William W. Kwok

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.

Molecular Basis for HLA-DQ Associations With IDDM

Autoimmune diabetes is the clinical end point for a sequential cascade of immunologic events that occur in a genetically susceptible individual. Structural and functional analysis of the HLA class II susceptibility genes in IDDM suggests likely molecular mechanisms for several of the key steps in this cascade of autoimmune events. We outline a pathway in which the HLA-DQ genes associated with IDDM bias the immunologic repertoire toward autoimmune specificities, creating an autoimmune-prone individual, followed by amplification and triggering events that promote subsequent immune activation. There are several direct links between genetics and autoimmune disease in this pathway: the developmental maturation of T-cells in a genetically susceptible individual occurs through molecular interactions between the T-cell receptor and the HLA-peptide complex. Selection of T-cells with receptors likely to contribute to autoreactivity may preferentially occur in the context of specific HLA-DQ alleles that are diabetes prone, because of inefficiencies in the peptide-MHC structural interactions of these molecules. Subsequent activation of these T-cells in the context of recognizing islet-associated antigens can trigger a poorly regulated immune response that results in progressive islet destruction. These subsequent diabetes-specific events are also directed by specific HLA genes, most prominently by the binding of specific antigenic peptides by the disease-associated HLA molecules. In this sequential cascade, opportunities for environmental influences and modulation by non-HLA genes are identified that likely act in concert with the predominant genetic susceptibility contributed by the HLA molecules themselves. Clarification of the steps in this pathway extends our understanding of the prevailing role of HLA genes in IDDM pathogenesis and suggests opportunities to intervene at discrete initiating, disease-promoting, or regulatory steps in IDDM development.

HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.

High Level of PD-1 Expression on Hepatitis C Virus (HCV)-Specific CD8+ and CD4+ T Cells during Acute HCV Infection, Irrespective of Clinical Outcome

ABSTRACT We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.

Antibiotic-refractory Lyme arthritis is associated with HLA-DR molecules that bind a Borrelia burgdorferi peptide

An association has previously been shown between antibiotic-refractory Lyme arthritis, the human histocompatibility leukocyte antigen (HLA)–DR4 molecule, and T cell recognition of an epitope of Borrelia burgdorferi outer-surface protein A (OspA163–175). We studied the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes in 121 patients with antibiotic-refractory or antibiotic-responsive Lyme arthritis and correlated these frequencies with in vitro binding of the OspA163–175 peptide to 14 DRB molecules. Among the 121 patients, the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes were similar to those in control subjects. However, when stratified by antibiotic response, the frequencies of DRB1 alleles in the 71 patients with antibiotic-refractory arthritis differed significantly from those in the 50 antibiotic-responsive patients (log likelihood test, P = 0.006; exact test, P = 0.008; effect size, Wn = 0.38). 7 of the 14 DRB molecules (DRB1*0401, 0101, 0404, 0405, DRB5*0101, DRB1*0402, and 0102) showed strong to weak binding of OspA163–175, whereas the other seven showed negligible or no binding of the peptide. Altogether, 79% of the antibiotic-refractory patients had at least one of the seven known OspA peptide–binding DR molecules compared with 46% of the antibiotic-responsive patients (odds ratio = 4.4; P < 0.001). We conclude that binding of a single spirochetal peptide to certain DRB molecules is a marker for antibiotic-refractory Lyme arthritis and might play a role in the pathogenesis of the disease.

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.

Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence

Early after symptom onset, HCV-specific CD4+ T cell responses are primed and detectable in patients regardless of clinical outcome, but without early antiviral therapy these T cells become exhausted or deleted in chronically infected patients.

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-γ. 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.

Healthy Human Subjects Have CD4+ T Cells Directed against H5N1 Influenza Virus

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4+ T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4+ T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4+ T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-γ, TNF-α, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.

Differentiation stage determines pathologic and protective allergen-specific CD4+ T cell outcomes during specific immunotherapy

BACKGROUND The main obstacle to elucidating the role of CD4(+) T cells in allergen-specific immunotherapy (SIT) has been the absence of an adequately sensitive approach to directly characterize rare allergen-specific T cells without introducing substantial phenotypic modifications by means of in vitro amplification. OBJECTIVE We sought to monitor, in physiological conditions, the allergen-specific CD4(+) T cells generated during natural pollen exposure and during allergy vaccination. METHODS Alder pollen allergy was used as a model for studying seasonal allergies. Allergen-specific CD4(+) T cells were tracked and characterized in 12 subjects with alder pollen allergy, 6 nonallergic subjects, and 9 allergy vaccine-treated subjects by using peptide-MHC class II tetramers. RESULTS Allergen-specific CD4(+) T cells were detected in all of the subjects with alder pollen allergy and nonallergic subjects tested. Pathogenic responses--chemoattractant receptor homologous molecule expressed on T(H)2 lymphocytes (CRTH2) expression and T(H)2 cytokine production--are specifically associated with terminally differentiated (CD27(-)) allergen-specific CD4(+) T cells, which dominate in allergic subjects but are absent in nonallergic subjects. In contrast, CD27(+) allergen-specific CD4(+) T cells are present at low frequencies in both allergic and nonallergic subjects and reflect classical features of the protective immune response with high expression of IL-10 and IFN-γ. Restoration of a protective response during SIT appears to be due to the preferential deletion of pathogenic (CD27(-)) allergen-specific CD4(+) T cells accompanied by IL-10 induction in surviving CD27(+) allergen-specific CD4(+) T cells. CONCLUSIONS Differentiation stage divides allergen-specific CD4(+) T cells into 2 distinct subpopulations with unique functional properties and different fates during SIT.