Fitness costs limit viral escape from cytotoxic T lymphocytes at a structurally constrained epitope

The intense selection pressure exerted by virus-specific cytotoxic T lymphocytes (CTL) on replicating human immunodeficiency virus and simian immunodeficiency virus results in the accumulation of CTL epitope mutations. It has been assumed that fitness costs can limit the evolution of CTL epitope mutations. However, only a limited number of studies have carefully examined this possibility. To explore the fitness costs associated with viral escape from p11C, C-M-specific CTL, we constructed a panel of viruses encoding point mutations at each position of the entire p11C, C-M epitope. Amino acid substitutions at positions 3, 4, 5, 6, 7, and 9 of the epitope significantly impaired virus replication by altering virus production and Gag protein expression as well as by destabilizing mature cores. Amino acid substitutions at position 2 of the epitope were tolerated but required reversion or additional compensatory mutations to generate replication-competent viruses. Finally, while amino acid substitutions at positions 1 and 8 of the p11C, C-M epitope were functionally tolerated, these substitutions were recognized by p11C, C-M-specific CTL and therefore provided no selection advantage for the virus. Together, these data suggest that limited sequence variation is tolerated by the region of the capsid encoding the p11C, C-M epitope and therefore that only a very limited number of mutations can allow successful viral escape from the p11C, C-M-specific CTL response.     

Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness

Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8(+) T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637), displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication.     

Preferential escape of subdominant CD8+ T cells during negative selection results in an altered antiviral T cell hierarchy

Negative selection is designed to purge the immune system of high-avidity, self-reactive T cells and thereby protect the host from overt autoimmunity. In this in vivo viral infection model, we show that there is a previously unappreciated dichotomy involved in negative selection in which high-avidity CD8(+) T cells specific for a dominant epitope are eliminated, whereas T cells specific for a subdominant epitope on the same protein preferentially escape deletion. Although this resulted in significant skewing of immunodominance and a substantial depletion of the most promiscuous T cells, thymic and/or peripheral deletion of high-avidity CD8(+) T cells was not accompanied by any major change in the TCR V beta gene family usage or an absolute deletion of a single preferred complementarity-determining region 3 length polymorphism. This suggests that negative selection allows high-avidity CD8(+) T cells specific for subdominant or cryptic epitopes to persist while effectively deleting high-avidity T cells specific for dominant epitopes. By allowing the escape of subdominant T cells, this process still preserves a relatively broad peripheral TCR repertoire that can actively participate in antiviral and/or autoreactive immune responses.     

Identification of residues necessary for clonally specific recognition of a cytotoxic T cell determinant.

The residues in an influenza nucleoprotein (NP) cytotoxic T cell determinant necessary for cytotoxic T cell (CTL) recognition, were identified by assaying the ability of hybrid peptides to sensitize a target cell to lysis. The hybrid peptides were formed by substituting amino acids from one determinant (influenza NP 147-158) for the corresponding residues of a second peptide (HLA CW3 171-182) capable of binding to a common class I protein (H-2Kd). Six amino acids resulted in partial recognition; however, the presence of a seventh improved the potency of the peptide. Five of the six amino acids were shown to be required for recognition. The spacing of the six amino acids was consistent with the peptide adopting a helical conformation when bound. The importance of each amino acid in CTL recognition and binding to the restriction element was investigated further by assaying the ability of peptides containing point substitutions either to sensitize target cells or to compete with the natural NP sequence for recognition by CTL. The T cell response was much more sensitive to substitution than the ability of the peptide to bind the restriction element. Collectively the separate strategies identified an approximate conformation and orientation of the peptide when part of the complex and permitted a potential location in the MHC binding site to be identified. The model provides a rationalization for analogues which have previously been shown to exhibit greater affinity for the class I molecule and suggests that the binding site in major histocompatibility complex (MHC) class I molecules might have greater steric constraints that the corresponding area of class II proteins.     

Genome-wide characterization of a viral cytotoxic T lymphocyte epitope repertoire

A genome-wide search using major histocompatibility complex (MHC) class I binding and proteosome cleavage site algorithms identified 101 influenza A PR8 virus-derived peptides as potential epitopes for CD8+ T cell recognition in the H-2b mouse. Cytokine-based flow cytometry, ELISPOT, and cytotoxic T lymphocyte assays reveal that 16 are recognized by CD8+ T cells recovered directly ex vivo from infected animals, accounting for greater than 70% of CD8+ T cells recruited to lung after primary infection. Only six of the 22 highest affinity MHC class I binding peptides comprise cytotoxic T lymphocyte epitopes. The remaining non-immunogenic peptides have equivalent MHC affinity and MHC-peptide complex half-lives, eliciting T cell responses when given in adjuvant and with T cell receptor-ligand avidity comparable with their immunogenic counterparts. As revealed by a novel high sensitivity nanospray tandem mass spectrometry methodology, failure to process those predicted epitopes may contribute significantly to the absent response. These results have important implications for rationale design of CD8+ T cell vaccines.     

Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33-41) and the nucleoprotein (NP396-404), while the gp276-286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-gamma enhanced the presentation of gp33-41, whereas presentation of the gp276-286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-gamma treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276-286 epitope. The ability to generate gp276-286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.     

Acute phase cytotoxic T lymphocyte escape is a hallmark of simian immunodeficiency virus infection

Cytotoxic T-lymphocyte (CTL) responses peak coincident with the decline in acute HIV viremia. Despite two reports of CTL-resistant HIV variants emerging during acute infection, the contribution of acute CTL escape to HIV pathogenesis remains unclear. Difficulties inherent in studying acute HIV infection can be overcome by modeling virus-host interactions in SIV-infected rhesus macaques. We sequenced 21 complete simian immunodeficiency virus (SIV)mac239 genomes at four weeks post-infection to determine the extent of acute CTL escape. Here we show that viruses from 19 of 21 macaques escaped from CTLs during acute infection and that these escape-selecting CTLs were responsive to lower concentrations of peptide than other SIV-specific CTLs. Interestingly, CTLs that require low peptide concentrations for stimulation (high 'functional avidity') are particularly effective at controlling other viral infections. Our results suggest that acute viral escape from CTLs is a hallmark of SIV infection and that CTLs with high functional avidity can rapidly select for escape variants.     

Evolution of CD8+ T cell immunity and viral escape following acute HIV-1 infection

Induction of HIV-1-specific CD8(+) T cells during acute infection is associated with a decline in viremia. The role CD8(+) effectors play in subsequently establishing viral set point remains unclear. To address this, we focused on two acutely infected patients with the same initial Tat-specific CD8(+) response, analyzing their CD8(+) T cell responses longitudinally in conjunction with viral load and sequence evolution. In one patient initiating treatment during acute infection, the frequencies of Tat-specific CD8(+) T cells gradually diminished but persisted, and the Tat epitope sequence was unaltered. By contrast, in the second patient who declined treatment, the Tat-specific CD8(+) T cells disappeared below detection, in conjunction with Gag-specific CD4(+) T cell loss, as plasma viremia reached a set point. This coincided with the emergence of an escape variant within the Tat epitope and an additional Vpr epitope. New CD8(+) T cell responses emerged but with no further associated decline in viremia. These findings indicate that, in the absence of treatment, the initial CD8(+) T cell responses have the greatest impact on reducing viremia, and that later, continuously evolving responses are less efficient in further reducing viral load. The results also suggest that T cell help may contribute to the antiviral efficiency of the acute CD8(+) T cell response.     

Partial escape of HIV-1 from cytotoxic T lymphocytes during chronic infection

Viral mutational escape from CD8(+) cytotoxic T lymphocytes (CTLs) is typically considered to be a dichotomous process and uncommon during chronic HIV-1 infection. Ex vivo passaging of HIV-1 from persons with chronic infection, however, revealed the evolution of many fixed substitutions within and around CTL-targeted regions, with an associated increase in replicative capacity. This indicates an evolution of mutations during chronic HIV-1 infection that trade replicative fitness for incomplete evasion of CTLs, or "partial escape."     

Naturally arising HIV-1 Nef variants conferring escape from cytotoxic T lymphocytes influence viral entry co-receptor expression and susceptibility to superinfection

HIV-1 Nef is a key factor for pathogenesis and is known to down-regulate functionally important molecules, including viral entry co-receptor CCR5 and CXCR4, from the surface of HIV-infected cells. Some of these Nef activities are mediated by the well-conserved proline-rich region of Nef, and this region is highly targeted by cytotoxic T lymphocytes (CTLs). In the present study, we asked whether Nef variants selected under CTL-mediated selective pressure in vivo may constrain these important Nef activities. The analysis of autologous nef sequences isolated from a cohort of total 235 subjects in Japan revealed that the subjects showing amino acid variations, such as Arg75Thr and Tyr85Phe, located within the proline-rich region were significantly over-represented by those having HLA-B*3501. CTL assays corroborated that these mutations conferred escape from HLA-B(?)3501-restricted CTLs. The Arg75Thr variant Nef selectively impaired CCR5, but not CXCR4, down-regulation activity from the cell surface; whereas the Tyr85Phe variant Nef affected neither CCR5 nor CXCR4 down-regulation activity. Moreover, the cells expressing the Arg75Thr variant Nef significantly impaired protection from superinfection by CCR5-tropic, but not CXCR4-tropic, viruses. These results highlighted the importance of certain Nef-specific CTLs in modulation of viral co-receptor down-regulation activity and protection from HIV-1 superinfection, providing us with additional insight into vaccine design.     

Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost

Escape from specific T-cell responses contributes to the progression of human immunodeficiency virus type 1 (HIV-1) infection. T-cell escape viral variants are retained following HIV-1 transmission between major histocompatibility complex (MHC)-matched individuals. However, reversion to wild type can occur following transmission to MHC-mismatched hosts in the absence of cytotoxic T-lymphocyte (CTL) pressure, due to the reduced fitness of the escape mutant virus. We estimated both the strength of immune selection and the fitness cost of escape variants by studying the rates of T-cell escape and reversion in pigtail macaques. Near-complete replacement of wild-type with T-cell escape viral variants at an immunodominant simian immunodeficiency virus Gag epitope KP9 occurred rapidly (over 7 days) following infection of pigtail macaques with SHIVSF162P3. Another challenge virus, SHIVmn229, previously serially passaged through pigtail macaques, contained a KP9 escape mutation in 40/44 clones sequenced from the challenge stock. When six KP9-responding animals were infected with this virus, the escape mutation was maintained. By contrast, in animals not responding to KP9, rapid reversion of the K165R mutation occurred over 2 weeks after infection. The rapidity of reversion to the wild-type sequence suggests a significant fitness cost of the T-cell escape mutant. Quantifying both the selection pressure exerted by CTL and the fitness costs of escape mutation has important implications for the development of CTL-based vaccine strategies.     

A self determinant recognizing T cell hybridoma

A T cell hybridoma (53(113)) obtained by fusion of BALB/c spleen cells and the BW 5147 lymphoma T cell line is described. This hybridoma recognizes mouse RBC (MRBC) and rat RBC, but not human, rabbit, guinea pig, or SRBC. The culture supernatant possesses hemagglutinating activity for the same indicator RBC. In addition to this, 53(113) cells are able to form protein A plaques in the presence of guinea pig complement and normal mouse serum (NMS) or purified mouse immunoglobulins (Ig). Because mouse Ig as well as sonicates from MRBC are able to inhibit the rosettes between the hybridoma cells and the MRBC, and because the sonicates inhibit protein A plaque formation, it seems likely that the same product can recognize a similar determinant expressed on MRBC and mouse Ig. The hypothesis that a 53(113) structure recognizes identical or cross-reactive carbohydrate determinants shared by murine Ig and C is considered.     

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.     

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

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.     

Characterization of a "heteroclitic" cytotoxic lymphocyte clone: heterogeneity of receptors or signals?

In this work we report the isolation of a cloned CTL that recognizes three genetically distinct specificities. Of interest is the fact that the hierarchy of the strength of these specificities is dependent on the functional assay used for their detection. This suggests that either the specificities are the result of interactions with independent receptors or the cell can modulate signals from cross-reactive antigens into different functional responses.     

Broadly increased sensitivity to cytotoxic T lymphocytes resulting from Nef epitope escape mutations

Nef is an HIV-1 protein that is absent in most retroviruses, yet its reading frame is highly maintained despite frequent targeting by CD8(+) CTL in vivo. Because Nef is not necessarily required for viral replication, this consistent maintenance suggests that Nef plays an important role(s) and substantial fitness constraints prevent its loss in vivo. The ability of Nef to down-regulate cell surface MHC class I (MHC-I) molecules and render infected cells resistant to CTL in general is likely to be an important contributing function. We demonstrate that mutational escape of HIV-1 from Nef-specific CTL in vitro leads to progeny virions that are increased in their susceptibility to CTL of specificities for proteins other than Nef. The escape mutants contain multiple nef mutations that impair the ability of the virus to down-regulate MHC-I through disruption of its reading frame as well as epitope point mutations. Given the rarity of nef frameshifts in vivo, these data support the concept that the ability to down-regulate MHC-I could be a key constraint for preservation of Nef in vivo.     

Immunodominance of an antiviral cytotoxic T cell response is shaped by the kinetics of viral protein expression

Lymphocytic choriomeningitis virus (LCMV) infection induces a protective CTL response consisting of gp- and nucleoprotein (NP)-specific CTL. We find that a small load of LCMV led to immunodominance of NP-CTL, whereas a large viral load resulted in dominance of gp-CTL. This is the first study describing that immunodominance is not fixed after infection with a given pathogen, but varies with the viral load instead. We assumed higher Ag sensitivity for NP-CTL, which would explain their preferential priming at low viral load, as well as their overstimulation resulting in selective exhaustion at high viral load. The higher Ag sensitivity of NP-CTL was due to faster kinetics of NP-epitope presentation. Thus, we uncover a novel factor that impinges upon immunodominance and is related to the kinetics of virus protein expression. We propose that CTL against early viral proteins swiftly interfere with virus replication, resulting in efficient protection. If these "early" CTL fail in immediate virus control, they are activated in the face of higher viral load compared with "late" CTL and are therefore prone to be exhausted. Thus, the observed absence of early CTL in persistent infections might not be the cause, but rather the consequence of viral persistence.     

Characteristics of cytotoxic thyroglobulin-specific T cell hybridomas

Clones of cytotoxic thyroid-specific T cell hybridomas were generated by fusion of thyroglobulin-primed, "in vitro"-boosted CBA lymph node cells with the AKR-derived lymphoma cell line BW 5147. One hundred and thirty one clones were obtained. Among them, 15 were able to induce the lysis of 51Cr-labeled syngeneic thyroid epithelial cells after 5 h of incubation at 37 degrees C. Two T cell clones, HTC1 and HTC2, were further studied. These clones, which exhibit cell surface characteristics of cytotoxic cells, were specific for only syngeneic thyroid cells (allogeneic thyroid cells or syngeneic epithelial cells were never lysed by these hybridomas). Moreover, by using Ag-pulsed syngeneic macrophages as targets, syngeneic cytotoxicity was shown to be specific for thyroglobulin and not for a nonrelated Ag. The lysis obtained with these autoreactive thyroid-specific T cell clones is restricted to class I major histocompatibility Ag. This property is assessed by both the blocking of syngeneic cytotoxicity toward thyroid epithelial cells or thyroglobulin-pulsed macrophages only by anti-class I mAb and by the detection of specific lysis of target cells exclusively when effector hybrid cells and target thyroid epithelial cells or thyroglobulin-pulsed macrophages shared at least class I major histocompatibility Ag.