Eddie A. James

Memory T cells in latent Mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a CXCR3+CCR6+ Th1 subset.

An understanding of the immunological footprint of Mycobacterium tuberculosis (MTB) CD4 T cell recognition is still incomplete. Here we report that human Th1 cells specific for MTB are largely contained in a CXCR3+CCR6+ memory subset and highly focused on three broadly immunodominant antigenic islands, all related to bacterial secretion systems. Our results refute the notion that secreted antigens act as a decoy, since both secreted proteins and proteins comprising the secretion system itself are targeted by a fully functional T cell response. In addition, several novel T cell antigens were identified which can be of potential diagnostic use, or as vaccine antigens. These results underline the power of a truly unbiased, genome-wide, analysis of CD4 MTB recognition based on the combined use of epitope predictions, high throughput ELISPOT, and T cell libraries using PBMCs from individuals latently infected with MTB.

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.

Assessment of Seasonal Influenza A Virus-Specific CD4 T-Cell Responses to 2009 Pandemic H1N1 Swine-Origin Influenza A Virus

ABSTRACT Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.

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.

Specific immunotherapy modifies allergen-specific CD4+ T-cell responses in an epitope-dependent manner

BACKGROUND Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T-cell epitope level is critical for the design of new allergy vaccine strategies. OBJECTIVE We sought to characterize allergen-specific T-cell responses linked with allergy or peripheral tolerance and to determine how CD4(+) T-cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy. METHODS Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy, and phenotype of the DR04:01-restricted TGP-specific T-cell responses in 10 subjects with grass pollen allergy, 5 nonatopic subjects, and 6 allergy vaccine-treated subjects was determined by using an ex vivo peptide-MHC class II tetramer approach. RESULTS CD4(+) T cells in allergic subjects are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. Allergen-specific immunotherapy was associated with preferential deletion of allergen-specific TH2 cells and without a significant change in the frequency of TH1/TR1 cells. CONCLUSIONS Preferential allergen-specific TH2 cell deletion after repeated high-dose antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy.

Citrulline‐Specific Th1 Cells Are Increased in Rheumatoid Arthritis and Their Frequency Is Influenced by Disease Duration and Therapy

Rheumatoid arthritis (RA) is thought to be a T cell–mediated disease, based on its strong association with HLA class II alleles, clinical responsiveness to T cell–directed therapies, and the presence of CD4+ T cells in rheumatoid joints. The presence of anti–citrullinated protein antibodies (ACPAs) in RA serum and the association of these antibodies with HLA–DR4 alleles implicate citrulline‐specific autoreactive T cells in the development and progression of RA. The goal of this study was to determine the characteristics and specificity of autoreactive T cell responses in RA.

Dissecting Mechanisms of Immunodominance to the Common Tuberculosis Antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ)

Diagnosis of tuberculosis often relies on the ex vivo IFN-γ release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB. However, understanding of the immunological mechanisms underlying their diagnostic use is still incomplete. Accordingly, we investigated T cell responses for the TB Ags included in the these assays and other commonly studied Ags: early secreted antigenic target 6 kDa, culture filtrate protein 10 kDa, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these Ags. We found striking variations in prevalence and magnitude of ex vivo reactivity, with culture filtrate protein 10 kDa being most dominant, followed by early secreted antigenic target 6 kDa and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence in a given epitope. In some cases, the identified epitopes were restricted by a single specific common HLA molecule (selective restriction), whereas in other cases, promiscuous restriction of the same epitope by multiple HLA molecules was apparent. Definition of the specific restricting HLA allowed to produce tetrameric reagents and showed that epitope-specific T cells recognizing either selectively or promiscuously restricted epitopes were predominantly T effector memory. In conclusion, these results highlight the feasibility of more clearly defined TB diagnostic reagent.

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood–derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2–DQ8 heterodimer–restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.