Between-host evolution of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1: an approach based on phylogenetically independent comparisons

In human immunodeficiency virus type 1 (HIV-1), mutations that escape from cytotoxic T-lymphocyte (CTL) recognition have been documented, and sequence analyses have provided indirect support for the hypothesis that natural selection has favored CTL escape mutants within an infected host. In spite of such evidence for within-host selection by CTL, it has been more difficult to determine how natural selection by host CTL has influenced long-term evolution of HIV-1. We used statistical analysis of published HIV-1 genomic sequences to examine the role of natural selection in between-host evolution of CTL epitopes. Based on a phylogenetic analysis, we identified 21 pairs of closely related genomes isolated from different hosts and examined the pattern of nucleotide substitution in genomic regions encoding well-characterized CTL epitopes. The results revealed that certain CTL epitopes have been subject to repeated positive selection across the population, while others are generally conserved. Furthermore, evidence of positive selection was associated with divergence from the canonical epitope sequence and with an enhanced frequency of convergent amino acid sequence changes in CTL epitopes. The results support the hypothesis that CTL-driven selection has been a major factor in the long-term evolution of HIV-1.     

Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction

Background  

Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein. It does so by integrating predictions of proteasomal cleavage, TAP transport efficiency, and MHC class I affinity. At least four other methods have been developed recently that likewise attempt to predict CTL epitopes: EpiJen, MAPPP, MHC-pathway, and WAPP. In order to compare the performance of prediction methods, objective benchmarks and standardized performance measures are needed. Here, we develop such large-scale benchmark and corresponding performance measures and report the performance of an updated version 1.2 of NetCTL in comparison with the four other methods.     

Quantitative study of cytotoxic T-lymphocyte immunotherapy for nasopharyngeal carcinoma

We suggest that tRNA actively participates in the transfer of 3D information from mRNA to peptides - in addition to its well-known, "classical" role of translating the 3-letter RNA codes into the one letter protein code. The tRNA molecule displays a series of thermodynamically favored configurations during translation, a movement which places the codon and coded amino acids in proximity to each other and make physical contact between some amino acids and their codons possible. This specific codon-amino acid interaction of some selected amino acids is necessary for the transfer of spatial information from mRNA to coded proteins, and is known as RNA-assisted protein folding.     

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.     

Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. "Functional avidity," defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD(50)), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD(50) to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD(50) for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD(50) required for CTL to achieve 50% efficiency of infected cell killing [KE(50)]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE(50) despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE(50), demonstrating epitope dependence of KE(50). Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE(50). Thus, defining KE(50) values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.     

Gamma interferon is not required for mucosal cytotoxic T-lymphocyte responses or heterosubtypic immunity to influenza A virus infection in mice

Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-gamma) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-gamma, we have investigated the mechanism of HSI by analyzing the role of IFN-gamma for HSI in IFN-gamma gene-deleted (IFN-gamma(-/-)) mice. It has been reported that IFN-gamma is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-gamma is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-gamma(-/-) mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-gamma(-/-) mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-gamma does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.     

Antireticular cytotoxic serum as an agent for stimulating the immunity system]

The changes in the immunological indices characterizing the T and B immunity system and also in biological resistance of atherosclerosis patients were studied under conditons of the use of the antireticular cytotoxic serum microdoses. A possibility of employing the ACS for stimulation of the immune system was demonstrated.     

Naturally occurring TAP-dependent specific T-cell tolerance for a variant of an immunodominant retroviral cytotoxic T-lymphocyte epitope

Upon immunization and restimulation with tumors induced by the endogenous AKR/Gross murine leukemia virus (MuLV), C57BL/6 mice generate vigorous H-2K(b)-restricted cytotoxic T-lymphocyte (CTL) responses to a determinant (KSPWFTTL) derived from the p15E transmembrane portion of the viral envelope glycoprotein. By contrast, the highly homologous determinant RSPWFTTL, expressed by tumor cells induced by Friend/Moloney/Rauscher (FMR) MuLV, is not immunogenic, even when presented to the immune system as vaccinia virus-encoded cytosolic or endoplasmic reticulum (ER)-targeted minigene products. Such minigene products are usually highly immunogenic since they bypass the need for cells to liberate the peptide or transport the peptide into the ER by the transporter associated with antigen processing (TAP). Using KSPWFTTL-specific CTLs that cross-react with RSPWFTTL, we previously demonstrated that presentation of RSPWFTTL from its natural viral gene product is TAP dependent. Here, we show first that C57BL/6 mice express mRNA encoding RSPWFTTL but not KSPWFTTL and second that the ER-targeted RSPWFTTL minigene product is highly immunogenic in C57BL/6 mice with a targeted deletion in TAP1. These findings provide the initial demonstration of TAP-dependent tolerance induction to a specific self peptide and demonstrate that this contributes to the differential recognition of RSPWFTTL and KSPWFTTL by C57BL/6 mice.     

Regulation of cytotoxic T-lymphocyte activation by L-lactate and pyruvate

The activation of cytotoxic T lymphocytes (CTL) in vivo was found to be strongly augmented by two injections of 0.2 ml 1 X 10(-1) M pyruvate in spite of the relatively high concentration of glucose (approximately 10(-2) M) in the blood. The repeated injection of 1 X 10(-1) M L-lactate, in contrast, was found to suppress cytotoxic responses in vivo. The activation of CTL and DNA synthesis in mixed lymphocyte cultures, on the other hand, was found to be suppressed by pyruvate (1 X 10(-2) M), and was substantially augmented by 1 X 10(-2) M L-lactate. The glucose concentration in the culture medium was also approximately 10(-2) M. Taken together, these results suggest that the utilization of glucose is relatively ineffective and that the respiratory metabolism is a more effective source of energy during the early T cell activation. The results suggest also that the aerobic glycolysis of macrophages and their release of L-lactate may contribute to their function as accessory cells in immune responses. The differences between the in vivo and in vitro results are discussed.     

Suppression of cytotoxic T-lymphocyte activation by pyruvate and L-alanine

The activation of cytotoxic T lymphocytes (CTL) in allogeneic mixed-lymphocyte cultures was found to be strongly inhibited if 1-3 X 10(-2) M L-alanine or the structurally and biochemically related substance pyruvate was present in the period from 7 to 19 or from 19 to 120 hr. The cytotoxic response was not inhibited when L-alanine or pyruvate was present during the first 7 hr of the culture period. L-Alanine produced also little or no suppression, if added on Day 3 of the culture. L-Lactate or D-alanine at similar concentrations was not suppressive during the entire culture period. The suppression by pyruvate and L-alanine was strongly reduced by 1 X 10(-4) M adenosine. Adenosine in combination with an interleukin-2 (IL-2)-containing EL-4-cell supernatant was even more effective. Pyruvate and alanine (1-3 X 10(-2) M) also inhibited the DNA synthesis in mixed-lymphocyte cultures on Day 5 by about 50%, but both substances had practically no effect on DNA synthesis in cultures that had been supplemented with an IL-2-containing EL-4 supernatant. They had also no effect on the IL-2-dependent proliferation of several T-cell clones or of concanavalin A-activated thymocytes. These relatively selective regulatory effects of pyruvate and L-alanine may be useful for the analysis of the biochemical pathways during lymphocyte activation and/or for a selective manipulation of the immune response.     

Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species

BACKGROUND: The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the (51)Cr release assay. The numerous disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species. METHODS: This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18((3)) (3, 3'-dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations. RESULTS: A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the (51)Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus. CONCLUSIONS: We provide evidence that the flow cytometric assay using DIOC18((3)) is highly reproducible and is suitable to measure different types of cell cytotoxicity.     

A novel assay for assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.

BACKGROUND: Assessment of CD8(+) T-cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV-specific cytotoxicity by multiparameter flow cytometry. METHODS: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE(hi)PI(-)) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The (51)Cr-release assay and IFN-gamma ELISpot were performed by standard procedures. RESULTS: A comparison with the Cr-release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01). CONCLUSION: This new assay provides both specific and sensitive results when employed for the detection of HIV-specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels.     

Evidence for an involvement of T4+ cytotoxic T cells in tumor immunity

Cell-mediated immunity against cancer cells primarily involves class I major histocompatibility complex (MHC)-restricted cytotoxic T cells and natural killer (NK) cells. To investigate whether T4+ cytotoxic T cells also have a role in tumor-specific immunity, mice were immunized with a B cell lymphoma. T cell hybridomas were constructed from the immune spleen cells and analyzed for their cytotoxic ability against the immunizing lymphoma. A T4+, Lyt-1+ hybridoma cell line was developed (103L2) which specifically killed the immunizing tumor cells but not normal B cells or a range of other tumor cells of B or non-B origin. This cytotoxic hybridoma cell line differed from Lyt-2+ cytotoxic T lymphocyte cells and NK cells, commonly identified with cytotoxicity, in a number of important ways. First, the cells were class II MHC restricted; second, interleukin-2 was released from activated effector cells; and finally but most importantly, innocent nonparticipating bystander cells were also killed. The significance of this observation was that normal cells were protected, although a broad range of tumor cell types, including tumor antigen-negative mutants, were killed. It is therefore conceivable that T4+ cytotoxic T cells might play an important role in tumor immunity through the direct recognition and lysis of tumor cells while any tumor variants, arising due to antigen loss, would remain susceptible through the bystander killing effect and normal cells would remain unaffected. These results strongly suggest that tumor-reactive T4+ cytotoxic T cells belong to a new category of effector cells with an important role in tumor-specific immunity.     

Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization.

DNA immunization offers a novel means to induce cellular immunity in a population with a heterogeneous genetic background. An immunorecessive cytotoxic T-lymphocyte (CTL) epitope in influenza virus nucleoprotein (NP), residues 218 to 226, was identified when mice were immunized with a plasmid DNA encoding a full-length mutant NP in which the anchor residues for the immunodominant NP147-155 epitope were altered. Mice immunized with wild-type or mutant NP DNA were protected from lethal cross-strain virus challenge, and the protection could be adoptively transferred by immune splenocytes, indicating the role of cell-mediated immunity in the protection. DNA immunization is capable of eliciting protective cellular immunity against both immunodominant and immunorecessive CTL epitopes in the hierarchy seen with virus infection.     

A new theory of cytotoxic T-lymphocyte memory: implications for HIV treatment

We use simple mathematical models to examine the dynamics of primary and secondary cytotoxic T-lymphocyte (CTL) responses to viral infections. In particular, we are interested in conditions required to resolve the infection and to protect the host upon secondary challenge. While protection against reinfection is only effective in a restricted set of circumstances, we find that resolution of the primary infection requires persistence of CTL precursors (GTLp), as well as a fast rate of activation of the CTLp. Since these are commonly the defining characteristics of CTL memory, we propose that CTL memory may have evolved in order to clear the virus during primary challenge. We show experimental data from lymphocytic choriomeningitis virus infection in mice, supporting our theory on CTL memory. We adapt our models to HIV and find that immune impairment during the primary phase of the infection may result in the failure to establish CTL memory which in turn leads to viral persistence. Based on our models we suggest conceptual treatment regimes which ensure establishment of CTL memory. This would allow the immune response to control HIV in the long term in the absence of continued therapy.     

Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes

Recently, it was shown that electrical DNA-chips in connection with a magnetic bead-based sandwich-hybridization assay can be a suitable alternative for the analysis of gene expression by monitoring the respective mRNA levels. In this study, we established an improved assay which allowed for a significantly shortened but sensitive detection of specific mRNAs. These improvements include the change to a solution-based sandwich-hybridization and the rearrangement of the used oligonucleotide probes. The introduction of a second labeled detection probe further increased the hybridization signals and, in turn, leads to a substantial time reduction of the detection protocol. The presented solution-based sandwich-hybridization protocol for the electrochemical detection of specific mRNAs requires about 60 min and the whole procedure, including sampling, cell disruption, and RNA isolation, approx. 80 min. The assay of this study was verified by nutrient-limited growth experiments and the analysis of selected starvation marker genes of the industrial host Bacillus licheniformis. The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements.     

Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells

The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation.     

The complete primary structure of the T -Cell receptor genes from an alloreactive cytotoxic human T-lymphocyte clone

The complete primary structure of the cDNAs encoding the and chains of the T-lymphocyte receptor for antigen from a human alloreactive, cytotoxic T-cell clone, L17, is presented. Sequence analysis of these genes reveals that both are related to immunoglobulins and are composed of variable, diversity (at least in the case of the Ti clone), joining, and constant region sequences. Comparison of the sequence of the -chain cDNA to that of previously sequenced mouse and human cDNAs suggests the presence of human T-cell receptor D-region sequences. Southern blot analysis confirms the finding that these cDNAs represent the functional receptor genes expressed by the L17 cytotoxic T-cell clone. The availability of these full-length T-cell receptor cDNA clones from a human T-lymphocyte clone of known antigen specificity should allow an analysis of the relationship between T-cell receptor structure and function.