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Biomedical subjects

W W Kwok

Publications and source records attributed to W W Kwok.

At least 19 recordsLinked to original sources
PubMed2001

Generation of high quantities of viral and tumor-specific human CD4+ and CD8+ T-cell clones using peptide pulsed mature dendritic cells.

CD4+ and CD8+ T cells are key components of immune response against tumors and viruses. Many techniques have been used to clone and expand these cells in vitro for purposes of immunotherapy. Here, we describe an improved method to obtain large quantities of tumor and virus-specific human CD4+ and CD8+ T-cell clones. T cells derived from peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detected by IFN-gamma intracellular staining and were cloned by limiting dilution. Specific polyclonal T-cell populations were derived for all epitopes presented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure, it was possible to obtain from 10(9) to 10(12) of each clone. Furthermore, clones could be maintained in culture in the presence of IL-2 for at least 1 month without losing their antigen-specific reactivity (e.g. cytokine secretion, cytolytic activity and proliferation). Importantly, a majority of the CD8+ T-cell clones recognized endogenously processed antigens. This method is of value for the purposes of adoptive anti-virus or anti-tumor immunotherapy.

Amino Acid Sequence↗
PubMed2001

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.

Animals↗
PubMed2001

Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.

T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.

CD4-Positive T-Lymphocytes↗
PubMed2001

CD8 CTL from genital herpes simplex lesions: recognition of viral tegument and immediate early proteins and lysis of infected cutaneous cells.

HSV-2 causes chronic infections. CD8 CTL may play several protective roles, and stimulation of a CD8 response is a rational element of vaccine design for this pathogen. The viral Ags recognized by CD8 T cells are largely unknown. It has been hypothesized that HSV inhibition of TAP may favor recognition of virion input proteins or viral immediate early proteins. We tested this prediction using HSV-specific CD8 CTL clones obtained from genital HSV-2 lesions. Drug and replication block experiments were consistent with specificity for the above-named classes of viral proteins. Fine specificity was determined by expression cloning using molecular libraries of viral DNA, and peptide epitopes recognized at nanomolar concentrations were identified. Three of four clones recognized the viral tegument proteins encoded by genes UL47 and UL49. These proteins are transferred into the cytoplasm on virus entry. Processing of the tegument Ag-derived epitopes was TAP dependent. The tegument-specific CTL were able to lyse HLA class I-appropriate fibroblasts after short times of infection. Lysis of keratinocytes required longer infection and pretreatment with IFN-gamma. Another clone recognized an immediate early protein, ICP0. Lymphocytes specific for these lesion-defined epitopes could be reactivated from the PBMC of additional subjects. These data are consistent with an influence of HSV immune evasion genes upon the selection of proteins recognized by CD8 CTL in lesions. Tegument proteins, identified for the first time as Ags recognized by HSV-specific CD8 CTL, are rational candidate vaccine compounds.

ATP-Binding Cassette Transporters↗
PubMed2001

Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset.

Antigen-specific T cells acquire a distinctive phenotype during activation, with characteristic acquisition of surface markers and patterns of gene expression. Early after antigen stimulation, CD4(+) T lymphocytes increase their surface density of the CD4 marker, a trait which has been used to identify antigen-activated cells. The recent development of MHC tetramer technologies has greatly improved the ability to detect HLA class I-restricted T cells specific for known antigen epitopes. We have recently extended these studies to human class II-restricted CD4(+) T cell responses and now describe antigen-specific T cell responses from human peripheral blood in which elevated CD4 expression levels in human T cells following antigen stimulation identify the activated and proliferating subset of cells. The CD4(high) population is substantially enriched in epitope-specific cells identified by class II tetramer staining and almost all tetramer-positive cells are contained within the CD4(high) population. T cell clones derived from the tetramer-positive, CD4(high) population demonstrate antigen specificity and maintain tetramer staining, while the substantial number of CD4(high) cells which fail to stain with tetramer appear to proliferate as a result of bystander activation. Epitope-specific components of a polyclonal immune response are directly visualized and quantitated by tetramer detection, providing a direct measure of the heterogeneity of the human immune response.

CD4 Antigens↗
PubMed2001

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin.

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

Adolescent↗
PubMed2000

Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

Amino Acid Sequence↗
PubMed2000

Beta 57-Asp plays an essential role in the unique SDS stability of HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC allele associated with protection from insulin-dependent diabetes mellitus.

Studies of the stability of HLA-DQ have revealed a correlation between SDS stability of MHC class II alphabeta dimers and insulin-dependent diabetes mellitus (IDDM) susceptibility. The MHC class II alphabeta dimer encoded by HLA-DQA1*0102/DQB1*0602 (DQ0602), which is a dominant protective allele in IDDM, exhibits the greatest SDS stability among HLA-DQ molecules in EBV-transformed B-lymphoblastoid cells and PBLs. DQ0602 is also uniquely SDS stable in the HLA-DM-deficient cell line, BLS-1. We addressed the molecular mechanism of the stability of DQ0602 in BLS-1. A panel of mutants based on the polymorphic differences between HLA-DQA1*0102/DQB1*0602 and HLA-DQA1*0102/DQB1*0604 were generated and expressed in BLS-1. An Asp at beta57 was found to be critical for SDS stability, whereas Tyr at beta30, Gly at beta70, and Ala at beta86 played secondary roles. Furthermore, the level of class II-associated invariant chain peptide bound to HLA-DQ did not correlate with SDS stability, suggesting that class II-associated invariant chain peptide does not play a direct role in the unique SDS stability of DQ0602. These results support a role for DQB1 codon 57 in HLA-DQ alphabeta dimer stability and IDDM susceptibility.

Alleles↗
PubMed2000

HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals: direct detection of immunodominant antigen-responsive cells.

Ag-specific CD4+ T cells are present in peripheral blood in low frequency, where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases. MHC tetramers, which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells, provide a novel approach for the detection and characterization of such rare cells. In this study, we utilized this technology to identify HLA DQ-restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection. Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers. VP16 Ag-specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals, representing two predominant epitope specificities. Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide, T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide.

Amino Acid Sequence↗
PubMed2000

Increased frequency of HLA class II alleles DRB1*0301 and DQB1*0201 in Lambert-Eaton myasthenic syndrome without associated cancer.

Lambert-Eaton myasthenic syndrome (LEMS) is a rare autoimmune neuromuscular disorder characterized by pathogenic autoantibodies directed against the presynaptic voltage-gated calcium channels (VGCC), resulting in a clinical syndrome of proximal muscular weakness and autonomic dysfunction. Sixty percent of LEMS cases are associated with cancer, most commonly small cell carcinoma of the lung. In the 40% of LEMS patients without carcinoma, the stimulus for the production of VGCC autoantibodies is unknown; however, these LEMS patients have multiple other organ-specific autoantibodies. To investigate the autoimmune basis of noncancer associated LEMS (NCA-LEMS), high resolution typing of major histocompatibility loci was performed in 23 patients with NCA-LEMS. NCA-LEMS was strongly associated with DRB1*0301 (p<0.0001) and DQB1*0201 (p<0.0001), suggesting that NCA-LEMS is an autoimmune disorder associated with the DR3-DQ2 extended haplotype.

Adolescent↗
PubMed2000

Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans.

Herpes stromal keratitis (HSK) is a prevalent and frequently vision-threatening disease associated with herpes simplex virus type 1 (HSV-1) infection. In mice, HSK progression occurs after viral clearance and requires T cells and neutrophils. One model implicates Th1-like CD4 T cells with cross-reactivity between the HSV-1 protein UL6 and a corneal autoantigen. HSK can be prevented by establishing specific immunological tolerance. However, HSK can also occur in T-cell receptor-transgenic X SCID mice lacking HSV-specific T cells. To study the pathogenesis of HSK in the natural host species, we measured local HSV-specific T-cell responses in HSK corneas removed at transplant surgery (n = 5) or control corneas (n = 2). HSV-1 DNA was detected by PCR in two specimens. HSV-specific CD4 T cells were enriched in three of the five HSK specimens and were not detectable in the control specimens. Reactivity with peptide epitopes within the tegument proteins UL21 and UL49 was documented. Responses to HSV-1 UL6 were not detected. Diverse HLA DR and DP alleles restricted these local responses. Most clones secreted gamma interferon, but not interleukin-5, in response to antigen. HSV-specific CD8 cells were also recovered. Some clones had cytotoxic-T-lymphocyte activity. The diverse specificities and HLA-restricting alleles of local virus-specific T cells in HSK are consistent with their contribution to HSK by a proinflammatory effect.

Adult↗
PubMed2000

Epstein-Barr virus entry utilizing HLA-DP or HLA-DQ as a coreceptor.

Epstein-Barr virus (EBV) glycoprotein gp350/gp220 association with cellular CD21 facilitates virion attachment to B lymphocytes. Membrane fusion requires the additional interaction between virion gp42 and cellular HLA-DR. This binding is thought to catalyze membrane fusion through a further association with the gp85-gp25 (gH-gL) complex. Cell lines expressing CD21 but lacking expression of HLA class II molecules are resistant to infection by a recombinant EBV expressing enhanced green fluorescent protein. Surface expression of HLA-DR, HLA-DP, or HLA-DQ confers susceptibility to EBV infection on resistant cells that express CD21. Therefore, HLA-DP or HLA-DQ can substitute for HLA-DR and serve as a coreceptor in EBV entry.

Cell Line↗
PubMed1999

MHC-peptide ligand interactions establish a functional threshold for antigen-specific T cell recognition.

Antigen-specific T cell recognition is dependent on the functional density of the TCR-ligand, which consists of specific MHC molecules and a specifically bound peptide. We have examined the influence of the affinity and concentration of exogenous peptide and the density of specific MHC molecules on the proliferation of a CD4+, DQA1*0501/DQB1*0201 (DQ2.1)-restricted, HSV-2-specific T cell clone. Using antigen peptide analogs with different mutations of known DQ2-anchor residues, T cell response was reduced in an peptide-affinity and - concentration specific manner. The decrease using weaker binding peptides was gradual as stimulation with a peptide with intermediate affinity yielded intermediate T cell proliferation and the poorest binding peptide induced an even weaker T cell response. MHC class II density on the APC was modified using DQ2 homo- and heterozygous B-LCLs as APCs, however this variation of MHC concentration had no effect on T cell proliferation. We interpret this as a reflection of a low threshold for activation of the T cell clone, in which peptide-MHC avidity is the over-riding determinant of the strength of ligand signal.

Alleles↗
PubMed1999

Peptide binding affinity and pH variation establish functional thresholds for activation of HLA-DQ-restricted T cell recognition.

Peptides derived from the HSV-2 VP16 protein were utilized for studies of peptide binding to DQ0302 molecules and T cell activation at both neutral and acidic pH. The native peptide VP16 430-444 contains an Asp at position 442, binds to DQ0302 strongly, with a Kd value of 50nM at acidic pH and very weakly, with a Kd value of greater than 10 microM at neutral pH. A truncated version of 430-444, i.e., VP16 433-442, binds with an affinity 10-fold lower compared to 430-444 at acidic pH, and binding at neutral pH was barely detectable. The homologous peptide 430-444,442A has an Asp to Ala substitution at position 442 and binds to DQ0302 with a Kd similar to 433-442. The short truncated analog 433-442A binds very poorly at both acidic and neutral pH. Both the wild type 430-444 and 433-442 peptides stimulated a HSV-specific T cell clone after a brief incubation with antigen presenting cells (APC) expressing DQ0302 at acidic pH. Much higher concentrations of wild type peptides were needed to activate T cells at neutral pH. In contrast, APC pulsed with Ala-substituted peptides 430-444,442A or 433-442A at neutral pH failed to stimulate the T cell clone, while APC pulsed at acidic pH and subsequently washed led to successful T cell activation. The Ala-substituted peptide was recognized by the T cell clone at neutral pH only when it was present in the APC culture throughout the stimulation process. While the MHC-peptide complexes formed with the native peptide are stable, complexes formed with the Ala-substituted peptide had a functional t1/2 of less than 4 hr at neutral pH.

Amino Acid Sequence↗
PubMed1999

Impaired binding of a DQ2 and DQ8-binding HSV VP16 peptide to a DQA1*0501/DQB1*0302 trans class II heterodimer.

DQalpha and DQbeta trans heterodimeric HLA-DQ molecules form in individuals heterozygous for the DQ2 and DQ8 specificities. Unique functions and disease associations have been postulated for such trans-dimers, which may be different from cis-encoded DQ molecules encoded by the corresponding haplotypes. We analyzed the ability of the trans-dimer encoded by HLA-DQA1*0501/DQB1*0302 to bind a peptide antigen which interacts with DQ molecules encoded by both parental haplotypes. Markedly impaired binding was observed, consistent with both the use of different anchor residues and with changes in levels of DQ cis-dimer availability for peptide binding interactions.

Animals↗
PubMed1999

MHC class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen.

Antigen-specific T helper cells present in peripheral blood at very low frequencies are capable of rapid clonal expansion during antigenic challenge. The exquisite specificity of this response provides for activation and expansion of a very select cohort of T cells, a feature we have used to directly identify and quantify human epitope-specific T helper cells from peripheral blood. Soluble tetramerized class II MHC molecules, loaded with an immunodominant peptide from hemagglutinin (HA) and labeled with fluorescent dyes, were constructed and used to directly identify antigen-specific T cells from influenza-immune individuals. After 7 days of proliferation in response to stimulation by HA peptide or whole influenza vaccine, cells staining positive with the HA tetramer had undergone between 6 and 9 divisions and were CD3(+), CD4(+), CD25(+), and CD8(-), characteristic of activated T helper cells responding to antigen. The HA epitope-specific component of the complex response to whole influenza vaccine represented a major subset of proliferating T helper cells. Soluble class II tetramers allow a direct approach for the analysis of immunodominant antigenic specificities. The identification of antigen-specific T helper cells in the peripheral blood provides a means for tracking the immune response against infectious agents and in autoimmune disease. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Antigens, Viral↗
PubMed1998

Exceptional stability of the HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus.

HLA-DQ alleles are closely associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM) but the immunologic mechanisms involved are not understood. Structural studies of the IDDM-susceptible allele, HLA-DQA1*0301/DQB1*0302, have classified it as a relatively unstable dimer, particularly at neutral pH. This is reminiscent of studies in the nonobese diabetic mouse, in which I-A(g7) is relatively unstable, in contrast to other murine I-A alleles, suggesting a correlation between unstable MHC class II molecules and IDDM susceptibility. We have addressed this question by analysis of dimer stability patterns among various HLA-DQ molecules. In EBV-transformed B-lymphoblastoid cell lines and PBL, the protein encoded by the IDDM-protective allele HLA-DQA1*0102/DQB1*0602 was the most SDS stable when compared with other HLA-DQ molecules, including HLA-DQA1*0102/DQB1*0604, a closely related allele that is not associated with protection from IDDM. Expression of six different HLA-DQ allelic proteins and three different HLA-DR allelic proteins in the bare lymphocyte syndrome cell line, BLS-1, revealed that HLA-DQA1*0102/DQB1*0602 is SDS stable even in the absence of HLA-DM, while other HLA class II molecules are not. These results suggest that the molecular property of HLA-DQ measured by resistance to denaturation of the alphabeta dimer in SDS may play a role in IDDM protection.

Alleles↗