Cytotoxic T Lymphocytes Recognition Research Articles

Sexual Transmission of Single Human Immunodeficiency Virus Type 1 Virions Encoding Highly Polymorphic Multisite Cytotoxic T-Lymphocyte Escape Variants

Antigenic variation inherent in human immunodeficiency virus type 1 (HIV-1) virions that successfully instigate new infections transferred by sex has not been well defined. Yet this is the viral "challenge" which any vaccine-induced immunity must deal with. Closely timed comparisons of the virus circulating in the "donor" and that which initiates new infection are difficult to carry out rigorously, as suitable samples are very hard to get in the face of ethical hurdles. Here we investigate HIV-1 variation in four homosexual couples where we sampled blood from both parties within several weeks of the estimated transmission event. We analyzed variation within highly immunogenic HIV-1 internal proteins encoding epitopes recognized by cytotoxic T lymphocytes (CTLs). These responses are believed to be crucial as a means of containing viral replication. In the donors we detected virions capable of evading host CTL recognition at several linked epitopes of distinct HLA class I restriction. When a donor transmitted escape variants to a recipient with whom he had HLA class I molecules in common, the recipient's CTL response to those epitopes was prevented, thus impeding adequate viral control. In addition, we show that even when HLA class I alleles are disparate in the transmitting couple, a single polymorphism can abolish CTL recognition of an overlapping epitope of distinct restriction and so confer immune escape properties to the recipient's seroconversion virus. In donors who are themselves controlling an early, acute infection, the precise timing of onward transmission is a crucial determinant of the viral variants available to compose the inoculum.

Functional Constraints of Influenza A Virus Epitopes Limit Escape from Cytotoxic T Lymphocytes

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M1(58-66). We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M1(58-66) epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes.

Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes

Cytotoxic T lymphocytes (CTL) are critical for control of lentiviruses, including equine infectious anemia virus (EIAV). Measurement of equine CTL responses has relied on chromium-release assays, which do not allow accurate quantitation. Recently, the equine MHC class I molecule 7-6, associated with the ELA-A1 haplotype, was shown to present both the Gag-GW12 and Env-RW12 EIAV CTL epitopes. In this study, 7-6/Gag-GW12 and 7-6/Env-RW12 MHC class I/peptide tetrameric complexes were constructed and used to analyze Gag-GW12- and Env-RW12-specific CTL responses in two EIAV-infected horses (A2164 and A2171). Gag-GW12 and Env-RW12 tetramer-positive CD8+ cells were identified in nonstimulated peripheral blood mononuclear cells as early as 14 days post-EIAV inoculation, and frequencies of tetramer-positive cells ranged from 0.4% to 6.7% of nonstimulated peripheral blood CD8+ cells during the 127-day study period. Although both horses terminated the initial viremic peak, only horse A2171 effectively controlled viral load. Neutralizing antibody was present during the initial control of viral load in both horses, but the ability to maintain control correlated with Gag-GW12-specific CD8+ cells in A2171. Despite Env-RW12 dominance, Env-RW12 escape viral variants were identified in both horses and there was no correlation between Env-RW12-specific CD8+ cells and control of viral load. Although Gag-GW12 CTL escape did not occur, a Gag-GW12 epitope variant arose in A2164 that was recognized less efficiently than the original epitope. These data indicate that tetramers are useful for identification and quantitation of CTL responses in horses, and suggest that the observed control of EIAV replication and clinical disease was associated with sustained CTL recognition of Gag-specific epitopes.

Immunogenetics: Safety in numbers

A recent report from Enrique Gonzalez et al provides a striking example of the influence that duplications of immune receptor genes can have on HIV susceptibility and progression to AIDS. The HIV virus utilises three major receptors of the immune system to beat this system from within. These are the CD4 coreceptor on CD4þ T-helper cells, the CCR5 chemokine receptor on T-cells and the DC sign receptor on antigenpresenting dendritic cells (DCs). The virus utilises these receptors to attack to, penetrate into and replicate in mainly CD4þ T cells and it eventually destroys and depletes them. In the case of DC sign, this appears to allow DCs to transport HIV towards central lymphoid organs to gain better access to T lymphocytes. Polymorphisms in the HIV components that interact with these receptors and polymorphisms in the host receptors themselves, as well as duplications of these genes can therefore be expected to influence susceptibility to HIV infection and progression to AIDS. In this new study, the authors show that duplications of the gene that encodes CCL3L1, the most potent known ligand for CCR5, confers marked resistance to HIV infection and progression towards AIDS, independent of ethnic background. CCL3L1 presumably has potent anti-HIV activity because its chemokine activity might block the interaction between HIV gp120 envelope protein and CCR5. As the authors speculate, another alternative is that CCL3L1 may induce ligand-mediated internalisation of CCR5, thereby reducing its availability for use by gp120. More research is obviously needed to clarify the precise mechanism(s) by which CCL3L1 low copy number predisposes to HIV persistence following infection and to progression towards AIDS in those persistently infected. Not surprisingly, CCL3L1 copy number and CCR5 genotypes were shown to have interactive effects. Earlier work had shown before that CCR5 haplotypes, including CCR5 promoter polymorphisms as well as coding polymorphisms in CCR2 (CCR2-V64I) and CCR5 (D32), influence the risk of acquiring HIV and the rate of disease progression. – 5 In the current work, CCR5 detrimental (det), that is, disease accelerating and nondetrimental (non-det) CCR5 genotypes, were further analysed with respect to low or high CCL3L1 copies (CCL3L1 or CCL3L1 ) in an HIV-positive adult cohort. This analysis allowed this cohort to be stratified into four mutually exclusive genetic risk groups. Interestingly, the theoretically highest risk group (CCL3L1 CCR5) showed a greater than three-fold greater risk of progressing rapidly to eight of 12 AIDS-defining illnesses. The important role that genetics has in susceptibility to retrovirus-induced disease has been well-known for decades, ever since the pioneering work of Frank Lilly and co-workers – 8 on murine viral leukemogenesis. The genes involved were found to affect viral host range and replication as well as the immune response. The earliest gene locus (cluster) found to affect susceptibility to murine viral leukemogenesis was in fact MHC. Even at this early stage, a gene dose effect was evident, because in animals with homozygous MHC-resistant haplotypes a protection against viral leukemogenesis was more profound than in heterozygous animals with a single MHC-resistant haplotype. MHC haplotypes are known to affect a variety of specific immune responses, including antiviral antibodies, CD4þ Thelper cell responses and cytotoxic T lymphocyte (CTL) responses. For example, the HLA B*5701 allele frequently shows up in cohorts of long-term asymptomatic HIV-infected individuals: a pattern that probably reflects CTL recognition of dominant and conserved HIV peptides that HLA B*5701 presents. In most individuals infected with HIV, the virus manages to immuno-escape from CTL responses by generating mutations in the viral peptides presented by HLA molecules. Viral structural constraints in the conserved HIV peptides presented by HLA B*5701 apparently do not allow the virus to escape from immune attack by mutations in these particular sequences. So now we have a good idea of the importance of polymorphisms in CCR5, CCL3L1 and HLA genes for susceptibility to retroviral diseases. However, many more genetic effects are lurking in the dark, awaiting exploration. Nonetheless, the susceptibility loci that have already been identified point towards the mechanisms that underlie this susceptibility and, moreover, suggest ways of therapeutically exploiting the chemokine and chemokine receptor pathways. Also, in vaccine design experience with the HLA B57 resistance allele indicates that HIV vaccines should aim at induction of T-cell responses against conserved epitopes that the HIV virus cannot afford to evade by generating escape mutants. Clearly, the challenges for geneticists, virologists and immunologists alike are enormous, to not only further document genetic control of HIV infection and progression towards AIDS, but also to design novel therapeutic interventions based on these insights’

Cross-presentation by intercellular peptide transfer through gap junctions

Major histocompatibility complex (MHC) class I molecules present peptides that are derived from endogenous proteins. These antigens can also be transferred to professional antigen-presenting cells in a process called cross-presentation, which precedes initiation of a proper T-cell response; but exactly how they do this is unclear. We tested whether peptides can be transferred directly from the cytoplasm of one cell into the cytoplasm of its neighbour through gap junctions. Here we show that peptides with a relative molecular mass of up to approximately 1,800 diffuse intercellularly through gap junctions unless a three-dimensional structure is imposed. This intercellular peptide transfer causes cytotoxic T-cell recognition of adjacent, innocent bystander cells as well as activated monocytes. Gap-junction-mediated peptide transfer is restricted to a few coupling cells owing to the high cytosolic peptidase activity. We present a mechanism of antigen acquisition for cross-presentation that couples the antigen presentation system of two adjacent cells and is lost in most tumours: gap-junction-mediated intercellular peptide coupling for presentation by bystander MHC class I molecules and transfer to professional antigen presenting cells for cross-priming.

A Novel Tissue-Restricted Minor Histocompatibility Antigen Resulting from Differential Expression Due to a Deletion/Insertion Polymorphism in the P2X5 Purinergic Receptor Gene.

Minor histocompatibility antigens (mHags) that are selectively expressed by hematopoietic cells, including hematopoietic tumor cells, represent attractive targets for T cell-based immunotherapy after MHC-matched allogeneic stem cell transplantation (SCT) to boost the graft-versus-tumor response without causing graft-versus-host disease. We isolated a CD8+ cytotoxic T lymphocyte (CTL) clone from a SCT recipient that recognized a novel HLA-B*0702-restricted mHag with lymphoid lineage-specific expression. In vitro CTL recognition experiments revealed that this mHag was expressed by EBV-transformed B cells, CD40-activated B cells and PHA-stimulated T cells, but not expressed by cells of myeloid origin and non-hematopoietic fibroblasts. Furthermore, testing a panel of HLA-B*0702 transduced cell lines showed that several B lymphoid tumor cell lines express this mHag, which was designated as Lymphoid Restricted Histocompatibility antigen-1 (LRH-1). To define the chromosomal localization of the LRH-1 coding gene, we used genetic linkage analysis using EBV-lymphoblastoid cell lines (LCL) form the Centre d'Etude Polymorphism Humain (CEPH) reference families that have been extensively mapped for thousands of genetic markers. EBV-LCL generated from individuals of six CEPH reference families were retrovirally transduced with HLA-B*0702 and tested for CTL recognition. After determining the LRH-1 segregation pattern, linkage and haplotype analysis revealed that the LRH-1 coding gene maps to a 1.64 Mb region on chromosome 17p13.2 between marker D17S1845 and D17S1584. A database search identified 19 candidate genes that are localized within this region. Real-time RT-PCR analysis revealed that 3 genes showed an expression profile that corresponds to the CTL recognition pattern of which the purinergic receptor gene P2X5 was the most promising candidate. Transfection of P2X5 cDNA cloned from a homozygous antigen-positive individual into 293-HLA-B*0702 cells resulted in efficient recognition by the CTL clone. Testing of different P2X5 transcript variants and deletion constructs revealed that the nucleotide sequence coding the antigenic peptide is located within exon 3 of P2X5 transcript variant 1. In this region, we identified a synthetic nonameric peptide that sensitized antigen-negative donor EBV-LCL to CTL recognition, with half-maximal recognition observed at a peptide concentration of 20 nM. To identify the basis for LRH-1 disparity, exon 3 of the P2X5 alleles of the donor and recipient were sequenced and compared. The antigen-negative donor contained a homozygous C-nucleotide deletion/insertion polymorphism (DIP) resulting in a frame-shift in the protein at the third residue of the epitope. Transfection of the donor P2X5 transcript variant 1 into 293-HLA-B*0702 cells confirmed that the C-nucleotide DIP within the donor allele was associated with resistance to CTL recognition. These results provide the first evidence that mHag can result from differential protein expression as a consequence of a homozygous DIP in a transcript variant of the encoding gene.

HLA-Cw6 Alloreactive CTL Recognizing HLA-CW6+ Myeloma Cells but Not Non-Myeloma HLA-Cw6+ Cells Are Peptide Dependent.

We investigated the generation of myeloma-specific cytotoxic T lymphocytes (CTL) from unrelated normal donors non-HLA matched with the myeloma cell line SBN. The aim was to obtain alloreactive CTL specific of peptides to identify genes preferentially expressed by to myeloma cells. After coculture of SBN with PBL of unrelated donor, the T-cell line obtained was cloned and each CTL was assessed against SBN and SB-EBV (B-EBV cell line obtained by infecting B cells of SB patient with EBV) simultaneously. Only the CTL recognizing SBN but not SB-EBV were kept for further study. Among more than 200 clones screened, we isolated two different CTL CD8+ recognizing SBN only (60% of specific lysis at effector: target ratio 5). Their cytotoxicity was blocked by antibodies against HLA-I molecules and more precisely by mAb against HLA-B/Cw molecules. Both CTL recognized also other MM cell lines but only those matched with SBN for HLA-Cw0602 (XG6, BCN) suggesting an HLA-Cw6 recognition. Furthermore, antiserum directed against HLA-Cw6 abrogated the recognition of SBN, XG6 and BCN. Both CTL were not cytotoxic against BC-EBV nor against 3 other B-EBV cell lines derived from HLA-Cw0602 donors. This unreactivity was not restricted to B-EBV cell lines since PBL from 2 normal HLA-Cw0602 donors were also not recognized (cytotoxicity, TNF secretion). We directly measured cell surface HLA-Cw6 expression of both B-EBV and myeloma cells with a scFv directed against HLA-Cw6 and we showed that HLA-Cw6 was more expressed by B-EBV cells as compared to myeloma cells (2fold). So, the lack of recognition of B-EBV cells was not related to HLA-Cw6 expression level. We also looked for KIR expression by the CTL since KIR molecules could have blocked B-EBV recognition that expressed more HLA-Cw6 molecules as compared to myeloma cells. No KIR molecules (p58.1, p58.2, CD94) were found to be expressed by the CTL.To investigate whether the recognition of myeloma cells was peptide dependent, we performed two experiments i.e., acid elution and cold target competition. Acid elution (glycine 0.3M, pH=2.5) abrogated myeloma recognition and cold target competition showed that B-EBV cells were not recognized by the CTL. These data suggest that the CTL recognize a peptide or a set of peptides restricted to myeloma cells or that the CTL recognition is activated by tumor cells only. Anyway, such alloreactive CTL could be helpful for GVL reaction in allotransplanted patients with myeloma.

The Immunogenicity of a Human Lymphoid Cell-Restricted Minor Histocompatibility Antigen Results from Differential Protein Translation.

Minor histocompatibility antigens (mHA) that are presented selectively on hematopoietic cells, including leukemic cells, but not on nonhematopoietic tissues represent attractive immunotherapeutic targets after HLA-matched allogeneic hematopoietic cell transplantation (HCT) to enhance the graft-versus-leukemia (GVL) effect without instigating graft-versus-host disease. We isolated from an HLA-matched HCT recipient a CD8+ cytotoxic T-lymphocyte (CTL) clone, KSN 7A7, which recognized a novel HLA-A3-restricted mHA with lymphoid-restricted presentation. KSN 7A7 demonstrated high-level in vitro cytolysis of a recipient EBV-transformed B lymphoblastoid cell line (BLCL) and low-level cytolysis of recipient PHA-stimulated T cells, but no recognition of recipient dermal fibroblasts or any donor cells. When tested against a panel of allogeneic HLA-A3+ leukemic cells, KSN 7A7 showed cytolysis against malignant cells of B-lymphoid origin but not of myeloid origin. To identify the epitope recognized by KSN 7A7 CTL, HLA-A3-associated peptides were extracted from a phenotypically mHA+ BLCL, fractionated by reversed-phase HPLC, and tested for the ability to reconstitute CTL recognition of mHA− donor BLCL. After 3 rounds of RP-HPLC, candidate peptide masses within biologically active fractions were selected using nanospray Fourier transform mass spectrometry, and the most abundant candidate peptide ion was sequenced by collision activated dissociation. A synthetic decameric peptide corresponding to this sequence sensitized donor BLCL to CTL lysis, with half-maximal lysis observed at a peptide concentration of 2 nM. A database search revealed that this peptide was encoded by an alternative transcript of the C22orf18 gene (LocusLink 79019), with the epitope derived from an alternative exon that is not known to be utilized by other transcripts of this gene. To identify the basis for differential CTL recognition of donor and recipient BLCL, donor and recipient C22orf18 alleles were sequenced and compared. The mHA− donor was homozygous for a C → T single nucleotide polymorphism (SNP) within the codon for the first residue of the epitope that in turn created a translation termination signal, suggesting that the antigenicity of this locus is a consequence of differential translation of the protein encoded by this transcript in donor and recipient cells. Direct sequencing and PCR-RFLP analysis of the region surrounding this SNP in C22orf18 alleles in 45 additional HLA-A3+ individuals revealed that the frequency of the T allele encoding the translation termination signal was 0.25. Phenotyping of BLCL from these 45 individuals for mHA expression confirmed that homozygosity for the T allele was associated with resistance to CTL lysis. RT-PCR analysis revealed that the C22orf18 alternative transcript is almost exclusively expressed in resting and activated CD19+ cells, with very low levels of expression detected in CD4+, CD8+, and CD14+ cells, and negligible expression in nonhematopoietic tissues. Thus, therapeutic strategies targeting this mHA could selectively enhance the GVL effect in HCT recipients with B-lymphoid lineage leukemias.

Competition Among Peptides in Melanoma Vaccines for Binding to MHC Molecules

The effectiveness of peptide-based cancer vaccines depends on the ability of peptides to bind to MHC molecules on the surface of antigen-presenting cells, where they reconstitute epitopes for cytotoxic T lymphocytes (CTLs). Multivalent vaccines have advantages over single-peptide vaccines; however, peptides may compete for binding to the same MHC molecules. In particular, it is possible that peptides with high affinity for MHC molecules prevent the binding of lower-affinity peptides. However, only small numbers of peptide/MHC complexes per cell are required for CTL recognition. Thus, the authors hypothesized that competition of peptides for MHC binding would not significantly reduce CTL recognition of individual peptides within a multiple-peptide mixture, and this hypothesis was tested by a series of experiments performed in vitro. In multiple experiments, two peptides with different affinities for HLA-A*0201 molecules were mixed at various concentrations and pulsed onto HLA-A2 cells, which were then evaluated for susceptibility to lysis by HLA-A*0201-restricted CTLs. CTL recognition of the melanoma peptides gp100(154-162) (KTWGQYWQV), gp100(280-288) (YLEPGPVTA), and tyrosinase(369-377D) (YMDGTMSQV) was maintained even when target cells were co-pulsed with equimolar concentrations of peptides with comparable or higher affinity for HLA-A2. In some cases, CTL recognition was maintained even when the higher-affinity peptide was present at concentrations several orders of magnitude higher than the target peptide. In addition, CTLs generated by in vitro stimulation with a peptide mixture developed reactivity to three different peptides, at a level comparable to that obtained by stimulation with each individual peptide separately. These data suggest that CTLs can respond to multiple peptides presented on the same antigen-presenting cells and justify further investigation, in clinical trials, of multiple-peptide cancer vaccines.

Structural constraints on viral escape from HIV- and SIV-specific cytotoxic T-lymphocytes.

Cytotoxic T lymphocytes (CTL) play a central role in controlling lentiviral infections in both humans and monkeys. While they contain the spread of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), CTL are not capable of fully eradicating virus following infection. Ongoing viral replication can therefore lead to the accumulation of viral mutations within CTL epitopes that can undermine cellular immune control of virus. Here we review the importance of CTL in controlling HIV/SIV infection and how immunologic pressure exerted by effector T cells selects for viral variants that escape CTL recognition. We review two examples of viral escape from CTL at highly conserved epitopes that illustrate the extraordinary capacity of lentiviruses to adapt to their immunologic environment despite structural constraints on the ability of the virus to accommodate mutations.

Recognition of influenza virus epitope variants by human CTL

Recently, an HLA-B*3501-restricted cytotoxic T-lymphocytes (CTL) epitope in the nucleoprotein (NP 418–426) of influenza A viruses was identified, which exhibited a high degree of variability. In the present study, the recognition of epitope variants by human cytotoxic T-lymphocytes (CTL) was investigated. Human CD8+ CTL clones were specific for NP 418–426 epitope variants within one subtype of influenza virus or cross-reactive with hetero-subtypic variants. Positions in the 9-mer were identified as determinants of CTL-specificity. The in vivo existence of T cells cross-reactive with homo- and hetero-subtypic variants of the epitope was confirmed with virus-specific polyclonal T cell populations obtained from HLA-B*3501+ blood donors. Based on CTL recognition patterns and the history of infection as assessed by serology, it was hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.

HIV evolution: CTL escape mutation and reversion after transmission.

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level.

Impaired processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape mechanisms from CTL immune pressure in human immunodeficiency virus type 1 infection.

Investigating escape mechanisms of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTLs) is essential for understanding the pathogenesis of HIV-1 infection and developing effective vaccines. To study the processing and presentation of known CTL epitopes, we prepared Epstein-Barr virus-transformed B cells that endogenously express the gag gene of six field isolates by adopting an env/nef-deletion HIV-1 vector pseudotyped with vesicular stomatitis virus G protein and then tested them for the recognition by Gag epitope-specific CTL lines or clones. We observed that two field variants, SLFNTVAVL and SVYNTVATL, of an A*0201-restricted Gag CTL epitope SLYNTVATL, and three field variants, KYRLKHLVW, QYRLKHIVW, and RYRLKHLVW, of an A24-restricted Gag CTL epitope KYKLKHIVW escaped from being killed by the CTL lines, despite the fact that they were recognized when the synthetic peptides corresponding to these variant sequences were exogenously loaded onto the target cells. Thus, their escape is likely due to the changes that occur during the processing and presentation of epitopes in the infected cells. Mutations responsible for this mode of escape were located within the epitope regions rather than the flanking regions, and such mutations did not influence the virus replication. The results suggest that the impaired antigen processing and presentation often occur in HIV-1 field isolates and thus are one of the major mechanisms that enable HIV-1 to escape from CTL recognition. We emphasize the importance of testing HIV-1 variants in an endogenous expression system.

Preventing the Spontaneous Modification of an HLA-A2-Restricted Peptide at an N-Terminal Glutamine or an Internal Cysteine Residue Enhances Peptide Antigenicity

The p68-derived peptide, QIVDVCHDV, was identified by a reverse immunology approach as capable of reconstituting an epitope recognized by the melanoma-reactive cytotoxic T lymphocyte (CTL) line VMM5. The peptide has not been demonstrated definitively on the cell surface by mass spectrometry; thus, it is not yet considered appropriate for use in human melanoma vaccines. Interestingly, however, the antigenicity of this peptide was affected by spontaneous modifications at two distinct residues. Spontaneous modification of the QIVDVCHDV peptide can occur at the cysteine residue at position 6 or at the N-terminal glutamine residue, and both modifications dramatically affect CTL recognition. Avoidance of an acidic environment prevents the conversion of the N-terminal glutamine residue to pyroglutamic acid, a conversion that inhibits binding of the peptide to HLA-A2 and diminishes recognition by CTLs. Substitution of asparagine for the N-terminal glutamine and substitution of serine for the cysteine were shown to enhance the binding of the peptide to HLA-A2 and to enhance the recognition of the peptide by CTLs. These findings suggest general strategies for enhancing the antigenicity of other peptides containing similar amino acids in their sequence.

Identification of Sequential Viral Escape Mutants Associated with Altered T-Cell Responses in a Human Immunodeficiency Virus Type 1-Infected Individual

Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8(+) cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8(+) CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope.

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.

Epstein–Barr virus nuclear antigen 1 (EBNA1) induced cytotoxicity in epithelial cells is associated with EBNA1 degradation and processing

Epstein–Barr virus nuclear antigen 1 (EBNA1) has a central role in the maintenance and segregation of the Epstein–Barr virus (EBV) episome and by virtue of a glycine–alanine repeat domain is prevented from being endogenously processed for recognition by HLA class I restricted cytotoxic T lymphocytes (CTLs). We found that EBNA1 expression resulted in growth inhibition and a G2/M arrest in human squamous epithelial cell lines (SCC12F, SVK) but not epithelial cell lines of glandular origin (Hela, Ad/AH). The cytotoxicity of EBNA1 was associated with EBNA1 degradation and both these effects were blocked in SCC12F cells expressing either the anti-apoptotic bcl-2 protein or the EBV homolog of bcl-2, BHRF1. The endogenous degradation of EBNA1 in SVK epithelial cells was associated with specific CTL recognition, an effect not evident in EBNA1-expressing Hela cells. Consistent with the inability of SVK cells to tolerate EBNA1 expression, studies with a recombinant EBV demonstrated that SVK cells are unable to maintain stable virus infection, whereas Hela cells are able to efficiently establish latent EBV infection. These data have important implications for both the cellular requirements necessary to sustain a stable EBV infection and for the possible role of CTL responses in controlling EBV infection of epithelial cells.

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys.

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.

Potential limitations in using minor histocompatibility antigen-specific cytotoxic T cells for targeting solid tumor cells

We have shown previously that KIAA0223, a gene encoding a minor histocompatibility antigen, HA-1, whose expression was believed to be restricted to the hematopoietic cells, is aberrantly expressed in some solid tumor cell lines. However, its significance in tumor immunity needs to be determined. Cytotoxic activity of HA-1 H-specific cytotoxic T lymphocytes (CTLs) was assessed against solid tumor cell lines expressing KIAA0223 using 51Cr release assays. Five of seven cell lines were lysed when HLA-A*0201 was adequately expressed. One of the two CTL-resistant cell lines became susceptible after treatment with IFN-γ and TNF-α, while the other was lysed only after pulsing with HA-1 H peptide. In most cell lines tested, HA-1 H peptide was properly generated and presented for recognition by the CTL. However, impaired antigen processing and presentation observed in this study may result in escape from CTL recognition in vivo, as well as in vitro, as observed in this study.

Cytotoxic T lymphocytes recognize and lyse chondrocytes under inflammatory, but not non-inflammatory conditions.

The human major histocompatibility complex (MHC) class I allele HLA-B27 is strongly associated with seronegative spondyloarthropathies including ankylosing spondylitis and reactive arthritis. Although of unknown aetiology, one hypothesis suggests that a cytotoxic T cell (CTL) response against a self-antigen at sites of inflammation, such as entheses or joints may be involved. The chondrocyte is one of the major specialized cell types found both in articular cartilage and cartilaginous entheses and therefore is a possible source of such an antigen. CTL recognition of these cells is a potential mechanism for inflammation and cartilage damage, both through direct lysis of chondrocytes and the secretion of pro-inflammatory cytokines such as tumour necrosis factor and interferon-gamma (IFN-gamma). We test the feasibility of this hypothesis by examining the ability of chondrocytes to present antigen to CTL in vitro. Chondrocytes isolated from the ribcages of mice did not constitutively express detectable levels of MHC class I by fluorescence-activated cell sorting analysis. In addition, they were resistant to lysis by alloreactive and influenza A virus nucleoprotein (NP)-specific CTL. However, treatment of chondrocytes with IFN-gamma up-regulated MHC class I expression and rendered the cells susceptible to lysis by CTL. Similarly, IFN-gamma-treated chondrocytes infected with influenza A virus were recognized by NP-specific CTL, though with variable efficiency. Thus, we suggest that under certain circumstances CTL-mediated lysis of chondrocytes is potentially a potent mechanism for cartilage damage in vivo, but that low levels of MHC class I on healthy chondrocytes protects from immune recognition in health.