The complementarity-determining region-like loops of CD8 alpha interact differently with beta 2-microglobulin of the class I molecules H-2Kb and thymic leukemia antigen, while similarly with their alpha 3 domains

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8alphaalpha homodimers with a 10-fold higher affinity than H-2K(b) class I molecules. To understand the molecular basis for this difference, we created a panel of CD8alpha mutants and tested the ability of the CD8alphaalpha homodimers to bind to H-2K(b) tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the alpha3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2K(b) class I sequences are highly conserved in the alpha3 domain of MHC class I, this suggests that CD8 contacts the alpha3 domain of TL and H-2K(b) in a similar manner. In contrast, mutations in residues on the A and B beta strands of CD8 that are involved in contact with beta(2)-microglobulin affected interaction with the H-2K(b) tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2K(b). The unique high affinity binding of TL with CD8alphaalpha is most likely a result of amino acid differences in the alpha3 domain between TL and H-2K(b), particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8alphaalpha-H-2K(b) cocrystal.     

Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8(+) T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL(1-11)-specific CD8(+) T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer(+) CD8(+) T cells allowed the isolation of tetramer(bright) and tetramer(dull) populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL-specific CTL as well as CTL that recognize a peptide (NY-ESO-1(157-165)) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8(+) T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.     

High frequency of autologous anti-melanoma CTL directed against an antigen generated by a point mutation in a new helicase gene

We have identified an Ag recognized by autologous CTL on the melanoma cells of a patient who enjoyed an unusually favorable clinical evolution. The antigenic peptide, which is presented by HLA-A28 molecules, is encoded by a mutated sequence in a new gene. This gene, which was named MUM-3, is expressed ubiquitously and shows homology with the RNA helicase gene family. Limiting dilution analysis indicated that at least 0.15% of the blood CD8 T cells were tumor-specific CTL precursors. The MUM-3 Ag was recognized by 90% of these CTL, indicating that it is the dominant target Ag of the tumor-specific CTL response. The high frequency of anti-MUM-3 CTL was confirmed with tetramers of soluble HLA-A28 molecules loaded with the antigenic peptide. MUM-3 tetramers stained 1.2% of blood CD8 cells, a frequency that has never been reported for T cells directed against a strictly tumor-specific Ag. To confirm these results, the CD8 T cells that were clearly labeled with tetramers were restimulated in clonal conditions. About 90% of these cells proliferated, and all the resulting clones proved lytic and MUM-3 specific. By improving the conditions used for the in vitro restimulation of CTL precursors by the tumor cells, the same frequency could be obtained in limiting dilution analysis. These results show that some cancer patients have a high frequency of circulating CTL that are directed against a strictly tumor-specific Ag. These CTL are responsive to restimulation in vitro and are easily detected with tetramers. Such responses may therefore be an achievable goal for therapeutic vaccination with tumor-specific Ags.     

Multiple Chlamydia pneumoniae antigens prime CD8+ Tc1 responses that inhibit intracellular growth of this vacuolar pathogen

CD8(+) T cells play an essential role in immunity to Chlamydia pneumoniae (Cpn). However, the target Ags recognized by Cpn-specific CD8(+) T cells have not been identified, and the mechanisms by which this T cell subset contributes to protection remain unknown. In this work we demonstrate that Cpn infection primes a pathogen-specific CD8(+) T cell response in mice. Eighteen H-2(b) binding peptides representing sequences from 12 Cpn Ags sensitized target cells for MHC class I-restricted lysis by CD8(+) CTL generated from the spleens and lungs of infected mice. Peptide-specific IFN-gamma-secreting CD8(+) T cells were present in local and systemic compartments after primary infection, and these cells expanded after pathogen re-exposure. CD8(+) T cell lines to the 18 Cpn epitope-bearing peptides were cytotoxic, displayed a memory phenotype, and secreted IFN-gamma and TNF-alpha, but not IL-4. These CTL lines lysed Cpn-infected macrophages, and the lytic activity was inhibited by brefeldin A, indicating endogenous processing of CTL Ags. Finally, Cpn peptide-specific CD8(+) CTL suppressed chlamydial growth in vitro by direct lysis of infected cells and by secretion of IFN-gamma and other soluble factors. These studies provide information on the mechanisms by which CD8(+) CTL protect against Cpn, furnish the tools to investigate their possible role in immunopathology, and lay the foundation for future work to develop vaccines against acute and chronic Cpn infections.     

Direct detection and magnetic isolation of Chlamydia trachomatis major outer membrane protein-specific CD8+ CTLs with HLA class I tetramers

We recently identified HLA class I-presented epitopes in the major outer membrane protein (MOMP) of Chlamydia trachomatis that elicit CTL responses in human genital tract infections. T cells possessing cytolytic activities specific for these epitopes could be detected following in vitro stimulation of peripheral blood CD8(+) T cells with peptides. In the present study we used HLA-A2 tetramers for detailed characterization of MOMP-specific CTL responses. Ex vivo tetramer analysis detected MOMP-specific T cells in the peripheral blood of infected individuals at significant frequencies (0.01-0.20% of CD8(+) T cells). After in vitro stimulation with peptides, the frequencies of MOMP peptide-specific T cells increased up to 2.34% of CD8(+) T cells in bulk cultures. In contrast, HLA-A2/MOMP tetramer-binding T cells were virtually undetectable in the peripheral blood from uninfected individuals, either ex vivo or after 3 wk of in vitro peptide stimulation of their T cells. Magnetically sorted, tetramer-bound T cells specifically lysed peptide-pulsed targets as well as C. trachomatis-infected epithelial cells with nearly 50-fold greater per cell efficiency than that of unsorted populations. This study provides conclusive evidence of in vivo induction of HLA class I-restricted CD8(+) CTL responses to C. trachomatis MOMP. Direct detection of these cells with tetramers will allow their further characterization without prior manipulation and facilitate monitoring of CTL responses during infections and in immunization trials with MOMP-based vaccines.     

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

High avidity antigen-specific CTL identified by CD8-independent tetramer staining

Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.     

Polyclonal CTL responses observed in melanoma patients vaccinated with dendritic cells pulsed with a MAGE-3.A1 peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3(168-176) peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 x 10(-6) to 9 x 10(-4) of CD8(+) blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.     

Genome-based in silico identification of new Mycobacterium tuberculosis antigens activating polyfunctional CD8+ T cells in human tuberculosis

Although CD8(+) T cells help control Mycobacterium tuberculosis infection, their M. tuberculosis Ag repertoire, in vivo frequency, and functionality in human tuberculosis (TB) remains largely undefined. We have performed genome-based bioinformatics searches to identify new M. tuberculosis epitopes presented by major HLA class I supertypes A2, A3, and B7 (covering 80% of the human population). A total of 432 M. tuberculosis peptides predicted to bind to HLA-A*0201, HLA-A*0301, and HLA-B*0702 (representing the above supertypes) were synthesized and HLA-binding affinities determined. Peptide-specific CD8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors identified 70 new M. tuberculosis epitopes. Using HLA/peptide tetramers for the 18 most prominently recognized HLA-A*0201-binding M. tuberculosis peptides, recognition by cured TB patients' CD8(+) T cells was validated for all 18 epitopes. Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α revealed mono-, dual-, as well as triple-positive CD8(+) T cells, indicating these M. tuberculosis peptide-specific CD8(+) T cells were (poly)functional. Moreover, these T cells were primed during natural infection, because they were absent from M. tuberculosis-noninfected individuals. Control CMV peptide/HLA-A*0201 tetramers stained CD8(+) T cells in M. tuberculosis-infected and noninfected individuals equally, whereas Ebola peptide/HLA-A*0201 tetramers were negative. In conclusion, the M. tuberculosis-epitope/Ag repertoire for human CD8(+) T cells is much broader than hitherto suspected, and the newly identified M. tuberculosis Ags are recognized by (poly)functional CD8(+) T cells during control of infection. These results impact on TB-vaccine design and biomarker identification.     

Peptide affinity for MHC influences the phenotype of CD8(+) T cells primed in vivo

Priming C57BL/6 mice with dominant antigenic peptides of ovalbumin (OVA) or bovine insulin (INS) in complete Freund's adjuvant generates antigen-specific, H-2K(b)-restricted, CD8(+) CTL. OVA-CTL produced type 1 cytokines IFN-gamma and TNF-alpha, whereas INS-CTL produced IL-5 and IL-10 with low levels of IL-4 and IFN-gamma. Here, we investigate whether differential binding affinities of the OVA and INS peptides to H-2K(b) influence the phenotype of the CD8(+) CTL. OVA(257-264) was found to have significantly higher binding affinity than the INS A-chain(12-21) toward K(b). Exchanging the MHC anchor residues between the OVA and INS peptides reversed the K(b) binding capacity of the altered peptides. The lower affinity, altered OVA peptides induced CTL that produced IL-5 and IL-10 in addition to IFN-gamma, whereas high binding affinity, altered INS peptides induced CTL that produced IFN-gamma but not IL-5 or IL-10. These data suggest that MHC binding affinity of peptides can regulate the phenotype of the resulting CD8(+) T cells.     

Antigen-specific CD8+ T cells mediate a peptide-induced fatal syndrome

Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2(121-130) peptide inhibits the development of antiviral CTL specific for the immunodominant D(b):VP2(121-130) epitope expressed during acute Theiler's murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8(+) T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8(+) T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.     

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.     

Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of KD < approximately 80 microM and approximately 35 microM, respectively, to label human CTLs at 37 degrees C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with KD values >200 microM. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.     

Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells

Appropriate activation of naive CD8(+) T cells depends on the coordinated interaction of these cells with professional APC that present antigenic peptides in the context of MHC class I molecules. It is accepted that dendritic cells (DC) are efficient in activating naive T cells and are unique in their capacity to prime CD8(+) T cell responses against exogenous cell-associated Ags. Nevertheless, it is unclear whether epitopes, derived from endogenously synthesized proteins and presented by MHC class I molecules on the surface of other APC including B cells and macrophages, can activate naive CD8(+) T cells in vivo. By infecting transgenic CD11c-DTR/GFP mice that allow conditional depletion of DC with lymphocytic choriomeningitis virus (LCMV), which infects all types of APC and elicits a vigorous CTL response, we unambiguously show that priming of LCMV-specific CD8(+) T cells is crucially dependent on DC, despite ample presence of LCMV-infected macrophages and B cells in secondary lymphoid organs.     

A dominant role for CD8+-T-lymphocyte selection in simian immunodeficiency virus sequence variation

CD8(+) T lymphocytes (CD8-TL) select viral escape variants in both human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. The frequency of CD8-TL viral escape as well as the contribution of escape to overall virus diversification has not been assessed. We quantified CD8-TL selection in SIV infections by sequencing viral genomes from 35 SIVmac239-infected animals at the time of euthanasia. Here we show that positive selection for sequences encoding 46 known CD8-TL epitopes is comparable to the positive selection observed for the variable loops of env. We also found that >60% of viral variation outside of the viral envelope occurs within recognized CD8-TL epitopes. Therefore, we conclude that CD8-TL selection is the dominant cause of SIV diversification outside of the envelope.