Tumor necrosis factor-alpha induces coordinated changes in major histocompatibility class I presentation pathway, resulting in increased stability of class I complexes at the cell surface.

It is demonstrated that similar to interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) induces coordinated changes at different steps of the major histocompatibility complex (MHC) class I processing and presentation pathway in nonprofessional antigen-presenting cells (APCs). TNF-alpha up-regulates the expression of 3 catalytic immunoproteasome subunits--LMP2, LMP7, and MECL-1--the immunomodulatory proteasome activator PA28 alpha, the TAP1/TAP2 heterodimer, and the total pool of MHC class I heavy chain. It was also found that in TNF-alpha--treated cells, MHC class I molecules reconstitute more rapidly and have an increased average half-life at the cell surface. Biochemical changes induced by TNF-alpha in the MHC class I pathway were translated into increased sensitivity of TNF-alpha--treated targets to lysis by CD8(+) cytotoxic T cells, demonstrating improved presentation of at least certain endogenously processed MHC class I--restricted peptide epitopes. Significantly, it was demonstrated that the effects of TNF-alpha observed in this experimental system were not mediated through the induction of IFN-gamma. It appears to be likely that TNF-alpha--mediated effects on MHC class I processing and presentation do not involve any intermediate messengers. Collectively, these data demonstrate the existence of yet another biologic activity exerted by TNF-alpha, namely its capacity to act as a coordinated multi-step modulator of the MHC class I pathway of antigen processing and presentation. These results suggest that TNF-alpha may be useful when a concerted up-regulation of the MHC class I presentation machinery is required but cannot be achieved by IFN-gamma. (Blood. 2001;98:1108-1115)

Modulation of intracellular Ca(2+) concentration by vitamin B12 in rat thymocytes.

We have studied several novel effects of vitamin B12 (cyanocobalamin) on cellular Ca(2+) homeostasis in rat thymocytes. We determined the effect of various concentrations of vitamin B12 on intracellular Ca(2+) concentration ([Ca(2+)]i) and parameters of Ca(2+)in signaling using the fluorescent dye Fura-2. The basal [Ca(2+)]i in Ca(2+)-containing media was 115 +/- 5 nM but in vitamin B12 (10 nM)-treated thymocytes [Ca(2+)]i was decreased to 60 +/- 15 nM (mean +/- SEM) during the first 5 min. The decline in [Ca(2+)]i was accompanied by an increase in the endoplasmic reticulum Ca(2+) store, presumably as a result of Ca-ATPase activation. At the same time 100 nM-10 mM B12 induced the accumulation of Ca(2+) in mitochondria. Somewhat higher concentrations of B12 (1-10 microM) had no effect on [Ca(2+)]i. A further increase in B12 concentration with range from 50 microM to 1 mM caused a dose-dependent elevation of [Ca(2+)]i from the basal level (115 +/- 5 nM) up to 200 +/- 50 nM in thymocytes, and this elevation was partially blocked in Ca(2+)-free media. This high concentration of vitamin B12 caused a gradual decrease of endoplasmic reticulum Ca(2+) stores by means of Ca-ATPase inhibition. The B12-induced increase in [Ca(2+)]i was not observed after depletion of intracellular Ca(2+) stores, induced by addition of 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ), an inhibitor of endoplasmic reticulum Ca (2+)-ATPase, concanavalin A, or arachidonic acid. These studies show that vitamin B12 regulates [Ca(2+)]i via several different mechanisms at different B12 concentrations. Participation of G proteins and calmodulin activity in B12-mediated [Ca(2+)]i increase is discussed.

Supermotif peptide binding and degeneracy of MHC: peptide recognition in an EBV peptide-specific CTL response with highly restricted TCR usage.

We have investigated the presentation and CTL recognition of an HLA A*1101-restricted CTL peptide epitope AVFDRKSDAK (AVF)(3), derived from the EBV nuclear antigen (EBNA) 4, in the context of alleles belonging to the A3-supertype, A*0101, 0301, 1101, 3101, 3301, and 6801. The peptide binds to a A*6801 molecule as efficiently as to A*1101. The A*6801:AVF complex is recognized by some A*1101-restricted AVF- specific CTL clones. However, A*6801-positive (A*6801+) EBV-transformed lymphoblastoid cell lines (LCLs) are not killed by the same effectors. Furthermore, two A*6801+ donors did not mount an AVF-specific CTL response in vitro and lacked detectable AVF-specific effectors. Thus, this epitope is either subdominant, or non-immunogenic in the context of A*6801. These characteristics correlate with low stability of this MHC:peptide complex in living cells. We also demonstrate that a highly conserved AVF-specific TCR that dominates the AVF-specific CTL response in the majority of A*1101+ individuals recognizes the A*6801 molecule as a crossreactive alloantigen. Therefore, deletion of AVF-specific T cells may contribute to the non-immunogenicity or subdominance of the peptide in A*6801+ individuals.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

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.

Defective presentation of MHC class I-restricted cytotoxic T-cell epitopes in Burkitt's lymphoma cells.

Defects of antigen processing/presentation have been suggested to play a role in the escape of Burkitt's lymphoma (BL) from cytotoxic T lymphocyte (CTL)-mediated rejection. Impaired presentation of an immunodominant HLA A11-restricted epitope from the resident or recombinant vaccinia virus-expressed Epstein-Barr virus nuclear antigen (EBNA)4 was demonstrated in the EBV-positive E95B-BL28 and its EBV-negative parental BL28 cell lines. We have investigated whether this was due to (i) impaired production of the antigenic peptide, (ii) poor peptide translocation into the ER lumen or (iii) inefficient maturation and transport of the MHC-peptide complexes at the cell surface. The defect was not overcome by cytosolic expression of a pre-formed epitope, suggesting that presentation of EBNA4 is not limited by inefficient production of the antigenic peptide. BL28 expressed 5- to 10-fold lower levels of the transporter associated with antigen presentation (TAP) 1 and TAP2 proteins and behaved poorly in a streptolysin-O-mediated peptide translocation assay, whereas E95B-BL28 showed higher TAP expression and virtually normal transporter function. Up-regulation of HLA A11 and reconstitution of TAP activity by treatment with IFN-gamma did not restore presentation of the resident EBNA4 in E95SB-BL28 and did not enhance presentation of the vaccinia virus-expressed intact protein or preformed epitope. Efficient maturation of class I molecules to Endo H-resistant species was demonstrated in pulse-chase experiments. Taken together, our findings identify a previously uncharacterized defect of antigen presentation which appears to affect events occurring after proteasomal degradation but before TAP-dependent peptide transport and MHC class I assembly and maturation.

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.

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.