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R N Germain

Publications and source records attributed to R N Germain.

At least 55 records · Page 3Linked to original sources
PubMed1996

Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists.

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.

Animals↗
PubMed1996

Partial signaling by CD8+ T cells in response to antagonist ligands.

Structural variants of an agonist peptide-major histocompatibility complex (MHC) molecule ligand can show partial agonist and/or antagonist properties. A number of such altered ligands appear to act as pure antagonists. They lack any detectable ability to induce T cell effector function and have been described as unable to induce calcium transients and turnover of inositol phosphates. This has been interpreted as an inability of these ligands to initiate any T cell receptor (TCR)-dependent signal transduction, with their antagonist properties ascribed to competition with offered agonist for TCR occupancy. Yet antagonists for mature CD8+ T cells can induce positive selection of thymocytes, implying active induction of T cell differentiation events, and partial agonists or agonist/antagonist combinations elicit a distinctive pattern of early TCR-associated tyrosine phosphorylation events in CD4+ T cells. We have therefore directly examined proximal TCR signaling in a CD8+ T cell line in response to various related ligands. TCR engagement with natural peptide-MHC class I agonist resulted in the same pattern of early TCR-associated tyrosine phosphorylation events as seen with CD4+ cells, including accumulation of both the p21 and p23 forms of phosphorylated zeta, phosphorylation of CD3 epsilon, and association of phosphorylated ZAP-70 with the TCR. Two antagonists that lacked the ability to induce any detectable CTL effector response (cytolysis, esterase release, gamma interferon secretion, interleukin-2 receptor alpha upregulation) were nevertheless found to also induce TCR-dependent phosphorylation events. In these cases, there was preferential accumulation of the p21 form of phospho-zeta without net phosphorylation of CD3 epsilon, as well as the association of nonphosphorylated ZAP-70 kinase with the receptor. These data show that variant ligands induce similar TCR-dependent phosphorylation events in CD8+ T cells as first observed in CD4+ cells. More importantly, they demonstrate that some putatively pure antagonists are actually a subset of partial agonists able to induce intracellular biochemical changes through the TCR. This delivery of a partial signal by antagonists raises the possibility that antagonism in some cases may result from active interference with stimulation of effector activity by agonist in mature T cells, while the same variant signal could selectively trigger intracellular events that allow positive without negative selection in thymocytes.

Amino Acid Sequence↗
PubMed1996

Peptides corresponding to CD4-interacting regions of murine MHC class II molecules modulate immune responses of CD4+ T lymphocytes in vitro and in vivo.

Immune responses mediated by CD4+ T cells depend on Ag-specific alpha beta TCRs that recognize the specific antigenic peptide presented by MHC class II molecules. Interactions between CD4 coreceptors and monomorphic regions of MHC class II molecules contribute to these responses. To examine whether immune reactions could be modulated by specifically interfering with CD4-MHC class II interactions, we have used, in various in vitro and in vivo assays, peptides that correspond to a region of MHC class II molecules previously shown to control interaction with CD4. Depending on the chemical nature and concentration of these peptides, they modulated Ag-specific responses of CD4+ T cells. At high concentrations, these peptides inhibited T cell responses in vitro. However, under conditions that can cause Ag-induced unresponsiveness, the peptides enhanced T cell responses. Also, primary in vivo immune responses to systemically administered soluble protein Ag, keyhole limpet hemocyanin, were enhanced when mice were treated with peptides corresponding to the CD4-interacting region of MHC class II molecules but not when treated with control peptides. Lymphokine profiles suggested that the peptides may favor the differentiation of Th1 cells, because lymphocytes from peptide-treated mice secreted more IL-2 and IFN-gamma than lymphocytes from nontreated or control-peptide-treated mice upon restimulation with Ag in vitro. These results demonstrate that MHC class II-derived peptides can directly interfere with interactions with CD4 and modulate T cell responses in vitro and in vivo.

Adjuvants, Immunologic↗
PubMed1996

Variant TCR ligands: new insights into the molecular basis of antigen-dependent signal transduction and T-cell activation.

Recent studies have identified peptide-MHC molecule ligands of alpha beta T-cell receptors with properties apparently distinct from classical agonists. These complexes, which are slight structural variants of the immunizing peptide or original presenting MHC molecule, have several novel properties. They can act as partial agonists able to induce only some and not other effector activities of the T cell, as antagonists able to inhibit T-cell functions stimulated by agonist ligand, or as mixed partial agonists/antagonists. Here we discuss the existing data suggesting that a simple receptor occupancy model does not account for the properties of these TCR ligands and review emerging data on qualitative differences in signal transduction following TCR engagement by priming versus variant complexes. We propose several non-exclusive models to explain both the biochemical and biological properties of variant ligands with partial agonist or antagonist properties.

Animals↗
PubMed1996

Unexpectedly complex regulation of CD4/CD8 coreceptor expression supports a revised model for CD4+CD8+ thymocyte differentiation.

CD4+ CD8+ TCRlo thymocytes are the precursors of CD4+ and CD8+ mature T cells, whose receptors show specific recognition of peptide-MHC class II and MHC class I complexes, respectively. How T cells emerge from the intrathymic differentiation process with selective expression of either CD8 molecule or CD4 molecule coordinated with the MHC class specificity of the TCR has been the subject of intense examination. Many previous studies of this question have been based on the assumption that extinction of CD4 or CD8 expression by the precursor thymocytes was a steady, uninterrupted process. Here we show that this is an incorrect assumption, with CD4 and CD8 expression undergoing an unexpectedly complex series of expression changes involving down-modulation, kinetically asymmetric up-regulation, and then selective loss. Based on these data, we propose a model for the differentiation pathway of alphabeta TCR thymocytes that explains previous, apparently contradictory findings and establishes useful parameters for future studies at the cellular and gene level.

Animals↗
PubMed1996

Comparison of thymocyte development in normal and invariant chain-deficient mice provides evidence that maturation-related changes in TCR and co-receptor levels play a critical role in cell fate.

Cells of invariant chain-deficient mice show a substantial decrease in cell surface MHC class II protein expression, as well as a change in the occupancy of the expressed class II molecules. Taking advantage of recent advances in phenotypic identification of transitional populations of developing thymocytes, the effects of these changes in MHC class II on positive and negative selection were reanalyzed. A marked (approximately 6-fold) reduction in CD4 single-positive mature cells was seen in H-2b mutant mice, yet there was little change in the number of CD4hiCD8intTCRint cells, a population containing the cells from which mature CD4+ cells derive. In normal mice expressing I-E and MMTV-encoded vSAG, V beta-specific negative selection occurred at a later point in the maturation pathway for cells showing greater expression of CD8 than CD4. In invariant chain-deficient mice, vSAG-mediated negative selection was diminished in general and what deletion still occurred was seen in more mature populations as compared to wild-type mice. Taken together, the decrease in MHC class II expression in invariant chain mutant mice and these alterations in the timing of thymocyte deletion provide strong support for an avidity model of negative selection. Perhaps more importantly, they emphasize the importance of the increasing TCR expression, the changing co-receptor levels and the movement from one antigen-presenting cell to another that accompany T cell maturation in determining the fate of developing thymocytes.

Animals↗
PubMed1995

Inhibition of invariant chain (Ii)-calnexin interaction results in enhanced degradation of Ii but does not prevent the assembly of alpha beta Ii complexes.

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.

Animals↗
PubMed1995

Involvement of both major histocompatibility complex class II alpha and beta chains in CD4 function indicates a role for ordered oligomerization in T cell activation.

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.

Animals↗
PubMed1995

Major histocompatibility complex class I presentation of peptides derived from soluble exogenous antigen by a subset of cells engaged in phagocytosis.

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.

Amino Acid Sequence↗
PubMed1995

Evidence for lineage commitment and initiation of positive selection by thymocytes with intermediate surface phenotypes.

Previous studies have shown that both MHC class I and class II molecules can stimulate CD4+CD8+ thymocytes to develop into TCRint cells with surface phenotypes, suggesting initial differentiation along either the CD4 or CD8 developmental pathways. In accord with the view that these cells have undergone lineage commitment, we show here that CD4intTCRint cells generated by recognition of either MHC class show the same partial down-regulation of CD8 from the level on precursor double positive thymocytes as do almost mature CD4intTCRhigh cells positively selected only by MHC class I recognition. As expected of cells undergoing positive selection, we find that either class I or class II MHC molecules on radioresistant thymic stromal cells alone are sufficient for generation of both types of transitional phenotype cells. Biases in V beta representation associated with allele-dependent positive selection are seen only in the TCRhigh and not the TCRint cohorts, however. These findings are consistent with the hypothesis that transitional phenotype thymocytes have begun but not completed positive selection, and that entry into the CD4 vs CD8 pathways is not uniquely determined by the specific coreceptor-MHC molecule interaction involved in precursor thymocyte activation.

Animals↗
PubMed1995

The biochemistry and cell biology of antigen presentation by MHC class I and class II molecules. Implications for development of combination vaccines.

T lymphocytes play a central role in adaptive immunity. They provide direct effector function, regulate the activity of non-antigen-specific effector cells such as macrophages, and control the production of antibodies by B cells. Thus, the proper stimulation of T cells is critical to effective vaccination. T cells bearing alpha beta receptors are stimulated by antigen-derived peptides displayed on cell surfaces bound to highly polymorphic, major histocompatibility complex-encoded glycoproteins. To elicit suitable T cell responses vaccines must, therefore, contain proteins or peptides derived from the organism against which protection is desired, the pathogen-derived peptides must be capable of interacting with the allelic forms of the MHC molecules expressed in the vaccinated individuals, and the vaccine components must be delivered in a manner that ensures they are made available for binding to the MHC molecules on appropriate antigen-presenting cells. This paper has reviewed the rules governing peptide binding to MHC molecules, the intracellular pathways of protein synthesis, protein degradation, and protein and peptide transport involved in bringing together antigenic peptides and MHC molecules, and the distinct function of MHC class I versus class II molecules. The implications of this knowledge for effective combined vaccine design and delivery were considered.

Animals↗
PubMed1995

Transient aggregation of major histocompatibility complex class II chains during assembly in normal spleen cells.

Many cell surface proteins exist as complexes of multiple subunits. It is well established that most such complexes are assembled within the endoplasmic reticulum (ER). However, the mechanistic details of the assembly process are largely unknown. We show here that alpha and beta subunits of major histocompatibility complex class II antigens in spleen cells of normal mice pass through a transiently aggregated phase in the ER prior to assembly with the invariant chain (Ii). Aggregates form immediately after synthesis and disappear concomitantly with assembly of mature alpha beta Ii complexes. In spleen cells lacking Ii, aggregates fail to be efficiently dissociated over time, implicating subunit assembly as a requirement for disaggregation. Two ER chaperones, BiP and calnexin, bind to newly synthesized class II MHC chains but do not contribute appreciably to the large size of the aggregates. Our observations suggest that some subunits of multisubunit complexes pass through a transient, dynamic high molecular weight aggregate phase during the physiological process of assembly. The results further suggest a novel role for Ii in promoting stable dissociation of preformed aggregates containing alpha and beta subunits rather than in preventing their formation.

Adenosine Triphosphate↗
PubMed1995

Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists.

Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses to TCR occupancy by these variant ligands.

Amino Acid Sequence↗
PubMed1995

Binding domain regulation of MHC class II molecule assembly, trafficking, fate, and function.

Major histocompatibility complex class II molecules are heterodimeric type I integral membrane glycoproteins whose primary function is the capture of fragments of antigen in the endocytic pathway, and the presentation of these peptides to CD4+ alpha beta TCR-bearing T cells. The biochemical features of the class II peptide binding domain optimize it for this function by allowing interaction with denatured proteins prior to extensive degradation in endosomes and lysosomes. These same properties pose problems for alpha beta heterodimer assembly, avoidance of non-productive interactions with self-proteins, intracellular transport and dimer stability. This review discusses how coevolution of alpha and beta chain binding domain polymorphism and the extrinsic control of binding site function by invariant chain occupancy deal with these problems and permit the efficient functioning of the class II presentation system.

Antigen Presentation↗
PubMed1995

Allelic differences affecting invariant chain dependency of MHC class II subunit assembly.

The conserved invariant chain associates with highly polymorphic alpha and beta subunits guiding class II transport through the secretory pathway. Early associations of these three polypeptides inside antigen-presenting cells are poorly understood. The present experiments provide a detailed picture of the structure and fate of class II alpha and beta subunits in invariant chain mutants possessing different MHC haplotypes. In the absence of invariant chain, A alpha bA beta b is predominantly expressed as free A alpha b and A beta b chains by both splenocytes and activated LPS/IL-4 blasts, confirming that A alpha bA beta b assembly is strongly dependent on invariant chain coexpression. A quite different situation exists with respect to other allelic products. In the absence of invariant chain, A alpha kA beta k, E alpha kE beta k, and A alpha dA beta d molecules assemble efficiently and are conformationally similar to mature wild-type heterodimers. The contribution of invariant chain to subunit assembly thus differs for allelic variants, suggesting that sequential associations of alpha, beta, and invariant chain may be affected by polymorphic differences.

Alleles↗
PubMed1995

Extensive trafficking of MHC class II-invariant chain complexes in the endocytic pathway and appearance of peptide-loaded class II in multiple compartments.

Major histocompatibility complex class II molecules bind and present to T cells fragments of protein antigens entering the endocytic pathway. Using normal B lymphoblasts, we have combined metabolic pulse-chase labelling, high resolution organelle fractionation, and immunoprecipitation to examine class II trafficking and antigen loading in a physiological model system. Most newly synthesized class II-invariant chain complexes first entered early endosomes, then accessed multiple discrete endocytic subcompartments cofractionating with late endosomes and immature lysosomes. Invariant chain was removed and peptide-loaded class II molecules appeared in each of these latter distinct organelles. These findings suggest that class II molecules traffic through much of the endocytic pathway, permitting capture of distinct determinants made available under differing conditions of pH and proteolytic activity.

Animals↗