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.     

Impacts of epitope expression kinetics and class I downregulation on the antiviral activity of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes

The determinants of CD8(+) cytotoxic T-lymphocyte (CTL) antiviral activity against human immunodeficiency virus type 1 (HIV-1) remain poorly defined. Although recent technological advances have markedly enhanced the ability to detect HIV-1-specific T cells, commonly used assays do not reveal their direct interaction with virus. We investigated two determinants of CTL antiviral efficiency by manipulating HIV-1 and measuring the effects on CTL suppression of viral replication in acutely infected cells. Translocation of a Gag epitope into the early protein Nef markedly increased the activity of CTL recognizing that epitope, in comparison to HIV-1 expressing the epitope normally in the late protein Gag. Because this epitope translocation resulted not only in earlier expression but also in loss of major histocompatibility complex class I downregulation by Nef, the activities of CTL against a panel of viral constructs differing in kinetics of epitope expression and class I downmodulation were compared. The results indicated that both the timing of epitope expression and the reduction of class I have profound effects on the ability of CTL to suppress HIV-1 replication in acutely infected cells. The epitope targeting of CTL and viral control of class I therefore likely play important roles in the ability of CTL to exert pressure on HIV-1.     

How many human immunodeficiency virus type 1-infected target cells can a cytotoxic T-lymphocyte kill?

The antiviral role of CD8+ cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. Specifically, the degree to which CTLs reduce viral replication by killing HIV-1-infected cells in vivo is not known. Here we employ mathematical models of the infection process and CTL action to estimate the rate that CTLs can kill HIV-1-infected cells from in vitro and in vivo data. Our estimates, which are surprisingly consistent considering the disparities between the two experimental systems, demonstrate that on average CTLs can kill from 0.7 to 3 infected target cells per day, with the variability in this figure due to epitope specificity or other factors. These results are compatible with the observed decline in viremia after primary infection being primarily a consequence of CTL activity and have interesting implications for vaccine design.     

The antiviral activity of HIV-specific CD8+ CTL clones is limited by elimination due to encounter with HIV-infected targets.

Adoptive immunotherapy of virus infection with viral-specific CTL has shown promise in animal models and human virus infections and is being evaluated as a therapy for established HIV-1 infection. Defining the individual obstacles for success is difficult in human trials. We have therefore examined the localization, persistence, and antiviral activity of HIV-1 gag-specific CTL clones in both HIV-1-infected and uninfected haplotype-matched human (hu)-PBL-SCID mice. Injection of gag-specific clones but not control CTL into HIV-1-infected hosts reduced plasma viremia by >10-fold but failed to eliminate the virus infection from most treated animals. The failure to eradicate virus did not reflect selection of escape variants because the gag epitope remained unmutated in virus isolates obtained after CTL therapy. Injection of carboxyfluorescein diacetate succinimide ester-labeled CTL demonstrated markedly different fates for gag-specific CTL in the presence or absence of HIV-1 infection. HIV-1-specific CTL rapidly disappeared in infected recipients, whereas they were maintained at high numbers in uninfected mice. By contrast, control CTL were long lived in both infected and uninfected recipients. Thus, interaction of CTL with virus-infected target cells in vivo leads not only to target destruction but also to the rapid disappearance of the infused CTL, and it limits the capacity of CTL therapy to eliminate HIV-1 infection.     

Functional adaptation of Nef to the immune milieu of HIV-1 infection in vivo

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4(+) T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.     

HIV-1 Gag Cytotoxic T Lymphocyte Epitopes Vary in Presentation Kinetics Relative to HLA Class I Downregulation

Although CD8⁺ cytotoxic T lymphocytes (CTLs) are protective in HIV-1 infection, the factors determining their antiviral efficiency are poorly defined. It is proposed that Gag targeting is superior because of very early Gag epitope presentation, allowing early killing of infected cells before Nef-mediated downregulation of human leukocyte antigen class I (HLA-I). To study Gag epitope presentation kinetics, three epitopes (SL9_77-85, KF11_162-172, and TW10_240-249) were genetically translocated from their endogenous location in the Rev-dependent (late) gag gene into the Rev-independent (early) nef gene with concomitant mutation of the corresponding endogenous epitopes to nonrecognized sequences. These viruses were compared to the index virus for CTL-mediated suppression of replication and the susceptibility of this antiviral activity to Nef-mediated HLA-I downregulation. SL9-specific CTLs gained activity after SL9 translocation to Nef, going from Nef sensitive to Nef insensitive, indicating that translocation accelerated infected cell recognition from after to before HLA-I downregulation. KF11-specific CTL antiviral activity was unchanged and insensitive to HLA-I downregulation before and after KF11 translocation, suggesting that already rapid recognition of infected cells was not accelerated. However, TW10-specific CTLs that were insensitive to Nef at the baseline became sensitive with reduced antiviral activity after translocation, indicating that translocation retarded epitope expression. Cytosolic peptide processing assays suggested that TW10 was inefficiently generated after translocation to Nef, compared to SL9 and KF11. As a whole, these data demonstrate that epitope presentation kinetics play an important role in CTL antiviral efficiency, that Gag epitopes are not uniformly presented early, and that the epitope context can play a major role in presentation kinetics.     

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

Frequent detection of escape from cytotoxic T-lymphocyte recognition in perinatal human immunodeficiency virus (HIV) type 1 transmission: the ariel project for the prevention of transmission of HIV from mother to infant

Host immunologic factors, including human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL), are thought to contribute to the control of HIV type 1 (HIV-1) replication and thus delay disease progression in infected individuals. Host immunologic factors are also likely to influence perinatal transmission of HIV-1 from infected mother to infant. In this study, the potential role of CTL in modulating HIV-1 transmission from mother to infant was examined in 11 HIV-1-infected mothers, 3 of whom transmitted virus to their offspring. Frequencies of HIV-1-specific human leukocyte antigen class I-restricted CTL responses and viral epitope amino acid sequence variation were determined in the mothers and their infected infants. Maternal HIV-1-specific CTL clones were derived from each of the HIV-1-infected pregnant women. Amino acid substitutions within the targeted CTL epitopes were more frequently identified in transmitting mothers than in nontransmitting mothers, and immune escape from CTL recognition was detected in all three transmitting mothers but in only one of eight nontransmitting mothers. The majority of viral sequences obtained from the HIV-1-infected infant blood samples were susceptible to maternal CTL. These findings demonstrate that epitope amino acid sequence variation and escape from CTL recognition occur more frequently in mothers that transmit HIV-1 to their infants than in those who do not. However, the transmitted virus can be a CTL susceptible form, suggesting inadequate in vivo immune control.     

Use of an In Vivo FTA Assay to Assess the Magnitude,Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination

Qualitative characteristics of cytotoxic CD8⁺ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.     

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.     

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

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

Discrepancy between in vitro measurable and in vivo virus neutralizing cytotoxic T cell reactivities. Low T cell receptor specificity and avidity sufficient for in vitro proliferation or cytotoxicity to peptide-coated target cells but not for in vivo protection.

The TCR-alpha beta of CTL recognize peptide Ag in association with MHC class I molecules. TCR binding should be highly specific to guarantee pathogen specificity and to avoid self-reactivity. Therefore, the in vivo relevance of T cells exhibiting cross-reactivities in vitro and the respective role of the TCR affinities involved are not clear. To analyze high and low avidity T cell activities both in vitro and in vivo, we investigated primary and clonal CTL responses specific for the lymphocytic choriomeningitis virus nucleoprotein 118-126 epitope in association with the two closely related H-2Ld or H-2Lq molecules. As expected, we found highly specific class I-allele-restricted CTL responses when antiviral protection or immunopathology in vivo and lysis of virus infected target cells in vitro were analyzed. In contrast, the CTL were MHC crossreactive and thus considerably less discriminatory against targets expressing high MHC-peptide densities and in proliferation assays. The data show that relatively high TCR avidities are required for virus neutralization in vivo, in contrast to in vitro analyses of peptide-coated target cells or proliferative T cell responses that may engage TCR of low avidity and broad specificity and therefore may not reflect biologically relevant TCR avidities.     

Recognition of a highly conserved region of human immunodeficiency virus type 1 gp120 by an HLA-Cw4-restricted cytotoxic T-lymphocyte clone.

Human immunodeficiency virus type 1 (HIV-1) isolates exhibit extensive sequence variation, particularly in the gp120 subunit of the envelope glycoprotein, and the degree of this variation has raised questions as to whether conserved regions of the HIV-1 envelope can be recognized by the host immune response. A CD8+ cytotoxic T-lymphocyte (CTL) clone specific for the HIV-1 envelope was derived by culturing peripheral blood mononuclear cells from an HIV-1 seropositive subject in the presence of a CD3-specific monoclonal antibody, interleukin-2, and irradiated allogeneic peripheral blood mononuclear cells. Lysis of target cells was restricted by an HLA-C molecule, Cw4, which has not been previously shown to present viral antigen to CTL. Mapping of the specificity of this CTL clone by using synthetic HIV-1 peptides localized the epitope to an 8-amino-acid region of gp120 (amino acids 376 to 383) which is conserved among approximately 90% of sequenced viral isolates. Examination of the recognition of variant peptides by this CTL clone demonstrated that a single, nonconservative amino acid substitution within the 8-amino-acid minimal epitope could abrogate lysis of targets incubated with the variant peptide. The identification of a CTL epitope in a highly conserved region of gp120 documents the ability of cellular immune responses of infected persons to respond to relatively invariant portions of this highly variable envelope glycoprotein. However, the ability of even a single-amino-acid change in gp120 to abolish lysis by CTL supports the hypothesis that sequence variation in HIV-1 may serve as a mechanism of immune escape. In addition, the identification of an HLA-C molecule presenting viral antigen to CTL supports a functional role for these molecules.     

High avidity cytotoxic T lymphocytes can be selected into the memory pool but they are exquisitely sensitive to functional impairment

High avidity cytotoxic T lymphocytes (CTL) are important in viral clearance and anti-tumor immunity, however, mechanisms for their optimal generation and maintenance in vivo remain unclear. Immunizing mice with an antibody-DNA vaccine encoding a single CTL epitope, induces a 100 fold higher avidity response than peptide vaccination with the identical epitope. The high avidity response is retained into memory and can be efficiently reactivated with an antibody-DNA boost. In contrast, reactivation of high avidity CTL with peptide, stimulated responses with a significant drop in avidity, suggesting loss or conversion of the high avidity CTL to lower avidity. Similarly, high avidity T cells maintained ex vivo were exquisitely sensitive to signaling with low doses of peptide (1 ng/ml) giving optimal TCR stimulation and resulting in retained avidity, proliferation and ability to kill specific targets. In contrast, high avidity T cells maintained ex vivo with supraoptimal TCR stimulation (10 μg/ml peptide) resulted in reduced avidity and failure to kill tumor cells. They also failed to proliferate, showed a significant increase in apoptosis and expressed high levels of the exhaustion marker programmed death-1 (PD-1) and low levels of the lymphocyte-activation gene 3 (LAG-3). This suggests high avidity T cells are recruited to the memory pool but can be lost by supraoptimal stimulation in vitro and in vivo. This is characterized by loss of function and an increase in cell death. The remaining CTL, exhibit low functional avidity that is reflected in reduced anti-tumor activity. This could contribute to failure of the immune system to control the growth of tumors and has implications for vaccination strategies and adoptive transfer of T cells.     

Overlapping epitopes in human immunodeficiency virus type 1 gp120 presented by HLA A, B, and C molecules: effects of viral variation on cytotoxic T-lymphocyte recognition.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) are thought to exert immunologic selection pressure in infected persons, yet few data regarding the effects of this constraint on viral sequence variation in vivo, particularly in the highly variable Env protein, are available. In this study, CD8+ HIV type 1 (HIV-1) envelope-specific CTL clones specific for gp120 were isolated from peripheral blood mononuclear cells of four HIV-infected individuals, all of which recognized the same 25-amino-acid (aa) peptide (aa 371 to 395), which is partially contained in the CD4-binding domain of HIV-1 gp120. Fine mapping studies revealed that two of the clones optimally recognized the 9-aa sequence 375 to 383 (SFNCGGEFF), while the two other clones optimally recognized the epitope contained in the overlapping 9-aa sequence 376 to 384 (FNCGGEFFY). Lysis of target cells by the two clones recognizing aa 375 to 383 was restricted by HLA B15 and Cw4, respectively, whereas both clones recognizing aa 376 to 384 were restricted by HLA A29. Sequence variation, relative to the IIIB strain sequence used to identify CTL clones, was observed in autologous viruses in the epitope-containing region in all four subjects. However, poorly recognized autologous sequence variants were predominantly seen for the A29-restricted clones, whereas the clones specific for SFNCGGEFF continued to recognize the predominant autologous sequences. These results suggest that the HLA profile of an individual may not only be important in determining the specificity of CTL recognition but may also affect the ability to recognize virus variants and suppress escape from CTL recognition. These results also identify overlapping viral CTL epitopes which can be presented by HLA A, B, and C molecules.     

Human antitumor CD8+ T cells producing Th1 polycytokines show superior antigen sensitivity and tumor recognition

Adoptive transfer of T cells expressing transgenic TCR with antitumor specificity provides a hopeful new therapy for patients with advanced cancer. To fulfill a large need for TCR with high affinity and specificity for various tumor entities, we sought to identify parameters for rapid selection of CTL clones with suitable characteristics. Twelve CTL clones displaying different Ag sensitivities for the same peptide-MHC epitope of the melanoma-associated Ag tyrosinase were analyzed in detail. Better MHC-multimer binding and slower multimer release are thought to reflect stronger TCR-peptide-MHC interactions; thus, these parameters would seem well suited to identify higher avidity CTL. However, large disparities were found comparing CTL multimer binding with peptide sensitivity. In contrast, CD8(+) CTL with superior Ag sensitivity mediated good tumor cytotoxicity and also secreted the triple combination of IFN-γ, IL-2, and TNF-α, representing a Th1 pattern often missing in lower avidity CTL. Furthermore, recipient lymphocytes were imbued with high Ag sensitivity, superior tumor recognition, as well as capacity for Th1 polycytokine secretion after transduction with the TCR of a high-avidity CTL. Thus, Th1 polycytokine secretion served as a suitable parameter to rapidly demark cytotoxic CD8(+) T cell clones for further TCR evaluation.     

Different effects of Nef-mediated HLA class I down-regulation on human immunodeficiency virus type 1-specific CD8(+) T-cell cytolytic activity and cytokine production

A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4(+) T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4(+) T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4(+) T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4(+) T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4(+) T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4(+) T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8(+) T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8(+) T cells are able to partially but not completely control HIV-1 replication in vivo.     

Altering effects of antigenic variations in HIV-1 on antiviral effectiveness of HIV-specific CTLs

The mutational escape of HIV-1 from established CTL responses is becoming evident. However, it is not yet clear whether antigenic variations of HIV-1 may have an additional effect on the differential antiviral effectiveness of HIV-specific CTLs. Herein, we characterized HIV-specific CTL responses toward Pol, Env, and Nef optimal epitopes presented by HLA-B*35 during a chronic phase of HIV-1 infection. We found CTL escape variants within Pol and Nef epitopes that affected recognition by TCRs, although there was no mutation within the Env epitope. An analysis of peptide-HLA tetrameric complexes revealed that CD8 T cells exclusively specific for the Nef variant were generated following domination by the variant viruses. The variant-specific cells were capable of killing target cells and producing antiviral cytokines but showed impaired Ag-specific proliferation ex vivo, whereas wild-type specific cells had potent activities. Moreover, clonotypic CD8 T cells specific for the Pol variant showed diminished proliferation, whereas Env-specific ones had no functional heterogeneity. Taken together, our data indicate that antigenic variations that abolished TCR recognition not only resulted in escape from established CTL responses but also eventually generated another subset of variant-specific CTLs having decreased antiviral activity, causing an additional negative effect on antiviral immune responses during a chronic HIV infection.     

Patterns of cytokine production in human immunodeficiency virus type 1 (HIV-1)-specific human CD8+ T cells after stimulation with HIV-1-infected CD4+ T cells

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.     

An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag.

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.