Capture of membrane components via trogocytosis occurs in vivo during both dendritic cells and target cells encounter by CD8(+) T cells.

Cytotoxic T lymphocytes recently stimulated by antigen-presenting cells (APC) display major histocompatibility class (MHC) I and II molecules inherited from APC. We have previously reported that, in vitro, transfer of MHC molecules and several other proteins occurs through trogocytosis, i.e. the active acquisition of target cell membrane fragments by T lymphocytes. Here, using the model of viral antigen LCMVgp33-41 recognition in transgenic P14 mice, we show that CD8(+) T cells perform trogocytosis in vivo, as detected by the capture of biotin- or fluorescence-labeled components of the APC surface. Trogocytosis occurs during interactions of CD8(+) T cells with at least two kinds of cells: target cells and dendritic cells (DC). In lymph nodes, CD8(+) T cells having performed trogocytosis with DC express the CD69 activation marker indicating that trogocytosis detects recently activated cells. Taken together, our findings suggest that trogocytosis may be a new in vivo marker of the recent interaction between a CD8(+) T cell and its cellular partners or targets.

Allosteric changes in the TCR/CD3 structure upon interaction with extra- or intra-cellular ligands.

T lymphocytes are activated by the interaction between the T-cell antigen receptor (TCR) and peptides presented by major histocompatibility complex (MHC) molecules. The avidity of this TCR-pMHC interaction is very low. Therefore, several hypotheses have been put forward to explain how T cells become specifically activated despite this handicap: conformational change model, aggregation model, kinetic segregation model, sequential interaction model and permissive geometry model. In the present paper, we conducted experiments to distinguish between the TCR-aggregation model and the TCR-conformational change model. The results obtained using a TCR capture ELISA with Brij 98-solubilized TCR molecules from normal or activated T cells showed that the ligand-TCR interaction causes structural changes in the CD3 epsilon cytoplasmic tail as well as in the extracellular TCR beta FG loop region. Size-fractionation experiments with Brij 98-solubilized TCR/CD3/co-receptor complexes from naïve or activated CD4(+) or CD8(+) T cells demonstrated that such complexes are found as either dimers or tetramers. No monomers or multimers were detected. We propose that: (1) ligand-TCR interaction results in conformational changes in the CD3 epsilon cytoplasmic tail leading to T-cell activation; (2) CD3 epsilon cytoplasmic tail interaction with intracellular proteins may dissociate pMHC and co-receptors (CD4 or CD8) from TCR/CD3 complexes, thus leading to the arrest of T-cell activation; and (3) T-cell activation appears to occur among dimers or tetramers of TCR/CD3/co-receptor complexes interacting with self and non-self (foreign) peptide-MHC complexes.

Interactions between CD8alphabeta and the TCRalphabeta/CD3-receptor complex.

CD8+ T cells recognize antigenic peptides bound to major histocompatibility complex (MHC) class I molecules on normal antigen-presenting cells (APC), as well as on virus-infected cells or tumour cells (pMHC). At least two receptor types participate in recognition of these complexes: T-cell receptor (TCR) alphabeta heterodimers and CD8alphabeta molecules. The former molecules react with antigenic peptide and variable regions of MHC class I molecules, whereas the latter molecules react with constant alpha3 regions of MHC class I molecules. As the avidity of both receptor-MHC interactions is low, it is believed that TCRalphabeta and CD8alphabeta heterodimers collaborate in T-cell recognition. We have established a TCR/CD3-CD8 capture ELISA, which can measure the interaction of pMHC with CD8alphabeta molecules and with TCR/CD3 complexes. The major findings are: (1) TCR/CD3 complexes derived from in vitro activated T cells and captured by anti-CD3 MoAb, do bind specific pMHC and (2) CD8+ T cells express at least three forms of CD8alphabeta molecules: single CD8alphabeta, CD3-CD8 and TCR/CD3-CD8 complexes. Only the latter complexes are associated with CD3zeta homodimers, and the quantity of TCR/CD3-CD8 complexes relative to total CD8alphabeta molecules appears to increase and to be selected into sucrose-gradient microdomains as a function of TCRalphabeta-mediated T-cell activation.

A new generation of Melan-A/MART-1 peptides that fulfill both increased immunogenicity and high resistance to biodegradation: implication for molecular anti-melanoma immunotherapy.

Intense efforts of research are made for developing antitumor vaccines that stimulate T cell-mediated immunity. Tumor cells specifically express at their surfaces antigenic peptides presented by MHC class I and recognized by CTL. Tumor antigenic peptides hold promise for the development of novel cancer immunotherapies. However, peptide-based vaccines face two major limitations: the weak immunogenicity of tumor Ags and their low metabolic stability in biological fluids. These two hurdles, for which separate solutions exist, must, however, be solved simultaneously for developing improved vaccines. Unfortunately, attempts made to combine increased immunogenicity and stability of tumor Ags have failed until now. Here we report the successful design of synthetic derivatives of the human tumor Ag Melan-A/MART-1 that combine for the first time both higher immunogenicity and high peptidase resistance. A series of 36 nonnatural peptide derivatives was rationally designed on the basis of knowledge of the mechanism of degradation of Melan-A peptides in human serum and synthesized. Eight of them were efficiently protected against proteolysis and retained the antigenic properties of the parental peptide. Three of the eight analogs were twice as potent as the parental peptide in stimulating in vitro Melan-specific CTL responses in PBMC from normal donors. We isolated these CTL by tetramer-guided cell sorting and expanded them in vitro. The resulting CTL efficiently lysed tumor cells expressing Melan-A Ag. These Melan-A/MART-1 Ag derivatives should be considered as a new generation of potential immunogens in the development of molecular anti-melanoma vaccines.

Pleiotropic effects of post-translational modifications on the fate of viral glycopeptides as cytotoxic T cell epitopes.

The fate of viral glycopeptides as cytotoxic T lymphocyte (CTL) epitopes is unclear. We have dissected the mechanisms of antigen presentation and CTL recognition of the peptide GP392-400 (WLVTNGSYL) from the lymphocytic choriomeningitis virus (LCMV) and compared them with those of the previously reported GP92-101 antigen (CSANNSHHYI). Both GP392-400 and GP92-101 bear a glycosylation motif, are naturally N-glycosylated in the mature viral glycoproteins, bind to major histocompatibility complex H-2D(b) molecules, and are immunogenic. However, post-translational modifications differentially affected GP92-101 and GP392-400. Upon N-glycosylation or de-N-glycosylation, a marked decrease in major histocompatibility complex binding was observed for GP392-400 but not for GP92-101. Further, under its N-glycosylated or de-N-glycosylated form, GP392-400 then lost its initial ability to generate a CTL response in mice, whereas GP92-101 was still immunogenic under the same conditions. The genetically encoded form of GP392-400, which on the basis of its immunogenicity could still be presented with H-2D(b) during the course of LCMV infection, does not in fact appear at the surface of LCMV-infected cells. Our results show that post-translational modifications of viral glycopeptides can have pleiotropic effects on their presentation to and recognition by CTL that contribute to either creation of neo-epitopes or destruction of potential epitopes.

Common antiviral cytotoxic t-lymphocyte epitope for diverse arenaviruses.

Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.

Structural and functional identification of major histocompatibility complex class I-restricted self-peptides as naturally occurring molecular mimics of viral antigens. Possible role in CD8+ T cell-mediated, virus-induced autoimmune disease.

Structural similarity (molecular mimicry) between viral epitopes and self-peptides can lead to the induction of autoaggressive CD4(+) as well as CD8(+) T cell responses. Based on the flexibility of T cell receptor/antigen/major histocompatibility complex recognition, it has been proposed that a self-peptide could replace a viral epitope for T cell recognition and therefore participate in pathophysiological processes in which T cells are involved. To address this issue, we used, as a molecular model of viral antigen, the H-2D(b)-restricted immunodominant epitope nucleoprotein (NP)-(396-404) (FQPQNGQFI) of lymphocytic choriomeningitis virus (LCMV). We identified peptide sequences from murine self-proteins that share structural and functional homology with LCMV NP-(396-404) and that bound to H-2D(b) with high affinity. One of these self-peptides, derived from tumor necrosis factor receptor I (FGPSNWHFM, amino acids 302-310), maintained LCMV-specific CD8(+) T cells in an active state as observed both in vitro in cytotoxic assays and in vivo in a model of virus-induced autoimmune diabetes, the rat insulin promoter-LCMV NP transgenic mouse. The natural occurrence and molecular concentration at the surface of H-2(b) spleen cells of tumor necrosis factor receptor I-(302-310) were determined by on-line micro-high pressure liquid chromatography/mass spectrometry and supported its biological relevance.

Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner.

Upon encounter of a CTL with a target cell carrying foreign Ags, the TCR internalizes with its ligand, the peptide-MHC class I complex. However, it is unclear how this can happen mechanistically because MHC molecules are anchored to the target cell's surface via a transmembrane domain. By using antigenic peptides and lipids that were fluorescently labeled, we found that CTLs promptly capture target cell membranes together with the antigenic peptide as well as various other surface proteins. This efficient and specific capture process requires sustained TCR signaling. Our observations indicate that this process allows efficient acquisition of the Ag by CTL, which may in turn regulate lymphocyte activation or elimination.

Molecular and functional dissection of the H-2Db-restricted subdominant cytotoxic T-cell response to lymphocytic choriomeningitis virus.

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.

Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells.

By stimulating human lymphocytes with an autologous renal carcinoma, we obtained CTL recognizing an antigen derived from a novel, ubiquitous protein. The CTL failed to lyse autologous EBV-transformed B cells, even though the latter express the protein. This is due to the presence in these cells of immunoproteasomes, which, unlike standard proteasomes, cannot produce the antigenic peptide. We show that dendritic cells also carry immunoproteasomes and fail to present this antigen. This may explain why the relevant CTL escape thymic deletion and are not regularly activated in the periphery. Lack of cleavage by the immunoproteasome was also observed for melanoma differentiation antigen Melan-A26-35/HLA-A2, currently used for antitumoral vaccination. For immunization with such antigens, proteins should be less suitable than peptides, which do not require proteasome digestion in dendritic cells.

Genetically encoded and post-translationally modified forms of a major histocompatibility complex class I-restricted antigen bearing a glycosylation motif are independently processed and co-presented to cytotoxic T lymphocytes.

The mechanisms by which antigenic peptides bearing a glycosylation site may be processed from viral glycoproteins, post-translationally modified, and presented by major histocompatibility complex class I molecules remain poorly understood. With the aim of exploring these processes, we have dissected the structural and functional properties of the MHC-restricted peptide GP92-101 (CSANNSHHYI) generated from the lymphocytic choriomeningitis virus (LCMV) GP1 glycoprotein. LCMV GP92-101 bears a glycosylation motif -NXS- that is naturally N-glycosylated in the mature viral glycoprotein, displays high affinity for H-2D(b) molecules, and elicits a CD8(+) cytotoxic T lymphocyte response. By analyzing the functional properties of natural and synthetic peptides and by identifying the viral sequence(s) from the pool of naturally occurring peptides, we demonstrated that multiple forms of LCMV GP92-101 were generated from the viral glycoprotein and co-presented at the surface of LCMV-infected cells. They corresponded to non-glycosylated and post-translationally modified sequences (conversion of Asn-95 to Asp or alteration of Cys-92). The glycosylated form, despite its potential immunogenicity, was not detected. These data illustrate that distinct, non-mutually exclusive antigen presentation pathways may occur simultaneously within a cell to generate structurally and functionally different peptides from a single genetically encoded sequence, thus contributing to increasing the diversity of the T cell repertoire.

Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses.

Single H2Kb, H2Db and double H2KbDb homozygous knockout (KO) mice were generated and their peripheral CD8+ T cell repertoires compared to that of C57BL/6 (B6) mice. Limited (10-20%, H2Db), substantial (30-50%, H2Kb) and profound (90%, H2KbDb) reduction of peripheral CD8+ T cells was observed in KO mice, without Vbeta diversity alteration. Classical class Ia molecules therefore ensure most but not all of the peripheral CD8+ T cell repertoire education. As expected, H2Kb but also H2Db KO mice developed choriomeningitis following intracranial infection by lymphocytic choriomeningitis virus with the same kinetics, lethality and CD8+ cell implication as wild-type B6 mice. By contrast, H2KbDb (class Ia-Ib+) KO mice survived. Choriomeningitis of H2Db KO mice was linked to the development of a subdominant (in normal B6 mice) H2Kb-restricted cytotoxic T lymphocyte response. Mice expressing a restricted set of histocompatibility class I molecules should represent useful tools to evaluate the immunological potentials of individual MHC class I molecules.

A structure-based approach to designing non-natural peptides that can activate anti-melanoma cytotoxic T cells.

Tumor antigens presented by major histocompatibility complex (MHC) class I molecules and recognized by CD8(+) cytotoxic T lymphocytes (CTLs) may generate an efficient antitumor immune response after appropriate immunization. Antigenic peptides can be used in vivo to induce antitumor or antiviral immunity. The efficiency of naked peptides may be greatly limited by their degradation in the biological fluids. We present a rational, structure-based approach to design structurally modified, peptidase-resistant and biologically active analogues of human tumor antigen MAGE-1.A1. This approach is based on our understanding of the peptide interaction with the MHC and the T cell receptor and its precise degradation pathway. Knowledge of these mechanisms led to the design of a non-natural, minimally modified analogue of MAGE-1.A1, [Aib2, NMe-Ser8]MAGE-1.A1, which was highly peptidase-resistant and bound to MHC and activated MAGE-1.A1-specific anti-melanoma CTLs. Thus, we showed that it is possible to structurally modify peptide epitopes to obtain analogues that are still specifically recognized by CTLs. Such analogues may represent interesting leads for antitumor synthetic vaccines.

Use of a high-affinity peptide that aborts MHC-restricted cytotoxic T lymphocyte activity against multiple viruses in vitro and virus-induced immunopathologic disease in vivo.

Binding of a specific peptide(s) from a viral protein to major histocompatibility complex (MHC) class I molecules is a critical step in the activation of CD8(+) cytotoxic T lymphocytes (CTLs). Once activated, CTLs can cause lethal disease in an infected host, for example, by killing virus-containing ependymal and ventricular cells in the central nervous system or viral protein-expressing beta cells in the pancreatic islets of Langerhans. Here we describe the usage of a designed (not natural) high-affinity peptide to compete with viral peptide(s)-MHC binding. This peptide blocks virus-induced CTL-mediated disease both in the CNS and in the pancreatic islets in vivo. Further, the blocking peptide aborts MHC-restricted killing of target cells by CTLs generated to three separate viruses: lymphocytic choriomeningitis virus, influenza virus, and simian virus 40.

In vivo treatment with a MHC class I-restricted blocking peptide can prevent virus-induced autoimmune diabetes.

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2Db for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the Db allele, which correlated directly with the affinity of the respective epitopes. Expression of the blocking peptide in the target cell lowered recognition of all Db-restricted LCMV epitopes. In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-gamma secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV Db-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on Db-restricted anti-self (viral) CTL. Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-gamma and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.

Analysis of the degradation mechanisms of MHC class I-presented tumor antigenic peptides by high performance liquid chromatography/electrospray ionization mass spectrometry: application to the design of peptidase-resistant analogs.

Peptide vaccines based on the use of MHC class I restricted epitopes are currently assayed for anti-tumor and anti-viral immunotherapy. With the aim of designing minimally modified, peptidase-resistant analogs, we developed a rational approach based on a detailed understanding of the degradation mechanism of peptides in serum. Degradation of murine tumor antigen P198 and human tumor antigen MAGE-3.A1 was followed by on line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). This method provided high precision and sensitivity for rapid and direct analysis of degradation fragments in a complex mixture and, very importantly, precise identification of transient degradation fragments present at low concentrations. The design of structurally modified analogs, and the analysis of their degradation by on-line HPLC/ESI-MS, allowed us to to demonstrate the efficiency of local modifications in the protection of a given peptide bond towards a specific peptidase activity.

The signal sequence of lymphocytic choriomeningitis virus contains an immunodominant cytotoxic T cell epitope that is restricted by both H-2D(b) and H-2K(b) molecules.

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates three well-characterized H-2D(b)-restricted immunodominant epitopes delineated in the NP, GP1, and GP2 proteins. Here we report that the H-2D(b)-restricted GP1 epitope GP33-41/43 (KAVYNFATC/GI) located in the signal sequence of LCMV is also the immunodominant epitope recognized by CTL at the surface of the same infected cells in the context of H-2K(b) restriction. The GP1 epitope bound to H-2D(b) and H-2K(b) molecules with comparable affinities. The respective binding processes involved different sets of peptide anchoring residues and required dramatically different conformations of the peptide backbone as well as rearrangement of residue side chains. The 10-mer peptide GP34-43 (AVYNFATCGI) was the optimal H-2K(b)-binding sequence and the 8-mer peptide GP34-41 (AVYNFATC) the minimal sequence for optimal H-2K(b)-restricted CTL recognition. Comparison of lytic activities of primary splenic anti-LCMV CTL from C57BL/6 (D(b+)/K(b+)), B10A.[5R] (D(b-)/K(b+)), and B10A.[2R] (D(b+)/K(b-)) mice against LCMV-infected or peptide-coated target cells expressing either one or the two MHC alleles revealed that the H-2K(b)-restricted component of the anti-GP1 CTL response was mounted independently of but as efficiently as its H-2D(b) counterpart. Analysis of the immune response against a GP1 variant that escapes CTL recognition showed that the GP1 epitope: (i) was likely the only immunodominant LCMV epitope in the context of H-2K(b), and (ii) could efficiently evade H-2D(b) and H-2K(b)-restricted CTL mediated lysis.

Measles virus recognizes its receptor, CD46, via two distinct binding domains within SCR1-2.

Measles virus (MV) enters cells by attachment of the viral hemagglutinin to the major cell surface receptor CD46 (membrane cofactor protein). CD46 is a transmembrane glycoprotein whose ectodomain is largely composed of four conserved modules called short consensus repeats (SCRs). We have previously shown that MV interacts with SCR1 and SCR2 of CD46. (M. Manchester et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2303-2307) Here we report mapping the MV interaction with SCR1 and SCR2 of CD46 using a combination of peptide inhibition and mutagenesis studies. By testing a series of overlapping peptides corresponding to the 126 amino acid SCR1-2 region for inhibition of MV infection, two domains were identified that interacted with MV. One domain was found within SCR1 (amino acids 37-56) and another within SCR2 (amino acids 85-104). These results were confirmed by constructing chimeras with complementary regions from structurally similar, but non-MV-binding, SCRs of decay accelerating factor (DAF; CD55). These results indicate that MV contacts at least two distinct sites within SCR1-2.