Junbao Yang

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.

Islet-Specific Glucose-6-Phosphatase Catalytic Subunit-Related Protein-Reactive CD4+ T Cells in Human Subjects

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is recognized as a major autoantigen for autoimmune type 1 diabetes (T1D) in the NOD mouse model. This study was undertaken to examine CD4+ T cell responses toward IGRP in human subjects. The tetramer-guided epitope mapping approach was used to identify IGRP-specific CD4+ T cell epitopes. IGRP23–35 and IGRP247–259 were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP13–25 and IGRP226–238 were identified as DRA1*0101/DRB1*0301-restricted epitopes. IGRP-specific tetramers were used to evaluate the prevalence of IGRP-reactive T cells in healthy and T1D subjects. More than 80% of subjects with either DRB1*0401 or DRB1*0301 haplotype have IGRP-specific CD4+ T cell responses for at least one IGRP epitope. IGRP-specific T cells from both healthy and T1D groups produce both γ-IFN and IL-10. DRA1*0101/DRB1*0401 IGRP247–259-restricted T cells also show cross-reactivity to an epitope derived from liver/kidney glucose-6-phosphatase. The detection of IGRP-reactive T cells in both type 1 diabetic subjects and healthy subjects and recent reports of other autoreactive T cells detected in healthy subjects underscore the prevalence of potentially autoreactive T cells in the peripheral immune system of the general population.

CD4+ T cells from type 1 diabetic and healthy subjects exhibit different thresholds of activation to a naturally processed proinsulin epitope.

Recent studies suggest that insulin is a primary autoantigen for type 1 diabetes. Several studies have identified preproinsulin (PPI) 76-90 as an immunodominant CD4+ T cell epitope. We developed a class II tetramer reagent using a modified PPI peptide with a lysine to serine substitution at position 88 (PPI 78-90(88S)) that has high binding affinity to DRA1*0101/DRB1*0401 (DR0401). Using this tetramer, positive responses were observed from both DR0401 healthy and type 1 diabetic subjects when T cells were stimulated with the PPI 78-90(88S) peptide. Seventy percent of these T cells proliferated in response to both the wild type PPI 76-90 and PPI 78-90(88S) peptides. However, when T cells were stimulated with wild type peptide and assayed with DR0401/PPI 78-90(88S), positive responses were only detected in the diabetic group but not in healthy subjects. When highly purified CD4+CD25-CD45RA+ T cells were stimulated with PPI 78-90(88S) peptide in the absence of antigen-presenting cells, T cells from the diabetic group were able to respond to peptide stimulation, while T cells from healthy subjects were not. These data suggest that T cells from type 1 diabetic subjects have a lower threshold of activation in response to PPI peptide stimulation as compared to healthy subjects.

Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire

The frequency of epitope-specific naive CD4+ T cells in humans has not been extensively examined. In this study, a systematic approach was used to examine the frequency of CD4+ T cells that recognize the protective Ag of Bacillus anthracis in both anthrax vaccine-adsorbed vaccinees and nonvaccinees with HLA-DRB1*01:01 haplotypes. Three epitopes were identified that had distinct degrees of immunodominance in subjects that had received the vaccine. Average naive precursor frequencies of T cells specific for these different epitopes in the human repertoire ranged from 0.2 to 10 per million naive CD4+ T cells, which is comparable to precursor frequencies observed in the murine repertoire. Frequencies of protective Ag-specific T cells were two orders of magnitude higher in immunized subjects than in nonvaccinees. The frequencies of epitope-specific memory CD4+ T cells in vaccinees were directly correlated with the frequencies of precursors in the naive repertoire. At the level of TCR usage, at least one preferred Vβ in the naive repertoire was present in the memory repertoire. These findings implicate naive frequencies as a crucial factor in shaping the epitope specificity of memory CD4+ T cell responses.

Autoreactive T cells specific for insulin B:11-23 recognize a low-affinity peptide register in human subjects with autoimmune diabetes

Significance The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains unclear. Studies in the nonobese diabetic mouse demonstrate recognition of a crucial insulin epitope presented in a weakly bound register. This work details a direct study of responses to this insulin B-chain peptide in humans. Responses were readily detected in subjects with type 1 diabetes. Furthermore, T-cell clones were shown to recognize the peptide presented in a weakly bound alternative register. These findings confirm the relevance of immune recognition of this segment of the insulin B-chain in human disease and highlight a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms. Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IAg7 in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a low-affinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the low-affinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.

Increased Frequencies of Myelin Oligodendrocyte Glycoprotein/MHC Class II-Binding CD4 Cells in Patients with Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG97–109(107E-S) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG97–109-reactive CD4+ T cells, isolated with DRB1*0401/MOG97–109 tetramers, and after a short-term culture of PMBCs with MOG97–109 peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG97–109(107E-S) tetramer+ cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG97–109-reactive T cells is greater in patients with MS as compared with healthy controls.

The Anthrax Vaccine Adsorbed Vaccine Generates Protective Antigen (PA)-Specific CD4+ T Cells with a Phenotype Distinct from That of Naïve PA T Cells

ABSTRACT Cellular immune responses against protective antigen (PA) of Bacillus anthracis in subjects that received the anthrax vaccine adsorbed (AVA) vaccine were examined. Multiple CD4+ T-cell epitopes within PA were identified by using tetramer-guided epitope mapping. PA-reactive CD4+ T cells with a CD45RA− phenotype were also detected by direct ex vivo staining of peripheral blood mononuclear cells (PBMC) with PA-specific tetramers. Surprisingly, PA-specific T cells were also detected in PBMC of nonvaccinees after a single cycle of in vitro PA stimulation. However, PA-reactive CD4+ T cells in nonvaccinees occurred at lower frequencies than those in vaccinees. The majority of PA-reactive T cells from nonvaccinees were CD45RA+ and exhibited a Th0/Th1 cytokine profile. In contrast, phenotyping and cytokine profile analyses of PA-reactive CD4+ T cells from vaccinees indicated that vaccination leads to commitment of PA-reactive T cells to a Th2 lineage, including generation of PA-specific, pre-Th2 central memory T cells. These results demonstrate that the current AVA vaccine is effective in skewing the development of PA CD4+ T cells to the Th2 lineage. The data also demonstrated the feasibility of using class II tetramers to analyze CD4+ cell responses and lineage development after vaccination.

CD4+ T cells recognize unique and conserved 2009 H1N1 influenza hemagglutinin epitopes after natural infection and vaccination

Influenza A/California/4/2009 (H1N1/09) is a recently emerged influenza virus capable of causing serious illness or death in otherwise healthy individuals. Serious outcomes were most common in young adults and children, suggesting that pre-existing heterologous immunity may influence the severity of infection. Using tetramers, we identified CD4(+) T-cell epitopes within H1N1/09 hemagglutinin (HA) that share extensive homology with seasonal influenza and epitopes that are unique to H1N1/09 HA. Ex vivo tetramer staining revealed that T cells specific for conserved epitopes were detectable within the memory compartment, whereas T cells specific for unique epitopes were naive and infrequent prior to infection or vaccination. Following infection, the frequencies of T cells specific for unique epitopes were 11-fold higher, reaching levels comparable to those of T cells specific for immunodominant epitopes. In contrast, the frequencies of T cells specific for conserved epitopes were only 2- to 3-fold higher following infection. In general, H1HA-reactive T cells exhibited a memory phenotype, expressed CXCR3 and secreted IFN-γ, indicating a predominantly Th1-polarized response. A similar Th1 response was seen in vaccinated subjects, but the expansion of T cells specific for HA epitopes was comparatively modest after vaccination. Our findings indicate that CD4(+) T cells recognize both strain-specific and conserved epitopes within the influenza HA protein and suggest that naive T cells specific for HA epitopes undergo significant expansion, whereas memory T cells specific for the conserved epitopes undergo more restrained expansion.

CD4+ T cells recognize diverse epitopes within GAD65: implications for repertoire development and diabetes monitoring.

Type 1 diabetes is associated with T‐cell responses to β‐cell antigens such as GAD65. Single T‐cell epitopes have been investigated for immune monitoring with some success, but multiple epitopes may be required to fully characterize responses in all subjects. We used a systematic approach to examine the diversity of the GAD65‐specific T‐cell repertoire in subjects with DRB1*04:01 haplotypes. Using class II tetramers, we observed responses to 15 GAD65 epitopes, including five novel epitopes. The majority were confirmed to be processed and presented. Upon stimulation with peptides, GAD‐specific responses were equally broad in subjects with diabetes and healthy controls in the presence or absence of CD25+ T cells, suggesting that a susceptible HLA is sufficient to generate a potentially autoreactive repertoire. Without depleting CD25+ cells, GAD113–132 and GAD265–284 responses were significantly stronger in subjects with diabetes. Although nearly every individual responded to at least one GAD65 epitope, most were seen in less than half of the subjects tested, suggesting that multiple epitopes are recommended for immune monitoring.

Expression of HLA-DP0401 Molecules for Identification of DP0401 Restricted Antigen Specific T Cells

Human histocompatibility leukocyte antigen (HLA)-DPA1*0103/DPB1*0401 (DP0401) is the most common HLA class II molecule and is present in approximately 45% of the Caucasian population. In this study, soluble HLA-DP0401 molecules were expressed as “empty’’ class II molecules in insect cells. Utilizing these soluble DP molecules and the Tetramer Guided Epitope Mapping (TGEM) approach, the influenza A Puerto Rico/8/34 matrix protein (MP) derived peptide MP41−60 VLMEWLKTRPILSPLTKGIL and the Clostridium tetani Tetanus Toxin (TT) derived peptide TT634−653 DKISDVSTIVPYIGPALNIV were identified as the DP0401 restricted MP and TT epitopes, respectively. In addition, T cells specific for the cancer testis antigen NY-ESO-1 and the breast/ovarian cancer over-expressing antigen Her-2/neu were detected in DP0401 subjects by DP0401 tetramers. The availability of HLA-DP0401 tetramers should facilitate the study of DP restricted T cell responses.