Erik Wambre

Assessment of Bet v 1-Specific CD4 + T Cell Responses in Allergic and Nonallergic Individuals Using MHC Class II Peptide Tetramers

In this study, we used HLA-DRB1*0101, DRB1*0401, and DRB1*1501 peptide tetramers combined with cytokine surface capture assays to characterize CD4+ T cell responses against the immunodominant T cell epitope (peptide 141–155) from the major birch pollen allergen Bet v 1, in both healthy and allergic individuals. We could detect Bet v 1-specific T cells in the PBMC of 20 birch pollen allergic patients, but also in 9 of 9 healthy individuals tested. Analysis at a single-cell level revealed that allergen-specific CD4+ T cells from healthy individuals secrete IFN-γ and IL-10 in response to the allergen, whereas cells from allergic patients are bona fide Th2 cells (producing mostly IL-5, some IL-10, but no IFN-γ), as corroborated by patterns of cytokines produced by T cell clones. A fraction of Bet v 1-specific cells isolated from healthy, but not allergic, individuals also expresses CTLA-4, glucocorticoid-induced TNF receptor, and Foxp 3, indicating that they represent regulatory T cells. In this model of seasonal exposure to allergen, we also demonstrate the tremendous dynamics of T cell responses in both allergic and nonallergic individuals during the peak pollen season, with an expansion of Bet v 1-specific precursors from 10−6 to 10−3 among circulating CD4+ T lymphocytes. Allergy vaccines should be designed to recapitulate such naturally protective Th1/regulatory T cell responses observed in healthy individuals.

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.

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.

Direct ex vivo analysis of allergen-specific CD4+ T cells

To the Editor: CD4+ T cells play essential roles in allergic sensitization and late-phase reactions. Previous studies identified epitopes within allergens such as the cat allergen Fel d 1.1-5 However, in some cases, HLA restrictions were not clearly defined. Recent studies have applied tetramers to define HLA-restricted epitopes, studying subjects with cat and other allergies.4 In general, previous reports analyzed single, previously characterized epitopes and, with 1 exception,4 analyzed cells after in vitro expansion, which can alter cell phenotypes. Recent advances in MHC class II tetramer analysis enhance the efficiency of epitope identification and provide the sensitivity to examine phenotypes without in vitro expansion.6,7 We applied these methods to study allergen specific CD4+ T-cell responses in subjects with cat allergy. To identify novel T-cell epitopes within Fel d 1, the Tetramer-Guided Epitope Mapping (TGEM) approach was applied to multiple subjects with allergy recruited with informed consent from the Virginia Mason Medical Center Allergy Clinic and Benaroya Research Institute. Overlapping peptides corresponding to both chains of Fel d 1 were pooled and used to stimulate T-cell cultures as described.6 Each peptide pool was loaded into purified class II molecules to generate tetramers.8 After 14 days, cultured cells were stained with corresponding pooled peptide loaded tetramers. Positive wells were stained again using tetramers loaded with single peptides (see this article’s Fig E1 in the Online Repository at www.jacionline.org). Applying this approach, we identified novel Fel d 1 epitopes for 6 HLA types (Table I). Binding predictions9 and experiments using shorter peptides defined minimal epitopes. Responses to these Fel d 1 peptides were absent in subjects without allergy (Fig E1). Table I Fel d 1 T cell epitopes To determine frequencies for Fel d 1 specific T cells, we used a direct enrichment protocol, essentially as described by Day et al.7 For 9 subjects with allergy and 5 subjects without allergy, 20 to 30 million uncultured PBMCs were stained with phycoerythrin tetramers for 100 minutes. Cells were then stained with surface anti-bodies (anti-CD3 fluorescein isothiocyanate and anti-CD4 allophycocyanin from eBioscience, San Diego, Calif; anti-CD14 peridinin-chlorophyll-protein [PerCP] and anti-CD19 PerCP from BD Pharmingen, San Diego, Calif), incubated with anti-phycoerythrin magnetic beads (Miltenyi Biotec, Auburn, Calif), washed, and enriched by using a magnetic column (Miltenyi Biotec). Bound, phycoerythrin-labeled cells were stained with ViaProbe (BD Bioscience, San Jose, Calif), flushed, collected, and enumerated by flow cytometry (FACS Caliber cytometer, BD Bioscience; FlowJo software, Tree Star, Ashland, Ore). Frequencies were calculated using the following formula Frequency=n/N where n designates the number of tetramer positive cells in the bound fraction and N designates the total number of CD4+ T cells in the sample. Fel d 1–specific CD4+ T-cell frequencies in subjects with allergy ranged from 1 in 7,000 to 1 in 300,000 (Fig 1, A). Frequencies in subjects without allergy were barely detectable (Fig 1, B). Frequency measurements were reproducible in that frequencies for samples from the same subjects obtained at least 2 months apart were not significantly different (P = .495, paired t test with Welch correction). FIG 1 Ex vivo staining of Fel d 1 CD4+ T cells with tetramers. A, PBMCs from subjects with cat allergy were stained with phycoerythrin-labeled Fel d 1 tetramers (Tet) followed by the anti-phycoerythrin bead enrichment protocol. Each panel shows a representative ... To determine the phenotype of Fel d 1–specific T cells, parallel samples were analyzed by direct enrichment, using antibodies against surface markers of interest instead of anti-CD3. This allowed direct phenotyping with minimal manipulation. Pheno-typing was carried out for 5 subjects with allergy (representative results shown in Fig 1, C). A comparison of the phenotype of Fel d 1–specific T cells and total CD4+ T cells for all 5 subjects is summarized in Fig 1, D. As shown, more than 90% of Fel d 1–specific T cells were CD45RO, CD28, CD62L, and CCR4–positive. Most cells were CXCR3 and CCR6–negative but showed heterogeneous expression of CRTH2 and CCR7. In comparison with total CD4+ cells, a higher percentage of Fel d 1–specific T cells expressed CCR4 and CRTH2, and a lower percentage expressed CXCR3. As an internal control, we examined the phenotype of influenza A–specific T cells (not shown). Influenza-specific T cells were CD45RO and CD28–positive, but were CXCR3-positive, CCR4-negative, and CRTH2-negative. Thus, allergen-specific T cells show a distinct phenotype compared with influenza-specific T cells and the total CD4+ T-cell population. In support of these phenotyping experiments, we conducted cytokine analysis of directly enriched Fel d 1–specific T cells using Miltenyi capture reagents after stimulation with tetramer, anti-CD28 (10 μg/mL), and anti-CD49d (2 μg/mL) for 4 hours at 37°C. Although 10% to 50% of the tetramer-positive cells secreted IL-5, no IFN-γ was detected (Fig 1, E). IL-5 secretion was limited to the tetramer positive population. To examine additional cytokines, cultured cells were reactivated by using plate-bound tetramer and soluble anti-CD28. Supernatants were harvested after 48 hours and assayed for cytokine content by using the Meso Scale Sector Imager, Gaithersburg, Md. In addition to IL-5, Fel d 1–specific T cells produced TH2 cytokines such as IL-13 and IL-4 (not shown). Previous reviews and studies have highlighted the attractive features of using MHC class II tetramers to simultaneously determine T-cell frequencies and phenotypes. Since the arrival of class II tetramers, technical advances have increased the utility of these reagents. First, the availability of additional alleles enables the study of larger segments of the population. Second, TGEM facilitates rapid identification of T-cell epitopes.6 Third, enrichment methods allow direct ex vivo detection of antigen-specific T cells.7 This study applied these methods to visualize and characterize Fel d 1–specific T cells in subjects with allergy. We identified epitopes restricted by multiple HLA-DR alleles. We observed frequencies ranging from 1 in 7,000 to 1 in 300,000 CD4+ T cells in subjects with allergy. In subjects without allergy, allergen-specific T-cell frequencies were near back-ground levels. However, tetramers may be limited in their ability to detect low-affinity cells. As such, tetramer measurements may underestimate T-cell frequencies in some cases. In contrast with many published studies, we have directly examined the phenotype of allergen-specific T cells. Our observations indicated that nearly all Fel d 1–specific T cells (ex vivo) exhibit a memory phenotype. Fel d 1–specific T cells showed heterogeneous expression of CCR7, a marker thought to be upregulated on central memory and downregulated on effector memory cells. A previous report observed enriched CCR4 expression by allergen-specific T cells.4 Our results were more dramatic in that almost all allergen-specific T cells were CCR4-positive (25%-30% of total CD4+ T cells were CCR4-positive). Expression of CCR4 is notable because CCR4 is a TH2 marker that has been associated with trafficking to nonlymphoid sites, including the skin and airway mucosa. Thus, high levels of CCR4 expression may lead to rapid recruitment into relevant sites for allergic immune responses. In contrast with CCR4 expression, the prostaglandin D2 receptor CRTH2 (another TH2 marker)10 was expressed at variable frequencies (17%-88%) among subjects with cat allergy. Although variable, these frequencies were always higher than total CD4+ T cells. Regardless of CRTH2 expression level, tetramer-based cytokine assays indicated high levels of IL-5 and low levels of IFN-γ. These cytokine results reinforced the surface phenotype results. Cytokine levels were robust, which is typical of effector T cells. The absence of CXCR3 and CCR6 expression indicates that these peripheral cells do not belong to TH1 or TH17 lineages. However, these lineages could be present within specific tissues or during stages of allergy that were not reflected in our samples. In conclusion, we used class II tetramers to rapidly define HLA-restricted epitopes within allergens and characterize allergen-specific T cells ex vivo. The application of tetramers to study allergen-specific CD4+ T cells may facilitate the development of peptide biologics for allergy treatment. Future studies using tetramers after immunotherapy should clarify the underlying mechanisms for allergen-specific immune tolerance.

Characterization of CD4+ T cell subsets in allergy.

Allergen specific T(H)2 cells are a key component of allergic disease, but their characterization has been hindered by technical limitations and lack of epitope data. Knowledge about the factors that drive the differentiation of naïve T cells into allergy-promoting T(H)2 cells and the influence of allergen specific immunotherapy on the phenotype and function of allergen-specific T cells have also been limited. Recent advances indicate that innate and adaptive immune factors drive the development of diverse subsets of allergen-specific T cells. While allergen-specific T cells are present even in non-allergic subjects, highly differentiated T(H)2 cells are present only in allergic subjects and their disappearance correlates with successful immunotherapy. Therefore, elimination of pathogenic T(H)2 cells is an essential step in tolerance induction.

Distinct characteristics of seasonal (Bet v 1) vs. perennial (Der p 1/Der p 2) allergen‐specific CD4+ T cell responses

Cite this as: E. Wambre, M. Bonvalet,V. B. Bodo, B. Maille`re, G. Leclert,H. Moussu, E. Von Hofe, A. Louise,A.‐M. Balazuc, D. Ebo, C. Hoarau,G. Garcia, L. Van Overtvelt and P. Moingeon, Clinical & Experimental Allergy, 2011 (41) 192–203.

A phenotypically and functionally distinct human TH2 cell subpopulation is associated with allergic disorders

A unique T helper cell signature in allergic patients isolates the pathogenic cells and provides a target for disease intervention. Defining damaging cells Although T helper type 2 (TH2) cells provide necessary protection from certain types of pathogens, they are also implicated in allergy pathogenesis. Until now, methods to distinguish pathogenic cells that are reactive to allergens from the rest of the TH2 population were very limited. Wambre et al. characterized a population of memory TH2 cells, termed TH2A, that were only found in allergic individuals. They were able to do so without the use of antigen-specific tetramers. These cells decreased in patients that benefited from allergen immunotherapy, indicating that targeting TH2A cells could disrupt allergic responses. Allergen-specific type 2 helper T (TH2) cells play a central role in initiating and orchestrating the allergic and asthmatic inflammatory response pathways. One major factor limiting the use of such atopic disease–causing T cells as both therapeutic targets and clinically useful biomarkers is the lack of an accepted methodology to identify and differentiate these cells from overall nonpathogenic TH2 cell types. We have described a subset of human memory TH2 cells confined to atopic individuals that includes all allergen-specific TH2 cells. These cells are terminally differentiated CD4+ T cells (CD27− and CD45RB−) characterized by coexpression of CRTH2, CD49d, and CD161 and exhibit numerous functional attributes distinct from conventional TH2 cells. Hence, we have denoted these cells with this stable allergic disease–related phenotype as the TH2A cell subset. Transcriptome analysis further revealed a distinct pathway in the initiation of pathogenic responses to allergen, and elimination of these cells is indicative of clinical responses induced by immunotherapy. Together, these findings identify a human TH2 cell signature in allergic diseases that could be used for response-monitoring and designing appropriate immunomodulatory strategies.

Allergen‐specific CD4+ T cell responses in peripheral blood do not predict the early onset of clinical efficacy during grass pollen sublingual immunotherapy

Surrogate biomarkers of efficacy are needed in support of allergen‐specific immunotherapy.

Single Cell Assessment of Allergen-Specific T Cell Responses with MHC Class II Peptide Tetramers: Methodological Aspects

Background: We report herein critical methodological principles for assessing, at a single cell level, allergen-specific T cell responses using MHC class II peptide tetramers. Methods: We developed MHC class II peptide tetramers to monitor T cell responses against the immunodominant Bet v 1141–155 peptide in individuals with either an HLA-DRB1*0101, DRB1*0401 or DRB1*1501 background. In vitro stimulation was performed with serially truncated versions of the Bet v 1141–155 epitope chemically conjugated to the Ii-Key peptide. Results: Identification of Bet v 1141–155 as a high-affinity epitope for multiple HLA-DRB1 allotypes led to the development of corresponding tetramers detecting Bet v 1141–155-specific T cells with a high specificity and sensitivity. Stimulation with Bet v 1141–155 Ii-Key conjugate peptides is the most efficient procedure to expand Bet v 1141–155-specific CD4+ T cells, allowing to detect such cells in both allergic and healthy individuals. MHC class II Bet v 1141–155 tetramer-positive T cells produce IFN-γ and IL-10 in healthy individuals, and IL-5 in allergic patients. Frequencies of Bet v 1-specific CD4+ T cells circulating in the blood of allergic or nonallergic individuals range from approximately 10–5 to 10–3 CD4+ T cells, outside or within the pollen season, respectively. Conclusions: MHC class II peptide tetramers are valuable tools to assess allergen-specific T cell responses, both qualitatively and quantitatively. Selection of a high-affinity T cell epitope, as well as optimization of in vitro stimulation conditions to expand rare T cell progenitors are critical success factors in those analyses.

Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

Conceptually, allergic responses may involve cross‐reactivity by antibodies or T‐cells. While IgE cross‐reactivity among grass‐pollen allergens has been observed, cross‐reactivity at the allergen‐specific T‐cell level has been less documented. Identification of the patterns of cross‐reactivity may improve our understanding, allowing optimization of better immunotherapy strategies.