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.     

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201⁺ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28_(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28_(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28_(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28_(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.     

Differences in the MHC-restricted self-recognition repertoire of intra-thymic and extra-thymic cytotoxic T lymphocyte precursors

The MHC specificity of TNP-specific pCTL from the thymus and spleen of F1 leads to parent chimeras was evaluated. It was found that in the presence of exogenously added helper factor IL 2, thymic pCTL were restricted to recognizing TNP only in association with host MHC determinants, whereas splenic pCTL recognized TNP in association with either host or donor MHC determinants. Thus, the spleens of F1 leads to parent chimeras contain a pCTL repertoire that is not present intrathymically. Data are presented which suggest that such pCTL did nevertheless differentiate into functional competence in the chimeric host. These results are consistent with extra-thymic differentiation as the mechanism by which such nonthymically restricted pCTL may have developed.     

Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present an immunodominant viral cytotoxic T lymphocyte epitope.

For controlling infection of the mouse with the lymphocytic choriomeningitis (LCM) virus, CD8+ CTL are essential. In the infected BALB/c mouse the arising LCM virus-specific CTL are exclusively restricted by the class I MHC-encoded molecule L; K- or D-restricted antiviral CTL cannot be detected. Thus, the infected L-deficient BALB/c mutant C-H-2dm2 should not be capable of eliminating the virus. The experimental evidence proves the contrary, which is explained by K- and D-restricted CTL that this mouse generates. Why such cells remain undetectable in BALB/c mice is currently unexplained, because there is no lack of precursors and the corresponding virus Ag is presented. Despite the absence of lytic activity in vitro, other than the one associated with L, transfusion of day 8-immune spleen cells from BALB/c into infected C-H-2dm2 (L-deficient) mice results in accelerated virus elimination from the organs of the latter, which was manifest as soon as 8 h after cell transfer. Furthermore, lytic activity did not attain measurable levels in the recipients' spleens. Obviously, this infection can be terminated by CD8+ T lymphocytes even when these cells' lytic activity is below detectability.     

In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones

SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen

Ran is considered to be a promising target for tumor-specific immunotherapy because its protein is exclusively expressed in tumor tissues, though its mRNA can be expressed in most normal tissues. In our study, we obtained four candidate wild-type epitopes designated Ran1, Ran2, Ran3, and Ran4, derived from the Ran antigen with the highest predicted affinity with MHC-I, indicated by affinity prediction plots and molecular dynamics simulation. However, in vitro affinity assays of these epitopes showed only a moderate affinity with MHC-I. Thus, we designed altered peptide ligands (APLs) derived from Ran wild-type epitopes with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. Of the eight tested peptides, the 1Y analog had the strongest binding-affinity and lowest-dissociation rate to HLA-A*0201. Additionally, we investigated the CTLs activities induced by Ran wild-type peptides and the APLs in human PBMCs and in HLA-A*0201/K^b transgenic mice. Ran1 1Y was superior to other APLs and wild-type peptides in eliciting epitope-specific CTL immune responses both in vitro and in vivo. In summary, a wild-type epitope of the tumor-specific antigen Ran, expressed broadly in many tumors, was identified and designated Ran1. An APL of Ran1, Ran1 1Y, was further designed and verified in vitro and in vivo and found to elicit a stronger Ran-specific CTL response, indicating a potential anti-tumor application in the future.     

In vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8(+) T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425-p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA-A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon-γ in healthy donors (HLA-A*02(+) purified protein derivative(+)). In cytotoxicity assays in vitro and in vivo, Rv3425-p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.     

Human autoantibodies modulate the T cell epitope repertoire but fail to unmask a pathogenic cryptic epitope

Abs can tune the responses of Ag-specific T cells by influencing the nature of the epitope repertoire displayed by APCs. We explored the interaction between human self-reactive T cells and human monoclonal autoantibodies from combinatorial Ig-gene libraries derived from autoimmune thyroiditis patients and specific for the main autoantigen thyroid peroxidase (TPO). All human mAbs extensively influenced the T cell epitope repertoire recognized by different TPO-specific T cell clones. The action of the human mAbs was complex, because sometimes the same Ab suppressed or enhanced the epitopes recognized by the 10 different TPO-specific T cell clones. The human mAbs could modulate the epitope repertoire when TPO was added exogenously and when expressed constitutively on the surface of APCs. However, they could not unmask an immunodominant cryptic TPO epitope. In this study, we show that human autoantibodies influence the activity of self-reactive T cells and prove their relevance in concealing or exposing epitopes recognized by self-reactive T cells. However, our results further stress the biological significance of the immunodominant cryptic epitope we have defined and its potential importance in the evolution of autoimmunity.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Acquisition of an anti-idiotypic cytotoxic T lymphocyte repertoire in B cell-transferred or tetraparental bone marrow chimeric mice

In previous studies we showed that major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) specific for the cross-reactive idiotype (CRI) of MOPC-104E myeloma protein could only be induced in BALB/c or BAB-14 mice which have the ability to produce the CRI, but not in C.AL-20 or C.B-20 mice which have no ability to produce the CRI. The strong correlation between CRI-specific CTL responder strains and CRI producers supports the idea that the VH gene products are intrinsic primary antigenic stimuli for the generation of the anti-idiotypic CTL. To investigate the role of B lymphocytes in the selection of T lymphocyte repertoire, the purified B cells of CRI producer strains were repeatedly injected into anti-CRI CTL nonresponder neonatal mice. CRI-specific CTL activity was successfully induced in the CRI nonproducer mice only when they were exposed to CRI producer strain B lymphocytes from neonatal life. When the CTL nonresponder adult mice received CRI producer B lymphocytes, the nonresponder phenotype was not changed into the responder phenotype. Inducibility of CRI-specific CTL was also analyzed in tetraparental bone marrow chimeras. When CRI nonproducer bone marrow cells repopulated along with CRI producer bone marrow cells, the anti-CRI CTL of CRI nonproducer origin were generated. Adaptive differentiation of haplotype preference was also observed. When these observations are taken collectively, we see that the anti-idiotypic T lymphocyte repertoire is not a genetically determined one, but rather that the repertoire of T lymphocytes strongly depends on the postnatal selection process through the intrinsic idiotypic repertoire of B lymphocytes, i.e., internal images.     

T-cell receptors from virus-specific cytotoxic T lymphocytes recognizing a single immunodominant nine-amino-acid viral epitope show marked diversity.

Following infection of the H-2d mouse by lymphocytic choriomeningitis virus, the newly generated cytotoxic T lymphocyte (CTL) response is focused to a single 9-amino-acid peptide sequence (epitope) of the virus. More than 96% of the primary, secondary, and clonal CTL respond to this lymphocytic choriomeningitis virus nucleoprotein epitope. This unique system affords the opportunity to evaluate the T-cell response to a single viral CTL epitope in a case in which the outcome of infection, either viral clearance or host death, is mediated by the CTLs. Specifically, the molecular structure of the T-cell receptors (TCRs) of CTLs responding to this epitope was analyzed. By using an anchored polymerase chain reaction, the TCR chains of three CTL clones cDNAs were amplified, sequenced, and found to have unique V alpha of V beta chains relative to each other as well as to lack restriction to any particular variable chain. These data indicate that the highly diverse antiviral CTL response is pleomorphic and probably provides an advantage to the host as it limits the emergence of viral variants that could more easily arise if the TCR response were homogeneous.     

CD4 cytotoxic T lymphocyte differentiation

In vitro allostimulated CD4+ human lymphocytes were cloned by micromanipulation and expanded for a short time in IL-2 conditioned medium. In the present study we observed that proliferative noncytotoxic cloned cells were able to acquire the specific cytolytic activity under some modification of the cloned cells restimulation cycle. We demonstrated that rIFN-alpha and -gamma are the agents responsible for the acquisition of specific lytic activity.     

Theiler's virus infection induces a specific cytotoxic T lymphocyte response

Theiler's virus, a murine picornavirus, persists in the central nervous system of susceptible mouse strains and causes chronic inflammation and primary demyelination. One of the current hypotheses is that demyelination is, at least in part, mediated by virus-specific cytotoxic T lymphocytes (CTL). However, it is generally assumed that picornaviruses do not induce CTL. In point of fact, their existence has only been demonstrated for Coxsackievirus B-3. To determine whether Theiler's virus induces a CTL response, we generated a murine mastocytoma cell line stably transfected with the coding region of the genome of Theiler's virus strain DA. Using these cells as targets we showed that infected DBA/2 mice, a susceptible strain, produce cytotoxic T lymphocytes. The cytotoxic activity was Theiler's-virus specific. It was for the most part mediated by CD8+ T lymphocytes and H-2 restricted. This is the first demonstration that a specific CTL response is generated during Theiler's virus infection.     

Suppression of cytotoxic T lymphocyte activation by L-ornithine

The effect of L-ornithine on several types of immune reactions was analyzed. L-ornithine was found to suppress the activation of cytotoxic T lymphocytes (CTL) in vivo and in vitro. This suppressive effect was not observed with the structural analogues D-ornithine, L-lysine, or putrescine or with the amino acids L-histidine or L-alanine. The concentration of 9 X 10(-3) M L-ornithine was found to mediate a practically complete suppression of the cytotoxic response in vitro if applied on day 0 or day 1 of the culture, but a comparably weak suppression if applied on day 3. The same concentration of L-ornithine had no effect on the production of the lymphokines interleukin 2 (IL 2) and gamma-interferon (IFN-gamma). This concentration of ornithine had also no substantial effect on several types of proliferative responses, including the allogeneic mixed lymphocyte reaction, the concanavalin A-activated IL 2-dependent proliferation of thymocytes, and IL 2-dependent proliferation of the T cell clone W-2. These observations suggest that L-ornithine inhibits selectively the differentiation of CTL effector cells. By the criteria tested, the immunosuppressive effect of L-ornithine is more selective than that of cyclosporine A, which was previously found to suppress not only the activation of cytotoxic activity but also proliferative responses and the production of the lymphokines IL 2 and IFN-gamma.     

Detection and characterization of cytotoxic T lymphocyte precursors in the murine intestinal intraepithelial leukocyte population

Highly purified preparations of intraepithelial leukocytes (IEL) were obtained from the small intestinal mucosa. Leukocytes from the lamina propria (LPL) were isolated and phenotypically compared with IEL to verify that IEL were minimally contaminated by LPL. Because approximately 80% of IEL expressed the Lyt-2 antigen usually associated with cytotoxic/suppressor T lymphocytes, we wished to determine if precursors for cytotoxic T cells were present in this population. In order to generate cytotoxic cells, IEL and spleen cells from CBA/J mice (H-2k) were co-cultured with irradiated allogeneic spleen cells (H-2d or H-2b) in a one-way mixed leukocyte reaction (MLR). Four to six days later, the cultured cells were assayed against 51Cr-labeled H-2d or H-2b tumor or Con A-stimulated lymphoblast target cells, and the specificity of alloantigen-stimulated IEL and spleen cells was compared. The cytotoxic cells derived from both tissues displayed antigen-specific lysis of the allogeneic targets. Treatment of effector cells, generated from intraepithelial or splenic precursors, with monoclonal antibodies against Thy-1.2, Lyt-1.1, or Lyt-2.1 antigens plus complement, decreased cytotoxicity 85 to 100%, even though only 20 to 50% of the cells were lysed. The alloantigen specificity and surface antigen phenotype of the cultured IEL cells were identical to those of spleen cells and allowed us to conclude that IEL contained a cytotoxic T lymphocyte precursor (CTLp). Further characterization showed that, like spleen, the intraepithelial CTLp was Thy-1+ and Lyt-1+ and their sedimentation velocity was the same but differed from intraepithelial natural killer cells. Although 80% of IEL were Lyt-2+, the frequency of CTLp in the IEL population was estimated to be threefold to fivefold lower than in spleen, and the Lyt-2+ cells were shown not to be an enriched source of CTLp. Thus, the function of the majority of the IEL is still not known. However, there exists within this population CTLp, which may be capable of being stimulated with luminal antigens.     

Fine mapping of an H-2Kk restricted cytotoxic T lymphocyte epitope in SV40 T antigen by using in-frame deletion mutants and a synthetic peptide

The CTL response to SV40 in C3H/HeJ mice is directed against the tumor (T) Ag and is H-2Kk restricted. CTL specific for both the amino terminus (residues 1-271) and the carboxyl terminus (residues 512-708) of the T Ag molecule have been detected, and we have previously cloned CTL of both specificities. In this paper we show that the panel of 10 CTL clones specific for the C-terminal region includes clones specific for three different epitopes, termed C1, C2, and C3. Epitopes C1 and C2 are conserved in the T Ag of the related papova viruses BK and SA12, and only epitopes C2 and C3 are present on SV40 transformed targets bearing the Kk mutant Kkml. Epitopes C1 and C2 were mapped to residues 563-576 by using in-frame deletion mutants of SV40 T antigen, and all clones specific for these two epitopes can lyse Kk bearing target cells in the presence of a synthetic peptide comprising residues 559-576. Kk and Kkml differ at residue 152, which is located in the Ag-binding pocket. Because epitopes C1 and C2 can be formed by the same antigenic peptide, but epitope C1 is not present on SV40 transformed Kkml cells, epitopes C1 and C2 must differ in the contribution made by residue 152 of the MHC class I molecule. These data show that CTL epitopes on transformed cells can be made up of Ag fragments, and strengthen the idea that this is a general phenomenon for both class I and class II restricted T cell epitopes.     

Prevention of cytotoxic T cell escape using a heteroclitic subdominant viral T cell determinant

High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b). The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity.     

Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors

The role and induction requirements of helper T lymphocyte responses to herpes simplex virus type 1 (HSV-1) was examined. Splenocytes from mice that had been primed in vivo with infectious HSV-1 can be restimulated in vitro with live or partially UV-inactivated HSV-1 to generate high levels of herpes virus-specific cytotoxic T lymphocyte (CTL) activity. By comparison, naive splenocytes or splenocytes taken from mice primed with heat-inactivated HSV-1 failed to generate CTL after in vitro viral stimulation. In addition, infectious HSV-primed splenocytes can be rendered unresponsive to secondary in vitro restimulation by pretreatment with anti-Lyt-1 antiserum plus complement. Spleen cells were taken from mice that had been primed and restimulated in vivo with infectious HSV-1. Two days after the second priming, splenocytes were prepared and irradiated. These cells were capable of assisting in the generation of CTL to varying degrees in all of the above unresponsive populations of cells. The irradiated cells did not produce detectable levels of CTL activity when cultured alone with antigen. Also, if the irradiated splenocytes were treated with anti-Lyt-1 plus complement before their addition to cultures, all restorative activity was ablated. In contrast, irradiated splenocytes from mice that had been primed and restimulated in vivo with either heat-inactivated or UV-inactivated HSV-1 were unable to provide help to naive or helper-depleted cultures. The failure to supply helper activity appears not to involve the preferential activation of suppressor cells, as evidenced by cell mixing experiments and the addition of concentrated, antigen-stimulated spleen cell supernatant fluids to secondary anti-HSV-1 splenocyte cultures. Proliferative assays using interleukin 2- (IL 2) dependent cell lines as a measure of relative helper activity indicated that the inactivated forms of HSV-1 were incapable of effectively enlisting helper activity. These experiments therefore suggest that the observed failure of heat-inactivated or UV-inactivated HSV-1 preparations to induce anti-HSV CTL responses reflects the inability of the HSV-1-specific subset of helper T lymphocytes to recognize these forms of the antigen.