimulate CTL responses might be structurally similar to, or linked with, the H-2K or H- 2D molecules on the tumor surface. It has been previously demonstrated in allogenic models that antisera specific for the appropriate H-2K or H-2D products present on a target cell could specifically block CTL-mediated lysis (1,9). This report demonstrates that certain anti-H-2 alloantisera specific for the target tumor cells can block lysis of those target cells mediated by syngeneic tumor-specific CTL effector cells.
Assuntos
Antígenos de Neoplasias , Antígenos de Histocompatibilidade , Soros Imunes , Neoplasias Experimentais/imunologia , Linfócitos T/imunologia , Animais , Especificidade de Anticorpos , Testes Imunológicos de Citotoxicidade , Feminino , Isoanticorpos , Linfoma/imunologia , Sarcoma de Mastócitos/imunologia , CamundongosRESUMO
Feedback regulation of the primary humoral immune response to sheep erythrocytes (SRBC) was studied in vitro. Whole spleen cells or spleen cell subpopulations were incubated with antigen for 4 d under Mishell-Dutton conditions (education) and the surviving cells tested for regulatory activity in fresh anti-SRBC spleen cell cultures assayed by measuring plaque-forming cells on day 4. The data indicate that (a) whole spleen cells educated with SRBC exert potent antigen-specific suppression in the assay culture, (b) surface Ig- (sIg-) cells (T cells) prepared by either nylon-wool separation or fractionation on rabbit anti-mouse-Ig-coated polystyrene Petri dishes failed to generate suppressive activity when educated alone, in 2-mercaptoethanol, or in the presence of additional macrophages, (c) surface Ig (sIg+) (B) cells educated alone also failed to generate suppressor cells, and (d) mixing sIg- (T) and sIg+, Lyt 123- (B) cells reconstituted the ability to induce suppressor cells under these conditions. The antigen-primed cell actually required to transfer suppression was also characterized by separating cells using anti-Ig coated dishes, by fluorescence-activated cell sorting and by anti-Lyt treatment. All these methods clearly identified sIg+ (B) and not sIg+ (T) cells as the important educated cells. It is concluded that under our conditions, T cell-dependent B cells triggered by antigen during primary in vitro cultures cause potent specific feedback suppression of humoral responses. Possible mechanisms for this suppression, including antigen blockade or anti-idiotypic responses, are discussed.
Assuntos
Linfócitos B/imunologia , Cooperação Linfocítica , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Superfície/análise , Comunicação Celular , Células Cultivadas , Eritrócitos/imunologia , Retroalimentação , Feminino , Isoantígenos/análise , Masculino , Camundongos , Receptores de Antígenos de Linfócitos B/análiseRESUMO
4-Hydroxy-3-nitrophenyl (NP) derivatized syngeneic spleen cells injected intravenously stimulate maturation of an antigen-binding, idiotype-bearing induction-phase suppressor cell population, as well as an idiotype-binding anti-idiotype-bearing effector-phase suppressor cell population. Both cell types are present simultaneously in the spleen cell population 7-d after their induction. Furthermore, the cell population with antigen-binding properties can, in the presence of NP-derivatized syngeneic cells, induce a population of effector suppressor cells. The precursors of the effector suppressor population are not sensitive to concentrations of cyclophosphamide which prevented the generation of induction phase suppressor cells. These data provide direct evidence in support of the theory of network regulation of immune suppression. X
Assuntos
Haptenos/imunologia , Idiótipos de Imunoglobulinas , Nitrofenóis/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Ciclofosfamida/farmacologia , Epitopos , Hipersensibilidade Tardia/imunologia , Imunização , Camundongos , Fenilacetatos , Baço/imunologia , Baço/transplante , Transplante HomólogoRESUMO
The contributions of A alpha and A beta chains, and of subregions of A beta, to Ia-restricted recognition of antigen by Th lymphocytes were analyzed using a panel of L cells transfected with various pairs of A alpha b,d, or k genes and recombinant or wild-type A beta b,d, or k genes. The A beta genes included all possible exchanges of the whole NH2-terminal (beta 1) domain or halves of the beta 1 domain among these three allelic A beta genes. The Ia+ L cells derived from such transfections were used as antigen-presenting cells with a 21 member panel of responding Ia-restricted T hybridoma cells of differing nominal antigen specificity and Ia-restriction. Special care was taken to account for quantitative variation in levels of Ia expression throughout the experiments. The results of this analysis reveal that (a) only 2 of the 21 Th cells recognized Ia molecules involving either a nonparental A alpha or a nonparental A beta chain, and in both cases the degeneracy extended to only one of the two other alleles tested. This suggests that allele specific contributions from both A alpha and A beta chains are important in restricted recognition for most, if not all I-A-restricted Th cells. (b) In no case did substitution of the A beta 2 domain from either of the alternative haplotypes lead to any functionally detectable effects, demonstrating that polymorphisms in the A beta 1 domain can entirely account for the restriction imposed on Th cell responses by the entire A beta chain. (c) For 90% of the cells tested, replacement of the NH2-terminal portion of the beta 1 domain with an allogeneic segment led to Ia molecules unable to elicit Th responses. Furthermore, of all the cells permissive of the substitution of one or other half of the beta 1 domain, only two permitted the substitution of sequence from both alternative haplotypes. Taken together, these data strongly suggest that antigen recognition by most, if not all, I-A-restricted Th cells involves contributions from both halves of the A beta 1 domain. These data suggest that the role of I-A molecules in restricted Th cell recognition of antigen depends on conformational determinants unique to a particular combination of polymorphic alpha and beta chains, and that multiple such sites exist on a single Ia molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , DNA/genética , DNA Recombinante , Relação Dose-Resposta Imunológica , Epitopos , Genes , Genótipo , Hibridomas/imunologia , Células L , Camundongos , Polimorfismo Genético , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , TransfecçãoRESUMO
Clonal activation of CD4+ and CD8+ T lymphocytes depends on binding of peptide-major histocompatibility complex (MHC) molecule complexes by their alpha/beta receptors, eventually resulting in sufficient aggregation to initiate second messenger generation. The nature of intracellular signals resulting from such T cell receptor (TCR) occupancy is believed to be independent of the specific structure of the ligand being bound, and to vary quantitatively, not qualitatively, with the concentration of ligand offered and the affinity of the receptor for the peptide-MHC molecule complex. In contrast to the expectations of this model, the analysis of the response of a T helper type 1 clone to mutant E alpha E beta k molecules in the absence or presence of a peptide antigen revealed that peptide inhibited the interleukin 2 (IL-2) response to an otherwise allostimulatory mutant form of this MHC class II molecule. The inhibition was not due to competition for formation of alloantigen, it required TCR recognition of peptide-mutant MHC molecule complexes, and it decreased IL-2 production without affecting receptor-dependent IL-3, IL-2 receptor alpha, or size enlargement responses. This preferential reduction in IL-2 secretion could be correlated with the costimulatory signal dependence of this cytokine response, but could not be overcome by crosslinking the CD28 molecule on the T cell. These results define a new class of TCR ligands with mixed agonist/antagonist properties, and point to a ligand-related variation in the quality of clonotypic receptor signaling events or their integration with other signaling processes. It was also found that a single TCR ligand showed greatly different dose thresholds for the elicitation of distinct effector responses from a cloned T cell population. The observations that changes in ligand structure can result in qualitative alterations in the effects of receptor occupancy and that quantitative variations in ligand density can be translated into qualitative differences in T cell responses have important implications for models of intrathymic selection and control of the results of active immunization.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Células Clonais , Grupo dos Citocromos c/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Interleucina-2/antagonistas & inibidores , Interleucina-2/fisiologia , Interleucina-3/fisiologia , Células L , Ligantes , Camundongos , Dados de Sequência Molecular , Mutação , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
We have observed that a peptide corresponding to an immunodominant epitope of the HIV-1 envelope protein recognized by class I MHC-restricted CD8+ CTL can also induce T cell help for itself. The help is necessary for restimulation of CTL precursors in vitro with peptide alone in the absence of exogenous lymphokines, can be removed by depletion of CD4+ T cells, and can be replaced by exogenous IL-2. Whereas the CTL in BALB/c or B10. D2 mice are restricted by the class I molecule Dd, the Th cells are restricted by the class II molecule Ad, and the help can be blocked by anti-Ad mAb. To examine the genetic regulation of the induction of help, we studied B10.A mice that share the class I Dd molecule, but have different class II molecules, Ak and Ek. Spleen cells of immune B10.A mice behave like CD4-depleted BALB/c spleen cells in that they cannot be restimulated in vitro by the peptide alone, but can with peptide plus IL-2. Therefore, in the absence of exogenous lymphokines, peptide-specific help is necessary for restimulation with this immunodominant CTL epitope peptide, and in H-2d mice, this peptide stimulates help for itself as well as CTL. We speculate on the implications of these findings for the immunodominance of this peptide in H-2d mice, and for the selective advantage of pairing certain class I and class II molecules in an MHC haplotype.
Assuntos
Antígenos CD4/imunologia , Epitopos/imunologia , Produtos do Gene env/genética , HIV-1/imunologia , Complexo Principal de Histocompatibilidade/genética , Precursores de Proteínas/genética , Linfócitos T Citotóxicos/imunologia , Animais , Genes MHC Classe I , Proteína gp160 do Envelope de HIV , Camundongos , Camundongos Endogâmicos BALB C , Mapeamento por Restrição , Baço/citologia , TransfecçãoRESUMO
The I-Abm12 mutation has been used extensively to study the relationship between structure and function of murine class II major histocompatibility molecules. I-Abm12 differs from I-Ab by three amino acid replacements in the A beta chain, and the proposed structural model of the I-Abm12 molecule places these three amino acid substitutions along one of the alpha-helices where they may affect both antigen and TCR binding. Two of the substitutions, Ile----Phe67 and Thr----Lys71, are thought to point into the binding site, whereas the third substitution, Arg----Gln70, is thought to point up and hence, be available for binding to the TCR. These predicted orientations are consistent with serologic analysis of the bm12 molecule, which demonstrates that residue 70 is uniquely accessible to mAbs distinguishing I-Ab from I-Abm12. In this study we have determined the influence of each of these amino acid substitutions on the ability of the resulting molecules to stimulate a panel of I-Abm12 (allo) reactive T cell hybridomas. Our experiments indicate that reversion of the amino acid at position 70 from Gln (I-Abm12) to Arg (I-Ab) interferes with allorecognition by 33 of 35 I-Abm12-reactive hybridomas. On the other hand, many hybrids can tolerate amino acid substitutions at positions 67 or 71. Single amino acid substitutions at position 67, 70, or 71 are recognized by only a minority of I-Abm12-specific hybrids and usually the reactivity is greatly diminished. These data are most consistent with the idea that the amino acid at position 70 directly interacts with the TCR during allorecognition. The additional effects of residues 67 and 71 are consistent with a contribution by bound peptide to the allorecognition process.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Mutação , Aminoácidos/análise , Animais , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/genética , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/análise , Relação Estrutura-Atividade , TransfecçãoRESUMO
Intravenous (i.v.) injection of high amounts of soluble proteins often results in the induction of antigen-specific tolerance or deviation to helper rather than inflammatory T cell immunity. It has been proposed that this outcome may be due to antigen presentation to T cells by a large cohort of poorly costimulatory or IL-12-deficient resting B cells lacking specific immunoglobulin receptors for the protein. However, previous studies using T cell activation in vitro to assess antigen display have failed to support this idea, showing evidence of specific peptide-major histocompatibility complex (MHC) class II ligand only on purified dendritic cells (DC) or antigen-specific B cells isolated from protein injected mice. Here we reexamine this question using a recently derived monoclonal antibody specific for the T cell receptor (TCR) ligand formed by the association of the 46-61 determinant of hen egg lysozyme (HEL) and the mouse MHC class II molecule I-Ak. In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61-Ak complexes on their surface. DC readily isolated from spleen also display this TCR ligand on their surface. Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells. These results demonstrate that in the absence of activating stimuli, both lymphoid DC and antigen-unspecific B cells present to a similar extent class II-associated peptides derived from soluble proteins in extracellular fluid. The numerical advantage of the TCR ligand-bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.
Assuntos
Apresentação de Antígeno , Linfócitos B/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Linfonodos/imunologia , Muramidase/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Linfócitos B/metabolismo , Citometria de Fluxo , Imuno-Histoquímica , Ligantes , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Receptores de Antígenos de Linfócitos T/imunologia , Soroalbumina Bovina/imunologia , Solubilidade , Baço/citologia , Baço/imunologiaRESUMO
Invariant chain (Ii) associates with newly synthesized class II molecules in the endoplasmic reticulum (ER), an interaction that has been shown to interfere with peptide binding to class II molecules. The class II-associated invariant chain peptide (CLIP) region (residues 81-104) of Ii is believed to mediate this inhibition by engaging the binding domain of class II like an antigenic peptide. Together, these findings have given rise to a model in which CLIP association with the class II groove acts to prevent inappropriate presentation of peptides imported into the ER for association with major histocompatibility complex class I molecules. However, the properties of class II molecules synthesized by cells lacking coexpressed Ii are at least superficially inconsistent with this paradigm in that they do not show clear evidence of peptide acquisition. At the same time, we have previously shown the shortest form of Ii still containing CLIP to play an essential role in regulation of early class II molecule assembly and transport in the secretory pathway. Using covalent peptide technology, we now show that occupancy of the class II binding site in the ER regulates class II trafficking to the Golgi complex, an event that is the locus of the major defect in cells of Ii-deficient mice. These data argue that CLIP occupies the class II binding site, not to prevent interaction with short peptides meant for class I, but rather to maintain the structural integrity of class II molecules that are labile without engaged binding regions, and that would also associate with intact proteins in the ER if left unoccupied. By these means, CLIP occupancy of the class II binding site promotes effective export of useful class II molecules for endocytic peptide acquisition.