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Bruce D Walker

Publications and source records attributed to Bruce D Walker.

At least 73 records · Page 4Linked to original sources
PubMed2004

Heat shock protein-mediated cross-presentation of exogenous HIV antigen on HLA class I and class II.

Strong CD4(+) and CD8(+) T cell responses are considered important immune components for controlling HIV infection, and their priming may be central to an effective HIV vaccine. We describe in this study an approach by which multiple CD4(+) and CD8(+) T cell epitopes are processed and presented from an exogenously added HIV-1 Gag-p24 peptide of 32 aa complexed to heat shock protein (HSP) gp96. CD8(+) T cell recognition of the HSP/peptide complex, but not the peptide alone, was inhibited by brefeldin A, suggesting an endoplasmic reticulum-dependent pathway. This is the first report to describe efficient processing and simultaneous presentation of overlapping class I- and class II-restricted epitopes from the same extracellularly added precursor peptide complexed to HSP. Given previous reports of the strong immunogenicity of HSP/peptide complexes, the present data suggest that HSP-complexed peptides containing multiple MHC class I- and class II-restricted epitopes represent potential vaccine candidates for HIV and other viral infections suitable to induce effective CTL memory by simultaneously providing CD4 T cell help.

AIDS Vaccines↗
PubMed2004

HIV-1 Nef is preferentially recognized by CD8 T cells in primary HIV-1 infection despite a relatively high degree of genetic diversity.

OBJECTIVE: To compare the magnitude, breadth and protein specificity of HIV-1-specific CD8 T-cell responses against the clade B consensus sequence during primary and chronic HIV-1 infection and to analyze the impact of viral diversity on the localization of detected responses. METHODS: HIV-1-specific CD8 T-cell responses against the clade B consensus sequence in individuals with acute (n = 10), early (n = 19) and chronic (n = 10) infection were longitudinally assessed using an interferon-gamma EliSpot assay. RESULTS: CD8 T-cell responses against clade B consensus sequences were preferentially directed against central regions of Nef during primary HIV-1 infection, despite a relatively higher degree of genetic diversity compared with other subsequently targeted regions. In subjects with acute and early infection, Nef-specific CD8 T-cell responses against the consensus Nef sequence represented 94 and 46% of the total magnitude of HIV-1-specific CD8 T-cell responses, respectively. Subjects with untreated chronic infection exhibited broadly diversified CD8 T-cell responses against more conserved viral regions, with only 17% of virus-specific T-cell responses targeting Nef. The initial immunodominance of Nef persisted in individuals with treated acute infection, but shifted rapidly to Gag, Env and Pol in subjects with continuous antigen exposure. CONCLUSION: These data show that despite relatively high sequence variability, viral regions within the clade B consensus sequence of Nef are preferentially recognized during primary HIV-1 infection. Later diversification of responses to other proteins during prolonged antigen exposure provides evidence of the initial preferential immunogenicity of Nef epitopes compared to similarly conserved regions within other viral proteins.

Adult↗
PubMed2004

CD8+ T lymphocyte responses target functionally important regions of Protease and Integrase in HIV-1 infected subjects.

BACKGROUND: CD8+ T cell responses are known to be important to the control of HIV-1 infection. While responses to reverse transcriptase and most structural and accessory proteins have been extensively studied, CD8 T cell responses specifically directed to the HIV-1 enzymes Protease and Integrase have not been well characterized, and few epitopes have been described in detail. METHODS: We assessed comprehensively the CD8 T cell responses to synthetic peptides spanning Protease and Integrase in 56 HIV-1 infected subjects with acute, chronic, or controlled infection using IFN-gamma-Elispot assays and intracellular cytokine staining. Fine-characterization of novel CTL epitopes was performed on peptide-specific CTL lines in Elispot and 51Chromium-release assays. RESULTS: Thirteen (23%) and 38 (68%) of the 56 subjects had detectable responses to Protease and Integrase, respectively, and together these targeted most regions within both proteins. Sequence variability analysis confirmed that responses cluster largely around conserved regions of Integrase, but responses against a large, highly conserved region of the N-terminal DNA-binding domain of Integrase were not readily detected. CD8 T cell responses targeted regions of Protease that contain known Protease inhibitor mutation residues, but strong Protease-specific CD8 T cell responses were rare. Fine-mapping of targeted epitopes allowed the identification of three novel, HLA class I-restricted, frequently-targeted optimal epitopes. There were no significant correlations between CD8 T cell responses to Protease and Integrase and clinical disease category in the study subjects, nor was there a correlation with viral load. CONCLUSIONS: These findings confirm that CD8 T cell responses directed against HIV-1 include potentially important functional regions of Protease and Integrase, and that pharmacologic targeting of these enzymes will place them under both drug and immune selection pressure.

Journal Article↗
PubMed2004

Impact of intrapeptide epitope location on CD8 T cell recognition: implications for design of overlapping peptide panels.

BACKGROUND: Antigen-specific CD8 T cells following infection or immunization are typically assessed by measuring interferon-gamma production after stimulation with overlapping peptides spanning the region of interest. The effect of epitope location within such peptides is not known but may influence recognition. OBJECTIVE: To examine if peptides containing the appropriate C-terminal anchor amino acid residue would provide more sensitive detection of T cell responses. The impact was examined of epitope location within overlapping peptides on recognition of epitope-specific CD8 T cell responses. METHODS: C-terminal amino acid residues were analyzed in well-defined optimal epitopes for HIV, Epstein-Barr virus, cytomegalovirus and influenza and in peptide-binding motifs. Recognition of known epitopes within longer synthesized peptides by peripheral blood mononuclear cells or CD8 T cell lines was tested using interferon-gamma Elispot at various peptide concentrations. RESULTS: Only 9 of 20 amino acids served as the C-terminal anchor position in 96% of described optimal epitopes and in 95% of peptide-binding motifs. A CD8 T cell response to an epitope within a longer peptide is best detected when the epitope is situated at the C-terminal end of the longer peptide, both when using peptides designed to include the optimal epitope at every possible position and when comparing responses towards optimal epitopes and corresponding overlapping peptides in a larger group of subjects. CONCLUSION: When using overlapping peptides to screen for CD8 T cell responses, more sensitive detection will be achieved using known C-terminal anchor amino acid residues at the C-terminus.

Amino Acid Motifs↗
PubMed2004

Immune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection.

Mutations within cytotoxic T lymphocyte (CTL) epitopes impair T cell recognition, but escape mutations arising in flanking regions that alter antigen processing have not been defined in natural human infections. In human histocompatibility leukocyte antigen (HLA)-B57+ HIV-infected persons, immune selection pressure leads to a mutation from alanine to proline at Gag residue 146 immediately preceding the NH2 terminus of a dominant HLA-B57-restricted epitope, ISPRTLNAW. Although N-extended wild-type or mutant peptides remained well-recognized, mutant virus-infected CD4 T cells failed to be recognized by the same CTL clones. The A146P mutation prevented NH2-terminal trimming of the optimal epitope by the endoplasmic reticulum aminopeptidase I. These results demonstrate that allele-associated sequence variation within the flanking region of CTL epitopes can alter antigen processing. Identifying such mutations is of major relevance in the construction of vaccine sequences.

Alleles↗
PubMed2004

HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8(+) T cells producing both interferon-gamma and tumor necrosis factor-alpha.

CD8(+) T cells play a crucial role in the control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. Recent data suggest that HIV-1-specific CD8(+) T cell subsets may differ in their ability to exert these effector functions. Here, we directly compared the cytokine secretion patterns and cytotoxic capacity of HIV-1-specific CD8(+) T cells, using a flow-cytometric cytotoxicity assay based on caspase-3 activation in dying target cells. These experiments revealed considerable intraindividual and interindividual differences among epitope-specific T-cell effector functions: while the frequency of HIV-1-specific CD8(+) T cells secreting interferon-gamma but no tumor necrosis factor-alpha (TNF-alpha) following antigenic stimulation was only weakly correlated to their cytotoxic activity (R = 0.05, P =.57), a subset of CD8(+) T cells secreting both inter-feron-gamma and TNF-alpha was substantially more strongly associated with cytotoxicity (R = 0.67, P <.001). This subset of CD8(+) T cells also exhibited stronger intracellular perforin expression and more pronounced direct ex vivo HIV-1-specific cytoxicity than CD8(+) T cells secreting solely interferon-gamma following sorting of these subpopulations according to their cytokine profile. These results suggest that HIV-1-specific cytotoxicity of CD8(+) T cells is preferentially mediated by a subset of CD8(+) T cells secreting both interferon-gamma and TNF-alpha.

Adult↗
PubMed2004

HIV-1 specific CD8+ T cells with an effector phenotype and control of viral replication.

Most people infected with HIV-1 cannot control viral replication despite the presence of virus-specific CD8+ T cells. It has been postulated that this inability is related to the failure of these cells to mature into fully differentiated effector cells. We tested this hypothesis by comparing the maturation phenotype of virus-specific CD8+ T cells in people who could control viral replication off anti-retroviral therapy with those who could not. In five patients with treated acute HIV-1-infection, structured treatment interruption (STI) induced control of viral replication was associated with expansion of virus-specific CD8+ T cells with a fully differentiated effector phenotype. These effector cells were also expanded in treatment-naive chronically infected individuals who spontaneously controlled viral replication, and augmented expression of perforin was noted in both settings. Our data show that full maturation of virus-specific CD8+ T cells is possible in the context of HIV-1-infection, and suggest that such maturation might be important in viral control.

Antiretroviral Therapy, Highly Active↗
PubMed2004

Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8+ T cells.

Recent studies using MHC class I tetramers have shown that CD8(+) T cell responses against different persistent viruses vary considerably in magnitude and phenotype. At one extreme, hepatitis C virus (HCV)-specific CD8(+) T cell responses in blood are generally weak and have a phenotype that is perforin low and CCR7 high (early memory). At the other, specific responses to CMV are strong, perforin high, and CCR7 low (mature or effector memory). To examine the potential mechanisms behind this diversity, we compared CMV-specific responses in HCV-infected and healthy individuals. We find a striking difference in the phenotype of CMV-specific CD8(+) T cells between these groups. In the HCV-infected cohort, CMV-specific CD8(+) T cells lost markers associated with maturity; they had increased expression of CCR7 and reduced expression of Fas and perforin. They nevertheless responded to Ag in vitro in a manner similar to controls, with strong proliferation and appropriate acquisition of effector memory markers. The reduction in mature CD8 T cells in HCV-infected individuals may arise through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. Whatever the mechanism, HCV has a pervasive influence on the circulating CD8(+) T cell population, a novel feature that may be a hallmark of this infection.

Adult↗
PubMed2004

High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection.

BACKGROUND & AIMS: Cellular immune responses are thought to play a key role in the resolution of primary HCV infection. Although it has been consistently shown that CD4+ T-cell responses are maintained in those with spontaneous resolution but lost in those with persistent infection, the role of CD8+ T-cell responses remains controversial. Previous studies have largely focused on limited HLA alleles and predefined CD8+ T-cell epitopes, and, thus, comprehensive studies remain to be performed. METHODS: To understand the composition of the immune response associated with spontaneous resolution, we comprehensively mapped CD8+ T-cell responses in 20 HLA-diverse persons with resolved HCV infection, using HCV peptides spanning the entire genome. We analyzed the magnitude, breadth, function, and phenotype using ELISpot, class-I tetramers, intracellular cytokine staining, and cytolytic assays. We studied in parallel HCV-specific responses and viral sequence variation in persistent infection. RESULTS: Responses in individuals with resolved infection were strong and broad with robust proliferation in response to antigen. Responses in those persistently infected were rarely detected ex vivo and, when present, were narrowly directed and weak. However, they also proliferated in vitro. Dominant target epitopes differed among individuals in both cohorts, despite frequently shared HLA-alleles. CONCLUSIONS: These data indicate that persisting, strong CD8+ T-cell responses are observed in the majority of persons with resolved HCV infection and provide support for strategies to boost CD8+ T-cell responses for the prevention or treatment of HCV infection but also highlight the diversity of responses that may need to be elicited to provide protection.

CD8-Positive T-Lymphocytes↗
PubMed2004

Defective immune function of primary effusion lymphoma cells is associated with distinct KSHV gene expression profiles.

Primary effusion lymphomas (PEL) are uniformly infected with Kaposi's sarcoma-associated herpesvirus (KSHV), and thus likely present both tumor and viral antigens to the immune system. In order to grow unrestricted and cause disease, multiple immune evasion strategies may be utilized by PEL to evade immune surveillance. Using six well-established PEL cell lines and comparing these to Epstein-Barr virus-transformed B cell lines and peripheral blood B cells, significant differences were found in the surface expression of molecules involved in antigen presentation, T cell activation and cell-cell adhesion. Significantly reduced stimulation of cytotoxic T lymphocytes, lowered sensitivity to natural killer cell-mediated lysis and impaired function as antigen presenting cells in mixed leukocyte reactions were found for three PEL cell lines with particularly low CD54, CD58 and CD81 expression. Comparative microarray analysis demonstrated specific patterns of KSHV-encoded gene expression that were associated with the different immune functions of these cell lines. Thus, the present data suggest that distinct patterns of KSHV gene expression may be associated with particular phenotypic and functional characteristics of PEL cells, which may influence PEL pathogenesis.

Adult↗
PubMed2004

Fine specificity and cross-clade reactivity of HIV type 1 Gag-specific CD4+ T cells.

Despite growing evidence that HIV-1-specific CD4(+) T helper (Th) cells may play a role in the control of viremia, discrete Th cell epitopes remain poorly defined. Furthermore, it is not known whether Th cell responses generated using vaccines based on clade B virus sequences will elicit immune responses that are effective in regions of the world where non-clade B viruses predominate. To address these issues we isolated CD4(+) T cell clones from individuals with vigorous HIV-1-specific Th cell responses and identified the minimum epitopes recognized. The minimum peptide length required for induction of CD4(+) T cell proliferation, IFN-gamma secretion, and cytolytic activity ranged from 9 to 16 amino acids in the five epitopes studied. Cross-clade recognition of the defined epitopes was examined for variant peptides from clades A, B, C, D, and AE. Over half the variant epitopes (17 of 32) exhibited impaired recognition, defined as less than 50% of the IFN-gamma secretion elicited by B clade consensus sequence. There was no evidence for antagonistic activity mediated by the variant peptides, and despite strong responses there was no escape of autologous virus from Th responses in the epitopes we studied. Abrogated recognition of variant CD4(+) T cell epitopes presents a potential obstacle to vaccine development.

Amino Acid Sequence↗
PubMed2004

Differences in the expressed HLA class I alleles effect the differential clustering of HIV type 1-specific T cell responses in infected Chinese and caucasians.

China is a region of the world with a rapidly spreading HIV-1 epidemic. Studies providing insights into HIV-1 pathogenesis in infected Chinese are urgently needed to support the design and testing of an effective HIV-1 vaccine for this population. HIV-1-specific T cell responses were characterized in 32 HIV-1-infected individuals of Chinese origin and compared to 34 infected caucasians using 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence in an IFN-gamma ELISpot assay. All HIV-1 proteins were targeted with similar frequency in both populations and all study subjects recognized at least one overlapping peptide. HIV-1-specific T cell responses clustered in seven different regions of the HIV-1 genome in the Chinese cohort and in nine different regions in the caucasian cohort. The dominant HLA class I alleles expressed in the two populations differed significantly, and differences in epitope clustering pattern were shown to be influenced by differences in class I alleles that restrict immunodominant epitopes. These studies demonstrate that the clustering of HIV-1-specific T cell responses is influenced by the genetic HLA class I background in the study populations. The design and testing of candidate vaccines to fight the rapidly growing HIV-1 epidemic must therefore take the HLA genetics of the population into account as specific regions of the virus can be expected to be differentially targeted in ethnically diverse populations.

Alleles↗
PubMed2004

Major histocompatibility complex class I-intercellular adhesion molecule-1 association on the surface of target cells: implications for antigen presentation to cytotoxic T lymphocytes.

Polarization and segregation of the T-cell receptor (TCR) and integrins upon productive cytotoxic T-lymphocyte (CTL) target cell encounters are well documented. Much less is known about the redistribution of major histocompatibility complex class I (MHC-I) and intercellular adhesion molecule-1 (ICAM-1) proteins on target cells interacting with CTLs. Here we show that human leucocyte antigen-A2 (HLA-A2) MHC-I and ICAM-1 are physically associated and recovered from both the raft fraction and the fraction of soluble membranes of target cells. Conjugation of target cells with surrogate CTLs, i.e. polystyrene beads loaded with antibodies specific for HLA-A2 and ICAM-1, induced the accumulation of membrane rafts, and beads loaded with ICAM-1-specific antibodies caused the selective recruitment of HLA-A2 MHC-I at the contact area of the target cells. Disruption of raft integrity on target cells led to a release of HLA-A2 and ICAM-1 from the raft fraction, abatement of HLA-A2 polarization, and diminished the ability of target cells bearing viral peptides to induce a Ca(2+) flux in virus-specific CTLs. These data suggest that productive engagement of ICAM-1 on target cells facilitates the polarization of MHC-I at the CTL-target cell interface, augmenting presentation of cognate peptide-MHC (pMHC) complexes to CTLs. We propose that ICAM-1-MHC-I association on the cell membrane is a mechanism that enhances the linkage between antigen recognition and early immunological synapse formation.

Antigen Presentation↗
PubMed2004

Migration of antigen-specific T cells away from CXCR4-binding human immunodeficiency virus type 1 gp120.

Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.

Animals↗
PubMed2004

Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection.

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8(+) T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes.

Amino Acid Sequence↗
PubMed2004

Comprehensive analysis of human immunodeficiency virus type 1-specific CD4 responses reveals marked immunodominance of gag and nef and the presence of broadly recognized peptides.

Increasing evidence suggests that human immunodeficiency virus type 1 (HIV-1)-specific CD4 T-cell responses contribute to effective immune control of HIV-1 infection. However, the breadths and specificities of these responses have not been defined. We screened fresh CD8-depleted peripheral blood mononuclear cells (PBMC) from 36 subjects at different stages of HIV-1 infection for virus-specific CD4 responses by gamma interferon enzyme-linked immunospot assay, using 410 overlapping peptides spanning all HIV-1 proteins (based on the clade B consensus sequence). HIV-1-specific CD4 responses were identified in 30 of the 36 individuals studied, with the strongest and broadest responses detected in persons treated in acute infection who underwent treatment interruption. In individuals with identified responses, the total number of recognized HIV-1 peptides ranged from 1 to 36 (median, 7) and the total magnitude of responses ranged from 80 to >14,600 (median, 990) spot-forming cells/10(6) CD8-depleted PBMC. Neither the total magnitude nor the number of responses correlated with viremia. The most frequent and robust responses were directed against epitopes within the Gag and Nef proteins. Peptides targeted by >/=25% of individuals were then tested for binding to a panel of common HLA-DR molecules. All bound broadly to at least four of the eight alleles tested, and two bound to all of the HLA-DR molecules studied. Fine mapping and HLA restriction of the responses against four of these peptides showed a combination of clustering of epitopes and promiscuous presentation of the same epitopes by different HLA class II alleles. These findings have implications for the design of immunotherapeutic strategies and for testing candidate HIV vaccines.

Adult↗
PubMed2004

The immune response to AIDS virus infection: good, bad, or both?

A potent antigen-specific T cell response to HIV infection can contribute to the control of viral replication and is therefore beneficial to the host. However, HIV-mediated increases in generalized T cell activation also appear to accelerate both viral replication and CD4+ T cell depletion. A new study in the JCI attempts to experimentally distinguish the beneficial versus harmful aspects of this immune response.

Adjuvants, Immunologic↗