Biomedical subjects

V Levitsky

Publications and source records attributed to V Levitsky.

26 records · Page 2Linked to original sources

PubMed1998

The clonal composition of a peptide-specific oligoclonal CTL repertoire selected in response to persistent EBV infection is stable over time.

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.

Amino Acid Sequence↗

PubMed1998

Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines.

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.

B-Lymphocyte Subsets↗

PubMed1997

Natural variants of the immunodominant HLA A11-restricted CTL epitope of the EBV nuclear antigen-4 are nonimmunogenic due to intracellular dissociation from MHC class I:peptide complexes.

EBV isolates from human populations with a high frequency of HLA A11 evade recognition by CTLs specific for an immunodominant A11-restricted epitope derived from the EBV nuclear antigen 4 (EBNA-4). We have previously described four nonimmunogenic variants of this epitope carrying single amino acid substitutions in the anchor residues of the peptide. We have now investigated the antigenicity, A11 binding capacity, endoplasmic reticulum translocation, endogenous processing, and presentation of these variants. The nonimmunogenic peptides were either unable to bind to HLA A11 or formed complexes of significantly lower stability compared with the immunogenic epitope. The latter peptides were produced in relatively large amounts by endogenous processing of EBNA-4 and associated with A11 molecules almost as efficiently as the immunogenic epitope, but the complexes failed to accumulate at the cell surface. The defect was not reversed by incubation of lymphoblastoid cell lines carrying the variant EBV strains at 26 degrees C. CTL lysis of HLA A11 positive targets was achieved by expressing one of the nonimmunogenic peptides through a vaccinia recombinant. However, the amount of peptide required for CTL sensitization exceeded, by at least 30-fold, that required for recognition of the immunogenic epitope. Collectively, these results suggest that complexes containing the nonimmunogenic peptides are formed but are then destroyed intracellularly. Thus, a specialized sorting mechanism seems to contribute in shaping the repertoire of peptides presented to T lymphocytes.

Cell Line, Transformed↗

PubMed1997

Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus.

The T cell receptor (TCR) repertoires of cytotoxic responses to the immunodominant and subdominant HLA A11-restricted epitopes in the Epstein-Barr virus (EBV) nuclear antigen-4 were investigated in four healthy virus carriers. The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT). Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis. Blocking of cytotoxic function by antibodies specific for the CD8 coreceptor indicated that, while AVF-specific TCRs are of high affinity, the oligoclonal response to the IVT epitope includes both low- and high-affinity TCRs. Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

Amino Acid Sequence↗

PubMed1996

The life span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class I-restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4.

We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) A11-restricted cytotoxic T lymphocyte (CTL) epitopes derived from amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation of lymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences of CTL efficiency since clones specific for the two epitopes lysed equally well A11-positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT-specific effectors, suggesting that a higher density of A11-IVT complexes is presented at the cell surface. In accordance, 10-50-fold higher amounts of IVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350-12,800 IVT and 8-760 AVF molecules per cell. Peptide-mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of A11 expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal efficiency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface turnover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of approximately 3.5 as compared to approximately 2 h for molecules induced at 26 degrees C in the absence of exogenous peptides and >12 h for IVT-containing complexes. This difference in persistence is likely to determine the representation of individual class I-restricted CTL epitopes within the cell surface pool of molecules, and may be an important factor contributing to their immunogenicity.

Amino Acid Sequence↗

PubMed1996

Solvent exposed side chains of peptides bound to HLA A*1101 have similar effects on the reactivity of alloantibodies and specific TCR.

Peptides can affect the recognition of MHC class I molecules by allospecific antibodies. Two explanations have been proposed for this phenomenon. The 'conformational change' hypothesis suggests that peptide binding affects the availability of serologic determinants in the class I alpha1 and alpha2 domains while the 'peptide-side-chain effect' predicts that solvent exposed residues in the peptide are part of the serologic epitope. We have tested these possibilities by examining the recognition of peptide loaded HLA A*1101 molecules expressed in transporters associated with antigen processing (TAP)-deficient cell lines by three A11-specific mAb, and by comparing the effect of peptide analogues on the recognition of A11 complexes containing peptide epitopes from the Epstein-Barr virus nuclear antigen EBNA4 by antibodies and cytotoxic T lymphocytes (CT). The AUF5.13 and HB164 antibodies showed selective recognition of A11 molecules bound to partially overlapping sets of peptides from viral or cellular origin. The peptide dependence of AUF5.13 was confirmed in reconstitution experiments where A11 molecules were refolded at the surface of TAP-deficient T2/A11 cells that had been cultured at 26 degrees C and treated at pH3. Molecular modelling and Ala scanning mutagenesis of the IVTDFSVIK (IVT) and AVFDRKSDAK (AVF) peptides demonstrated that solvent-exposed peptide side chains affect CTL recognition as well as antibody binding. Substitution of Phe-P5 or Ser-P6 of the IVT peptide with Arg or Lys inhibited AUF5.13 recognition while binding was induced by substitution of the Arg-P5 and Lys-P6 of the AVF peptide with Ala. The results suggest that some allospecific antibodies recognized the surface of MHC class I-peptide complexes in a fashion similar to the TCR. This may involve direct interaction with the peptide side chains as well as recognition of peptide-induced perturbations in the class I complex.

ATP-Binding Cassette Transporters↗

PubMed1995

Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1.

The Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA1) is expressed in latently EBV-infected B lymphocytes that persist for life in healthy virus carriers, and is the only viral protein regularly detected in all malignancies associated with EBV. Major histocompatibility complex (MHC) class I-restricted, EBNA1-specific cytotoxic T lymphocyte (CTL) responses have not been demonstrated. Using recombinant vaccinia viruses encoding chimaeric proteins containing an immunodominant human leukocyte antigen A11-restricted CTL epitope, amino acids 416-424 of the EBNA4 protein, inserted within the intact EBNA1, or within an EBNA1 deletion mutant devoid of the internal Gly-Ala repetitive sequence, we demonstrate that the Gly-Ala repeats generate a cis-acting inhibitory signal that interferes with antigen processing and MHC class I-restricted presentation. Insertion of the Gly-Ala repeats downstream of the 416-424 epitope inhibited CTL recognition of a chimaeric EBNA4 protein. The results highlight a previously unknown mechanism of viral escape from CTL surveillance, and support the view that the resistance of cells expressing EBNA1 to rejection mediated by CTL is a critical requirement for EBV persistence and pathogenesis.

Antigen Presentation↗

PubMed1994

T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues.

Epstein-Barr virus (EBV) is a B lymphotropic herpesvirus of humans that elicits strong HLA class I-restricted cytotoxic T lymphocyte (CTL) responses. An influence of such responses on virus evolution was first suggested by our finding that EBV isolates from the highly HLA A11-positive Papua New Guinea (PNG) population carried a lys-thr mutation at residue 424 of the nuclear antigen EBV-encoded nuclear antigen (EBNA4) that destroyed the immunodominant target epitope for A11-restricted CTL recognition. Here we turn to a much larger population, Southern Chinese, where the A11 allele is again present in over 50% of the individuals. Each of 23 EBV isolates analyzed from this population were also mutated in the EBNA4 416-424 epitope, the mutations selectively involving one of the two anchor residues in positions 2 (417 val-leu) or 9 (424 lys-asp, -arg or -thr) that are critical for A11-peptide interaction. The majority of the Chinese isolates and all 10 PNG isolates also carried mutations affecting positions 1 and 2 of the next most immunodominant A11-restricted epitope, EBNA4 residues 399-408. These changes clearly affected antigenicity since A11-positive lymphoblastoid cell lines (LCLs) carrying these mutant EBV strains were not recognized by A11-restricted CTLs raised against the prototype B95.8 virus. Furthermore, Chinese donors naturally infected with these mutant viruses did not mount detectable A11-restricted CTL responses on in vitro stimulation with autologous LCL cells carrying either the B95.8 or their endogenous EBV strain. In two different highly A11-positive populations, therefore, immune pressure appears to have selected for resident EBV strains lacking immunodominant A11-restricted CTL epitopes.

Amino Acid Sequence↗