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R N Germain

Publications and source records attributed to R N Germain.

At least 19 recordsLinked to original sources
PubMed1992

Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression.

We have examined the roles of peptide and beta 2-microglobulin (beta 2m) in regulating the conformation and expression level of class I molecules on the cell surface. Using a cell line synthesizing H-2Dd H chain and mouse beta 2m but defective in endogenous peptide loading, we demonstrate the ability of either exogenous peptide or beta 2m alone to increase surface H-2Dd expression at both 25 degrees C and 37 degrees C. Peptide and beta 2m show marked synergy in their abilities to increase surface class I expression, with minimal increases promoted by peptide in the absence of free beta 2m. Low temperature-induced molecules have indistinguishable rates of loss of beta 2m and alpha 1/alpha 2 domain conformational epitopes during culture at 37 degrees C. However, the rate of alpha 3 epitope loss is much slower, indicating a minimum of two steps in class I loss from the cell surface: 1) loss of beta 2m binding to H chain and unfolding of the alpha 1/alpha 2 region; then 2) denaturation, degradation, or internalization of the free H chains possessing alpha 3 epitopes. These data show for the first time that free H chains survive for a finite time on the membrane in a form capable of refolding into alpha 1/alpha 2 epitope positive molecules upon addition of beta 2m and peptide. This refolding in the presence of beta 2m and peptide can explain the reported requirement for both components in sensitizing cells for class I-dependent CTL lysis. It also indicates that such conformational changes in class I molecules are not strictly dependent on either newly synthesized H chains or on intracellular chaperons. The study of H chain-peptide-beta 2m interaction on the cell surface may be relevant to understanding intracellular peptide loading events.

Amino Acid Sequence↗
PubMed1992

Serum angiotensin-1 converting enzyme activity processes a human immunodeficiency virus 1 gp160 peptide for presentation by major histocompatibility complex class I molecules.

T cell stimulation by the human immunodeficiency virus 1 gp160-derived peptide p18 presented by H-2Dd class I major histocompatibility complex molecules in a cell-free system was found to require proteolytic cleavage. This extracellular processing was mediated by peptidases present in fetal calf serum. In vitro processing of p18 resulted in a distinct reverse phase high performance liquid chromatography profile, from which a biologically active product was isolated and sequenced. This peptide processing can be specifically blocked by the angiotensin-1 converting enzyme (ACE) inhibitor captopril, and can occur by exposing p18 to purified ACE. The ability of naturally occurring extracellular proteases to convert inactive peptides to T cell antigens has important implications for understanding cytotoxic T lymphocyte responses in vivo, and for rational peptide vaccine design.

Amino Acid Sequence↗
PubMed1992

MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8.

Interactions between major histocompatibility complex (MHC) molecules and the CD4 or CD8 coreceptors have a major role in intrathymic T-cell selection. On mature T cells, each of these two glycoproteins is associated with a class-specific bias in MHC molecule recognition by the T-cell receptor. CD4+ T cells respond to antigen in association with MHC class II molecules and CD8+ T cells respond to antigen in association with MHC class I molecules. Physical interaction between the CD4/MHC class II molecules and CD8/MHC class I molecules has been demonstrated by cell adhesion assay, and a binding site for CD8 on class I has been identified. Here we demonstrate that a region of the MHC class II beta-chain beta 2 domain, structurally analogous to the CD8-binding loop in the MHC class I alpha 3 domain, is critical for function with both mouse and human CD4.

Animals↗
PubMed1992

Induction of broadly cross-reactive cytotoxic T cells recognizing an HIV-1 envelope determinant.

An immunodominant determinant for cytotoxic T lymphocytes (CTLs) exists in the hypervariable portion of human immunodeficiency virus-1 (HIV-1) gp160. Three mouse CTL lines (specific for isolates MN, RF, and IIIB) were examined for recognition of homologous determinants from distinct isolates. Only MN-elicited CTLs showed extensive interisolate cross-reactivity. Residue 325 played a critical role in specificity, with MN-elicited CTLs responding to peptides with an aromatic or cyclic residue and IIIB-induced cells recognizing peptides with an aliphatic residue at this position. CTL populations with broad specificities were generated by restimulation of IIIB-gp160 primed cells with MN-type peptides that have an aliphatic substitution at 325. This represents an approach to synthetic vaccines that can generate broadly cross-reactive CTLs capable of effector function against a wide range of HIV isolates.

AIDS Vaccines↗
PubMed1992

Presentation of antigen by mixed isotype class II molecules in normal H-2d mice.

A panel of DBA/2 T cell hybridomas specific for the sperm whale myoglobin epitope 110-121 was found to recognize antigen presented by the mixed isotype class II molecule E alpha dA beta d. The response was blocked by monoclonal antibodies specific for E alpha and A beta d chains; in addition, the hybridomas responded to antigen presented by L cells expressing E alpha A beta d molecules, and made no response with L cells expressing I-Ad or I-Ed molecules. Two more groups of hybridomas isolated from DBA/2 and B10.D2 mice immunized with myoglobin also recognized peptide 110-121 presented by E alpha d A beta d. Thus, although it is expressed at biochemically undetectable levels on spleen cells, the E alpha d A beta d molecule is an important presenting element in normal H-2d mice making a conventional immune response to a protein antigen. These results suggest that high levels of class II expression are not a prerequisite for T cell activation.

Animals↗
PubMed1992

How MHC class II molecules work: peptide-dependent completion of protein folding.

It is now clear that peptides play a key role in stabilizing the structure of MHC class II molecules. Here, Scheherazade Sadegh-Nasseri and Ron Germain propose that newly synthesized MHC class II, and indeed class I, molecules behave like partially folded proteins, with peptides acting as a surrogate portion of the MHC polypeptide structure that is necessary for completion of conformational maturation.

Animals↗
PubMed1991

The molecular basis of class II MHC allelic control of T cell responses.

To identify the molecular basis for the effects of MHC molecule polymorphism on T cell responses, we have combined functional T cell response testing with measurements of peptide binding to the class II MHC molecules on transfected cells. Our studies identify a small subset of spatially localized polymorphic residues of the E alpha E beta dimer (strand residue beta 29, and helix residues beta 72 and beta 75) regulating cytochrome c peptide presentation by two distinct mechanisms. The first effect is on quantitative control of net peptide binding. The replacement of the valine found at position beta 29 in E beta k with the glutamic acid found in E beta b results in a selective loss of pigeon cytochrome peptide but not moth cytochrome peptide binding to the resultant mutant E alpha E beta k molecule. Reciprocally, the replacement of glutamic acid at beta 29 in E beta b with valine results in a gain of pigeon peptide binding. These changes in binding parallel changes in T cell responses in vitro to these peptide-E alpha E beta combinations and mirror the in vivo immune response gene phenotypes of mice expressing E alpha E beta k and E alpha E beta b. E alpha E beta s molecules, which have a beta 29 glutamic acid, are nevertheless able to bind and present pigeon cytochrome peptides, and this is due to changes in helix residues beta 72 and beta 75 that compensate for the negative effect of the beta 29 glutamic acid. The second activity is a critical change in the conformation of the peptide bound to the same extent by distinct MHC molecules, as revealed by changes in T cell responses to moth cytochrome peptides presented by two E alpha E beta molecules differing only at position beta 29. Both of these effects can be ascribed to a single polymorphic residue modeled to be inaccessible to TCR contact (beta 29), providing a striking demonstration of how MHC molecule polymorphism can modify T cell-dependent immune responses without direct physical participation in the receptor recognition event.

Animals↗
PubMed1991

MHC class I surface expression in embryo-derived cell lines inducible with peptide or interferon.

It has long been recognized that the absence of expression of products of the major histocompatibility complex (MHC) during early development might allow the fetus to escape recognition by maternal lymphocytes. In addition to the MHC class I heavy chain and beta 2-microglobulin, antigenic peptide is an essential structural component of the class I molecule. Indeed, there is evidence that MHC-linked genes encoding peptide transporter molecules and possibly components of a proteolytic complex are necessary for MHC class I assembly and stability at the cell surface. Here we demonstrate that embryonic cells in general show a defect in MHC class I assembly. Surface expression was rescued in the presence of an appropriate antigenic peptide, or by treatment with interferon. Consistent with this, HAM1 messenger RNA was not constitutively expressed, but was inducible by interferon, and during differentiation in vitro. Thus, tolerance of the fetal allograft may in part be controlled at the level of peptide-dependent MHC class I assembly.

Animals↗
PubMed1991

Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex alpha/beta chains.

Distinct combinations of class II major histocompatibility complex (MHC) alpha and beta chains show widely varying efficiencies of cell surface expression in transfected cells. Previous studies have analyzed the regions of the class II chains that are critically involved in this phenomenon of variable expression and have shown a predominant effect of the NH2-terminal domains comprising the peptide-binding site. The present experiments attempt to identify the post-translational defects responsible for this variation in surface class II molecule expression for both interisotypic alpha/beta combinations failing to give rise to any detectable cell membrane molecules (e.g., E alpha A beta k) and intraisotypic pairs with inefficient surface expression (e.g., A alpha d A beta k). The results of metabolic labeling and immunoprecipitation experiments using L cell transfectants demonstrate that in both of these cases, the alpha and beta chains form substantial amounts of stable intracellular dimers. However, the isotype- and allele-mismatched combinations do not show the typical post-translational increases in molecular weight that accompany maturation of the N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These data are consistent with active or passive retention of relatively stable and long-lived mismatched dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticulum itself. This retention accounts for the absent or poor surface expression of these alpha/beta combinations, and suggests that conformational effects of the mismatching in the NH2-terminal domain results in a failure of class II molecules to undergo efficient intracellular transport.

Alleles↗
PubMed1991

MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding.

Class II major histocompatibility complex molecules undergo a change in structure upon stable binding of peptide antigen. Analysis of the site and extent of this change among class II molecules of splenic antigen-presenting cells reveals the preference of class II for peptide acquisition outside the endoplasmic reticulum and indicates that the class II presentation system is not saturated with self peptides. There are numerous empty class II molecules on the cell surface and peptide antigen is evidently important in regulating surface class II expression.

Animals↗
PubMed1991

A role for peptide in determining MHC class II structure.

T lymphocytes recognize antigen-derived peptides associated with major histocompatibility complex (MHC) class I or class II proteins. Peptide is critical in class I heavy-chain folding and/or stable association with beta 2-microglobulin. Although data exist suggesting a relationship between class II structure and peptide association, no equivalent positive contribution of peptide to the folding state or stability of class II dimers has yet been demonstrated. We report here that most purified E alpha k E beta k molecules leaving low pH in the absence of specific peptide lack a compact, stable dimeric structure. Brief exposure to the appropriate peptide just before and during neutralization promotes this specific conformation in proportion to stably bound peptide, indicating that peptide is important in determining class II MHC structure. Our results also indicate that efficient generation of long-lived peptide-class II complexes involves two stages: initial peptide binding in an acidic environment, which enhances the ability of class II to enter a conformation, from which stabilization upon neutralization results in high-affinity binding of previously associated peptide.

Animals↗
PubMed1991

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.

Antigens, CD↗
PubMed1991

Invariant chain promotes egress of poorly expressed, haplotype-mismatched class II major histocompatibility complex A alpha A beta dimers from the endoplasmic reticulum/cis-Golgi compartment.

Invariant chain (Ii) is a nonpolymorphic, non-major histocompatibility complex (MHC)-encoded glycoprotein that rapidly associates with newly synthesized class II MHC alpha and beta chains in the rough endoplasmic reticulum. This oligomerization of Ii, alpha, and beta and their cotransport within the cell led to speculation that Ii was an essential alpha beta transport protein. However, direct tests failed to show an absolute requirement for Ii in class II MHC molecule transport. More recently, it has become clear that different class II alpha beta chain combinations vary greatly in their efficiency of cell-surface expression, based largely on the allelic origin of the alpha and beta amino-terminal regions. Because the previous tests of Ii for a role in class II molecule expression utilized efficiently expressed alpha beta combinations, we have reexamined this question with several haplotype-mismatched murine A alpha and A beta chain combinations of various potentials for cell-surface expression. Using a transient expression assay in Ii-negative COS cells, we find that many inefficiently expressed alpha beta combinations show marked augmentation of surface expression upon cosynthesis of Ii. This effect is absent or minimal with evolutionarily coselected, haplotype-matched chains that give efficient expression alone. Biochemical studies show that at least one component of the Ii effect is an increased egress of already formed alpha beta dimers from the rough endoplasmic reticulum/cis-Golgi. We suggest that these results reflect the interaction of Ii with the peptide-binding domain of the poorly expressed class II molecules, either aiding in maintenance of a transportable conformation or competing with endoplasmic reticulum retention proteins, and thus enhancing movement to the cell surface. These results suggest a complex and variable role for trans-associated alpha and beta chains in the immune responses of MHC heterozygotes and provide a method for examining Ii interaction with class II MHC molecules independent of measurement of peptide presentation to T cells.

Animals↗
PubMed1991

Split anergy in a CD8+ T cell: receptor-dependent cytolysis in the absence of interleukin-2 production.

Engagement of the antigen-specific receptor (TCR) of CD4+ T lymphocytes without a second (costimulatory) signal prevents the subsequent production of interleukin-2 (IL-2) by these cells. Because IL-2 is a key immunoregulatory lymphokine and is also produced by a subset of CD8+ T cells that are able to kill target cells, the effect of engaging the TCR of one such clone in the absence of costimulatory signals was examined. The capacity for TCR-dependent IL-2 production was lost, indicating comparable costimulator-dependent signaling requirements for IL-2 production in CD4+ and CD8+ T cells. However, TCR-mediated cytotoxicity was not impaired, implying that costimulation is required for only certain TCR-dependent effector functions.

Animals↗
PubMed1991

Excess beta 2 microglobulin promoting functional peptide association with purified soluble class I MHC molecules.

T lymphocytes expressing alpha beta receptors recognize antigenic peptide fragments bound to major histocompatibility complex class I or class II molecules present on the surface membranes of other cells. Peptide fragments are present in the two available HLA crystal structures and recent data indicate that peptide is required for the stable folding of the class I heavy chain and maintenance of its association with the class I light chain, beta 2-microglobulin (beta 2m), at physiological temperature. To explain how the exogenous peptide used to create targets for cytotoxic cells bearing CD8 antigen could associate with apparently peptide-filled extracellular class I molecules, we hypothesized that stable binding of exogenous peptide to mature class I molecules reflects either the replacement of previously bound peptide during the well documented beta 2m exchange process or the loading of 'empty' class I heavy chains dependent on the availability of excess beta 2m. In either case, free beta 2m should enhance peptide/class I binding. Using either isolated soluble class I molecules or living cells, we show here that free purified beta 2m markedly augments the generation of antigenic complexes capable of T-cell stimulation.

Cell-Free System↗
PubMed1990

Structurally interdependent and independent regions of allelic polymorphism in class II MHC molecules. Implications for Ia function and evolution.

Possible interactions between regions of allelic polymorphism in the alpha- and beta-chains of class II MHC molecules were examined by measuring the efficiency of surface expression and the reactivity with mAb of wild-type and recombinant A alpha A beta-chain pairs from the b, d, and k haplotypes. These studies revealed regions of polymorphism within the alpha- and beta-chains that interact with complementary regions in the other chain. Unexpectedly, almost all the variable segments of both the class II MHC alpha- and beta-chains either directly contributed to or were near sites of interchain interactions. The exception was the beta HV3 (hypervariable (HV] segment (residues 61-71), which appeared to neither participate in nor be affected by interchain interactions. This division of the MHC molecule into interacting vs independent regions of allelic structural variation suggests that mutagenesis experiments involving the beta HV3 segment can be analyzed in a straightforward manner, as such mutations appear unlikely to alter the conformation of other molecular segments. Furthermore, functions attributed to the beta HV3 segment either experimentally or by population analysis should have a high probability of transfer by beta HV3 exchange (either experimentally or evolutionarily), because epitopes assigned to this region of the molecule are not affected by sequences outside this segment. This is of special importance because of the apparent involvement of this region in defining a potential site of interaction with antigenic peptides and TCR. In contrast, almost all other variable segments of the MHC molecule appear to have the capacity to contribute to interactions involving at least one other variable segment. This suggests not only that the experimental analysis of the contributions of these regions to various functions requires a consideration of inter- and intrachain interaction, but also that the transfer of function by genetic exchange of these structurally dependent regions is unpredictable. Selection must therefore operate on these interacting HV segments in the context of the complete alpha beta heterodimer. These results support our earlier arguments for cis-co-evolution of alpha- and beta-chain polymorphism and the absence of selection for F1 (hybrid) class II molecules. Finally, asymmetries observed in the contributions of particular pairs of HV segments to the efficient expression of Ia alpha beta heterodimers provide a basis for understanding mechanistically how cis-co-evolution may have occurred.

Alleles↗