αS1-Casein elucidate major T-cell responses in cow's milk allergy

Cow's milk allergy (CMA) is the most common food allergy in children,1Spergel J.M. Natural history of cow's milk allergy.J Allergy Clin Immunol. 2013; 131: 813-814Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar and approximately 42% of children can outgrow their CMA by age 8 years.1Spergel J.M. Natural history of cow's milk allergy.J Allergy Clin Immunol. 2013; 131: 813-814Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Whey (β-lactoglobulin the most abundant) and casein proteins are the major milk allergens.2Skripak J.M. Matsui E.C. Mudd K. Wood R.A. The natural history of IgE-mediated cow's milk allergy.J Allergy Clin Immunol. 2007; 120: 1172-1177Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar The composite allergen casein consists of several isoforms: αS1-casein, αS2-casein, β-casein, and κ-casein.3Wal J.M. Cow's milk allergens.Allergy. 1998; 53: 1013-1022Crossref PubMed Scopus (160) Google Scholar IgE sensitization is particularly frequent against αS1-casein, inducing strong immediate or delayed allergic reactions.4Fiocchi A. Bouygue G.R. Albarini M. Restani P. Molecular diagnosis of cow's milk allergy.Curr Opin Allergy Clin Immunol. 2011; 11: 216-221Crossref PubMed Scopus (33) Google Scholar Previous studies have shown that patients with persistent CMA showed IgE reactivity to epitopes from the casein group, specifically to αS1-casein, as compared with patients who developed clinical tolerance.5Vila L. Beyer K. Jarvinen K.M. Chatchatee P. Bardina L. Sampson H.A. Role of conformational and linear epitopes in the achievement of tolerance in cow's milk allergy.Clin Exp Allergy. 2001; 31: 1599-1606Crossref PubMed Scopus (192) Google Scholar Thus, we hypothesized that patients with persistent, IgE-mediated CMA may have abnormal T-cell responses to casein. In addition, little is known about specific T-cell responses toward these allergens in adults and children with CMA and nonallergic subjects. A total of 26 allergic subjects, including 18 cow's milk (CM)-allergic subjects older than 8 years and 8 CM-allergic subjects 8 years or younger with persistent CMA (including subjects who are tolerant to baked milk products [n = 8]) were recruited for this study (see the Methods section in this article's Online Repository at www.jacionline.org). Characteristics of the recruited milk-allergic subjects are presented in Table E1 in this article's Online Repository at www.jacionline.org. Eighteen HLA-matched subjects including 13 nonatopic control subjects and 5 patients with peanut or walnut allergy without milk allergy were recruited as control subjects (Table E1). Tetramer-guided epitope-mapping approach was used to identify Bos d–specific CD4+ T-cell epitopes. A total of 23 epitopes were identified: 3 β-lactoglobulin-, 5 αS1-casein- (including 4 previously identified6Elsayed S. Eriksen J. Oysaed L.K. Idsoe R. Hill D.J. T cell recognition pattern of bovine milk alphaS1-casein and its peptides.Mol Immunol. 2004; 41: 1225-1234Crossref PubMed Scopus (0) Google Scholar), 3 αS2-casein-, 8 β-casein-, and 4 κ-casein-T-cell epitopes (see Fig E1 and Table E2 for HLA-restriction information and epitope specificity between cohorts in this article's Online Repository at www.jacionline.org). Seven of the current identified epitopes have been previously reported in Immune Epitope Database (IEDB). The antiphycoerythrin magnetic bead tetramer enrichment approach was used to determine the frequencies of Bos d–specific T cells ex vivo. Bos d epitope-specific memory (CD45RA−) CD4+ T cells were detected in high frequencies in allergic subjects, while lower frequencies were observed in nonallergic subjects (Fig 1, A and B). A significant difference in frequencies of Bos d–specific cells between CM-allergic subjects older than 8 years and children 8 years or younger with CMA was observed (Fig 1, A and B). Regarding each allergen individually, β-lactoglobulin, αS1-casein, αS2-casein, β-casein, and κ-casein-epitope-specific memory T-cell responses were detected in 6 of 23 (26%), 13 of 23 (57%), 7 of 23 (30%), 11 of 23 (48%), and 4 of 23 (17%) subjects with CMA, while responses for subjects 8 years or younger were 0 of 7 (0%), 4 of 7 (57%), 1 of 7 (14%), 3 of 7 (42%), and 0 of 7 (0%) and for subjects older than 8 years were 6 of 16 (38%), 9 of 16 (56%), 6 of 16 (38%), 8 of 16 (50%), and 4 of 16 (25%), respectively. Responses for subjects with baked milk tolerance were 3 of 7 (43%), 4 of 7 (57%), 3 of 7 (43%), 2 of 7 (29%), and 1 of 7 (14%) and for subjects with baked milk intolerance were 3 of 16 (19%), 9 of 16 (56%), 4 of 16 (25%), 9 of 16 (56%), and 3 of 16 (19%), respectively. These results show that αS1-casein and β-casein responses were the most prevalent Bos d–specific T-cell responses in subjects with CMA and suggest a possibility of epitope spreading in older subjects as well as differential allergen recognition between the baked milk sensitized and the baked milk tolerant subjects. CD154 upregulation assay was also used to examine the overall T-cell responses toward these allergens in 8 of 26 milk-allergic subjects. Strong αS1-casein–specific responses were also observed, confirming our MHC class II tetramer staining (see Fig E2 in this article's Online Repository at www.jacionline.org). In total, these results suggested that most milk-specific CD4+ T-cell responses in subjects with CMA were directed against αS1-casein and β-casein. The current data are also in agreement with a previous study that showed that the frequency of casein-specific T cells was higher in children with CMA than in children without CMA.7Michaud B. Aroulandom J. Baiz N. Amat F. Gouvis-Echraghi R. Candon S. et al.Casein-specific IL-4- and IL-13-secreting T cells: a tool to implement diagnosis of cow’s milk allergy.Allergy. 2014; 69: 1473-1480Crossref PubMed Scopus (16) Google Scholar Chemokine receptor and differentiation marker expression of Bos d–specific T cells was analyzed by ex vivo tetramer staining of PBMCs (Fig 1, C-E). Detectable Bos d–specific CD4+ T cells in allergic subjects were CCR4+, suggesting a TH2 phenotype. In addition, CRTH2+ and CD27− T cells were observed (Fig 1, C and D), which indicates a terminally differentiated TH2 phenotype.8Archila L.D. Jeong D. Pascal M. Bartra J. Juan M. Robinson D. et al.Jug r 2-reactive CD4 T cells have a dominant immune role in walnut allergy.J Allergy Clin Immunol. 2015; 136: 983-992.e7Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar CCR4+ T cells that coexpressed CCR6 and Integrin β7 were also observed in subjects with CMA and were more common in children 8 years or younger than in older subjects with persistent CMA, suggesting that a subpopulation of these cells account for a TH17-like response (Fig E3, A and B). CCR6+CRTH2+ T cells were detected but to a lesser degree (Fig E3, C and D). However, we did not observe differences between allergic adults and children for these populations. However, higher CCR7 and CXCR3 expression (Fig 1, C and E) was observed in subjects older than 8 years. The presence of more CXCR3+ and CCR7+ milk-specific cells imply that TH1 and TCM (central memory T cells as defined by expression of CD27 and CCR7) milk-specific cells are more common in CM-allergic subjects older than 8 years than in children 8 years or younger with CMA. No difference in frequencies and phenotypes among subjects with baked milk tolerance or intolerance was observed (data not shown). The CD154 upregulation assay was used to analyze the cytokine profiles of Bos d–specific T cells (Fig 2, A). In subjects with CMA, a dominant TH2 response was observed, which accounted for 58% of the overall response (Fig 2, B). Among the TH2 cytokine producers, cells can be classified into IL-4 producers (3%), IL-4/IL-13 (44%) producers, and IL-4/IL-5/IL-13 (11%) producers. We did not detect cells that produced IL-5 or IL-13 alone. Interestingly, a subdominant TH2/TH17 response was also observed, which accounted for 27% of the overall T-cell response (Fig 2, B). Among IL-17A producers, cells can be classified into IL-17A producers (4%), IL-4/IL-17A producers (2%), and IL-4/IL-13/IL-17A producers (21%). Interestingly, higher proportions of IL-4/IL-5/IL-13 producers (30%) and IL-4 producers (33%) were detected in CM-allergic subjects older than 8 years (Fig 2, C); in contrast, higher proportions of IL-4/IL-13/IL-17A producers (61%) were detected in children 8 years or younger (Fig 2, C). There was no difference in the cytokine profiles of baked milk tolerant and intolerant subjects (data not shown). Conversely, T cells from nonallergic subjects produced IFN-γ, IL-10, or both, with low IL-17A (Fig 2, A and B). These results confirmed the observed TH2 and TH2/TH17 phenotypes in our ex vivo experiments and our previous studies that these phenotypes could be a potential trademark in food allergy.8Archila L.D. Jeong D. Pascal M. Bartra J. Juan M. Robinson D. et al.Jug r 2-reactive CD4 T cells have a dominant immune role in walnut allergy.J Allergy Clin Immunol. 2015; 136: 983-992.e7Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar The present study implicates an important role of Bos d–specific T-cell responses in the persistence of CMA. In older children and adults with CMA, a committed TH2 response was observed (Fig 2, B, IL4/IL5/IL13 triple cytokine producers). However, in younger children with CMA, TH2/TH17 responses were more prevalent, suggesting that these T-cell populations are not fully committed into the TH2 phenotype and could explain loss of CMA in younger children. Moreover, TCM are less susceptible to deletion by allergen specific immunotherapy in a murine model.9Mackenzie K.J. Nowakowska D.J. Leech M.D. McFarlane A.J. Wilson C. Fitch P.M. et al.Effector and central memory T helper 2 cells respond differently to peptide immunotherapy.Proc Natl Acad Sci U S A. 2014; 111: E784-E793Crossref PubMed Scopus (20) Google Scholar Accumulation of CCR7+CD27+ Bos d-epitope–specific T cells (TCM) in adults might be indicative of CMA persistence and also complicate possible oral immunotherapy for CMA. Knowledge of CM-epitope–specific T-cell responses will be useful in devising novel strategies to halt and reverse the progression of CMA. We thank Kavitha Gilroy and Sylvia Posso for help with subject recruitment. Subjects were recruited from the Virginia Mason Medical Center Allergy Clinic and Benaroya Research Institute with informed consent and institutional review board (IRB) approval (IRB title “Allergen and T-cell reagent resources for the study of allergic diseases”; approval no. IRB7109). A total of 26 subjects with a documented record of milk allergy and a positive ImmunoCAP score for milk extract (>0.35 kU/L) (Phadia AB, Uppsala, Sweden) were recruited for this study. All patients had onset of CMA in infancy, typically with first known exposure to cow milk with symptoms consisting typically of urticaria and gastrointestinal discomfort and vomiting. Diagnosis of CMA was made by specific IgE testing to cow milk, either skin prick testing or serum IgE testing, and history of presentation of clinical symptoms. Most recent serum cow milk-specific IgE levels are presented in Table E1. Eighteen patients (12 >8-year-old and 6 ≤8-year-old) do not tolerate any form of milk and 8 patients (6 >8-year-old and 2 ≤8-year-old) tolerate baked form of milk protein. Patients with baked milk intolerance had either accidental ingestion-induced symptoms or failed challenge to baked milk. The 8 patients tolerating baked milk had accidental ingestion of fresh milk–induced symptoms, but passed a baked milk challenge. Thirteen nonatopic and 5 atopic subjects with no clinical symptoms to milk, a negative ImmunoCAP score, and HLA-matched were also recruited as controls for this study. The features of these subjects are presented in Table E1. DNA samples were HLA-typed using Dynal Unitray SSP Kits (Invitrogen, Carlsbad, Calif) according to the manufacturer's instructions. Peptide libraries were generated on the basis of β-lactoglobulin, αS1-casein, αS2-casein, β-casein, and κ-casein sequences. The libraries consisted of overlapping peptides spanning the entire allergen, which were 20 amino acids in length with a 12 amino acid overlap synthetized by Mimotopes (Clayton, Australia). Peptide-loaded HLA-DR and HLA-DQ proteins were generated, as previously described.E1Novak E.J. Liu A.W. Nepom G.T. Kwok W.W. MHC class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen.J Clin Invest. 1999; 104: R63-R67Crossref PubMed Scopus (327) Google Scholar, E2Kwok W.W. Liu A.W. Novak E.J. Gebe J.A. Ettinger R.A. Nepom G.T. et al.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.J Immunol. 2000; 164: 4244-4249Crossref PubMed Scopus (113) Google Scholar The tetramer-guided epitope-mapping procedure was conducted as previously described.E3Novak E.J. Liu A.W. Gebe J.A. Falk B.A. Nepom G.T. Koelle D.M. et al.Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.J Immunol. 2001; 166: 6665-6670Crossref PubMed Scopus (118) Google Scholar Briefly, PBMCs from subjects with CMA were stimulated for 2 weeks with pool peptides that consisted of 5 overlapping peptides spanning the entire allergen. After 2 weeks, PBMCs were screened with pMHC-II tetramers loaded with the pool of peptides that corresponded to the stimulated well; those with a positive signal were rescreened again but this time with pMHC-II tetramers loaded with single peptides. CD154+ detection assay was carried out as previously described.E4Archila L.D. Jeong D. Pascal M. Bartra J. Juan M. Robinson D. et al.Jug r 2-reactive CD4 T cells have a dominant immune role in walnut allergy.J Allergy Clin Immunol. 2015; 136: 983-992.e7Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Briefly, for detection of CD154+-reactive T cells, 15 to 35 million of freshly isolated PBMCs (at 7 × 106 cells/mL) in culture medium Gibco RPMI 1640 (Thermo Fisher, Waltham, Mass) + 10% pooled human serum + 1% PenStrep were stimulated with 5 μg/mL of synthesized peptide pools (at a final concentration of 47.5 μM for Bos d 5, 60 μM for Bos d 9, 62.5 μM for Bos d 10, 62.5 μM for Bos d 11, and 50 μM for Bos d 12) and 1 μg/mL anti-CD40 (Miltenyi Biotec, Auburn, Calif) for 3 hours (for frequency) and 6 hours (for intracellular cytokine staining) at 37°C. Cells were also mock stimulated with dimethyl sulfoxide (0.05% final concentration) as negative control. After stimulation, cells were stained with phycoerythrin-conjugated CD154 (Miltenyi Biotec, Auburn, Calif) and labeled with antiphycoerythrin magnetic beads (Miltenyi Biotec) for 20 minutes at 4°C. A 1/100 fraction of cells was saved for analysis. The other fraction was passed through a Miltenyi magnetic column; magnetically enriched cells were next stained with a panel of antibodies of interest for 20 minutes at room temperature. After staining, cells were stained again with Via-probe+ (BD Biosciences, East Rutherford, NJ) for 10 minutes at 4°C before flow-cytometry. Data acquisition was performed using a FACSCanto flow cytometer and data were analyzed using FlowJo (Tree Star, Ashland, Ore). Frequency was calculated as previously described for tetramer analysis.E5Kwok W.W. Roti M. Delong J.H. Tan V. Wambre E. James E.A. et al.Direct ex vivo analysis of allergen-specific CD4+ T cells.J Allergy Clin Immunol. 2010; 125: 1407-1409Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Ex vivo analysis with pMHC-II (Peptide/MHC class II) tetramers was carried out as previously described.E5Kwok W.W. Roti M. Delong J.H. Tan V. Wambre E. James E.A. et al.Direct ex vivo analysis of allergen-specific CD4+ T cells.J Allergy Clin Immunol. 2010; 125: 1407-1409Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar For ex vivo intracellular cytokine staining combined with CD154 activation assay, BD GolgiStop was added during stimulation (BD Biosciences) according to the manufacturer's instructions. After 10 minutes at room temperature, cells were then fixed with fixation buffer (eBioscience, San Diego, Calif) and washed twice with a permeabilization buffer (eBioscience). Cells were then stained with a panel of antibodies (eBioscience and BD Biosciences) directed against cytokines of interest for 20 minutes at room temperature; cells were washed and immediately analyzed in FACSCanto flow cytometer. Statistical analysis was performed using the tests indicated in the figure legends using Prism 5.0 software (GraphPad Software, La Jolla, Calif).Fig E2Frequencies of milk allergen reactive CD4+ T cells. A and B, Frequencies of β-lactoglobulin-, αS1-casein-, αS2-casein-, β-casein-, and κ-casein-reactive T cells in 8 subjects with milk allergy (adults, teenagers, and children >8 years of age n = 6; children ≤8 years of age n = 2) with CD154 assays after 3-hour Bos d peptide stimulation using freshly isolated PBMCs. Each data point represents the frequency of T cells reactive to each allergen. An ANOVA test (with Bonferroni correction) was used to compare all columns in the statistical analysis. NS, Not significant. *P < .05, **P < .01, and ***P < .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Phenotypes of CCR6+Bos d–specific T cells in milk-allergic subjects after ex vivo pMHC-II tetramer enrichment using freshly isolated PBMCs. A and B, Tetramer+CD45RA− T cells were gated against CCR4 and CCR6. Each data point represents results for surface expression in tetramer-positive T cells from 23 subjects with milk allergy: 15 adults, teenagers, and children older than 8 years (triangles) and 8 children 8 years or younger (downside triangles) with CMA. C and D, Tetramer-positive CD45RA− T cells were gated against CCR6 and CRTH2. Each data point represents results for surface expression in tetramer-positive T cells from 26 subjects with milk allergy: 18 adults, teenagers, and children older than 8 years (triangles) and 8 children 8 years or younger (downside triangles) with CMA. A t test was used in the statistical analysis. NS, Not significant. *P < .05, **P < .01, and ****P < .0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1HLA and allergic status of recruited subjectsIDAge (y)SexHLA (DRB1∗)sIgE milk (f256) (kU/L)Symptoms to milk ingestionTolerates baked milkMilk-allergic adults 139F04:01, 07:0110.3I, II, III, IV, VNo 234M07:01, 13:010.5V, VIIINo 322F04:01, 10:0141.2VIIINo 418M10:01, 12:014.63VIIINo 530F01:01, 09:010.53VIIIYes 620M03:01, 13:013.29III, IV, VNo 720M01:01, 03:0182.9I, IIYes 819M07:01, 13:0141.1IV, VNo 918M04:01, 12:0126.1VIIINoMilk-allergic teenagers and children >8 y 1015M04:01, 15:014.46I, II, IVNo 1110M13:01, 15:0135.1IVYes 1210M03:01, 12:0164.7III, IV, VNo 1313F03:01, 03:0164.5I, IINo 149M03:01, 07:0122.7II, IV, VNo 1511M07:01, 15:0112.1IV, VYes 1612F01:01, 04:05100II, IV, VNo 1715F10:01, 15:011.27II, IVYes 1813M12:01, 15:019.28II, III, IV, VIIIYesMilk-allergic children 8 y or younger 194M10:01, 15:0177.2I, II, IV, VNo 203M04:01, 10:010.8III, IV, VIIYes 215F07:01, 11:0151.7II, IV, VNo 223M03:01, 07:0122.7I, II, IVYes 233M03:01, 07:017.3II, IVNo 245M09:01, 10:0152.2III, IVNo 255M01:01, 15:012.86IVNo 268M04:01, 07:0115.3II, III, IV, VNoNonatopic subjects 2729M03:01, 15:010AbsentYes 2831F01:01, 15:010AbsentYes 2934F01:01, 01:030AbsentYes 3034F03:01, 11:010AbsentYes 3135F04:01, 14:010AbsentYes 3232F07:01, 07:010AbsentYes 3338M04:01, 04:010AbsentYes 3437F03:01, 07:010AbsentYes 3532M01:01, 04:020AbsentYes 3635M07:01, 13:020AbsentYes 3724M07:01, 13:020AbsentYes 383M01:010AbsentYes 3923F15:01, 10:010AbsentYesAtopic subjects without milk allergy 40∗Subjects also had history of peanut or walnut and positive IgE ImmunoCAP for peanut or walnut.8M10:01, 15:010AbsentYes 41∗Subjects also had history of peanut or walnut and positive IgE ImmunoCAP for peanut or walnut.12M01:01, 13:020AbsentYes 42∗Subjects also had history of peanut or walnut and positive IgE ImmunoCAP for peanut or walnut.10F07:01, 11:010AbsentYes 43∗Subjects also had history of peanut or walnut and positive IgE ImmunoCAP for peanut or walnut.12M11:01, 15:010AbsentYes 44∗Subjects also had history of peanut or walnut and positive IgE ImmunoCAP for peanut or walnut.10F01:01, 15:010AbsentYesI. Itchy mouth, lips, and/or pharynxII. Abdominal discomfort and/or diarrheaIII. Nausea or vomitingIV. Severe skin itching or hives, acute or angioedemaV. Rhinitis and/or conjunctivitis and/or respiratory compromiseVI. Dizziness (feeling loss of consciousness)VII. Syncope (loss of consciousness)VIII. Desaturation with respiratory compromiseF, Female; M, male.∗ Subjects also had history of peanut or walnut and positive IgE ImmunoCAP for peanut or walnut. Open table in a new tab Table E2Bos d 5, Bos d 9, Bos d 10, Bos d 11, and Bos d 12 CD4+ T-cell epitopesHLA DRB1 restrictionBos dAmino acid sequence01:0103:0104:0107:0109:0110:0111:0115:01DRB4DRB5DQ6Bos d 5 17-36∗Previously identified as HLA-II–restricted T-cell epitopes in www.iedb.org. Epitope Bos d 5 17-36 (33950, 223605); Bos d 5 129-148 (222188); Bos d 9 17-36 (31145, 38207); Bos d 9 105-124 (74689, 51432, 34491, 70444); Bos d 9 137-156 (13715, 13716, 26364, 26341); Bos d 9 145-164 (26341, 45542, 18443); Bos d 9 169-188 (10, 5711, 7690).GTWYSLAMAASDISLLDAQS1/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/1‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 5 129-148∗Previously identified as HLA-II–restricted T-cell epitopes in www.iedb.org. Epitope Bos d 5 17-36 (33950, 223605); Bos d 5 129-148 (222188); Bos d 9 17-36 (31145, 38207); Bos d 9 105-124 (74689, 51432, 34491, 70444); Bos d 9 137-156 (13715, 13716, 26364, 26341); Bos d 9 145-164 (26341, 45542, 18443); Bos d 9 169-188 (10, 5711, 7690).DDEALEKFDKALKALPMHIR0/4†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/11‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.1/3†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.3/3‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 5 137-156DKALKALPMHIRLSFNPTQL0/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/5‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 9 17-36∗Previously identified as HLA-II–restricted T-cell epitopes in www.iedb.org. Epitope Bos d 5 17-36 (33950, 223605); Bos d 5 129-148 (222188); Bos d 9 17-36 (31145, 38207); Bos d 9 105-124 (74689, 51432, 34491, 70444); Bos d 9 137-156 (13715, 13716, 26364, 26341); Bos d 9 145-164 (26341, 45542, 18443); Bos d 9 169-188 (10, 5711, 7690).§Previously identified CD4+ T-cell epitopes by Elsayed et al.E6NENLLRFFVAPFPEVFGKEK1/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/1‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 9 105-124∗Previously identified as HLA-II–restricted T-cell epitopes in www.iedb.org. Epitope Bos d 5 17-36 (33950, 223605); Bos d 5 129-148 (222188); Bos d 9 17-36 (31145, 38207); Bos d 9 105-124 (74689, 51432, 34491, 70444); Bos d 9 137-156 (13715, 13716, 26364, 26341); Bos d 9 145-164 (26341, 45542, 18443); Bos d 9 169-188 (10, 5711, 7690).§Previously identified CD4+ T-cell epitopes by Elsayed et al.E6KVPQLEIVPNSAEERLHSMK1/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/3‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 9 137-156§Previously identified CD4+ T-cell epitopes by Elsayed et al.E6GVNQELAYFYPELFRQFYQL0/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/1‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 9 145-164∗Previously identified as HLA-II–restricted T-cell epitopes in www.iedb.org. Epitope Bos d 5 17-36 (33950, 223605); Bos d 5 129-148 (222188); Bos d 9 17-36 (31145, 38207); Bos d 9 105-124 (74689, 51432, 34491, 70444); Bos d 9 137-156 (13715, 13716, 26364, 26341); Bos d 9 145-164 (26341, 45542, 18443); Bos d 9 169-188 (10, 5711, 7690).FYPELFRQFYQLDAYPSGAW2/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/2‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.1/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.3/4‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.0/0†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/1‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 9 169-188∗Previously identified as HLA-II–restricted T-cell epitopes in www.iedb.org. Epitope Bos d 5 17-36 (33950, 223605); Bos d 5 129-148 (222188); Bos d 9 17-36 (31145, 38207); Bos d 9 105-124 (74689, 51432, 34491, 70444); Bos d 9 137-156 (13715, 13716, 26364, 26341); Bos d 9 145-164 (26341, 45542, 18443); Bos d 9 169-188 (10, 5711, 7690).§Previously identified CD4+ T-cell epitopes by Elsayed et al.E6SGAWYYVPLGTQYTDAPSFS1/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/2‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.0/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/4‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.0/0†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/1‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.0/4†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/11‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 10 17-36QETYKQEKNMAINPSKENLC1/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/1‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 10 177-196PQYLKTVYQHQKAMKPWIQP1/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.3/4‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 10 185-204QHQKAMKPWIQPKTKVIPYV1/3†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/3‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 11 25-44RINKKIEKFQSEEQQQTEDE0/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/4‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 11 41-60TEDELQDKIHPFAQTQSLVY0/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/7‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.1/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/1‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 11 49-68IHPFAQTQSLVYPFPGPIPN0/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/5‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 11 65-84PIPNSLPQNIPPLTQTPVVV0/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/5‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 11 89-108QPEVMGVSKVKEAMAPKHKE1/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/5‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 11 113-132KYPVEPFTESQSLTLTDVEN0/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.7/7‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bosd 11 153-172PTVMFPPQSVLSLSQSKVLP0/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/7‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 11 193-212FLLYQEPVLGPVRGPFPIIV1/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.2/7‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 12 1-20AQEQNQEQPIRCEKDERFFS0/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/5‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.1/3†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/3‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 12 57-76LPYPYYAKPAAVRSPAQILQ1/3†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/3‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 12 105-124SFMAIPPKKNQDKTEIPTIN2/2†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.0/4‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.Bos d 12 153-172PEVIESPPEINTVQVTSTAV0/1†No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.1/5‡No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.∗ Previously identified as HLA-II–restricted T-cell epitopes in www.iedb.org. Epitope Bos d 5 17-36 (33950, 223605); Bos d 5 129-148 (222188); Bos d 9 17-36 (31145, 38207); Bos d 9 105-124 (74689, 51432, 34491, 70444); Bos d 9 137-156 (13715, 13716, 26364, 26341); Bos d 9 145-164 (26341, 45542, 18443); Bos d 9 169-188 (10, 5711, 7690).† No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk tolerance.‡ No. of responders and no. of subjects tested with the designated HLA-DR or HLA-DQ with baked milk intolerance.§ Previously identified CD4+ T-cell epitopes by Elsayed et al.E6Elsayed S. Eriksen J. Oysaed L.K. Idsoe R. Hill D.J. T cell recognitioin pattern of bovine milk alphaS1-casein and its peptides.Mol Immunol. 2014; 41: 1225-1234Crossref Scopus (21) Google Scholar Open table in a new tab I. Itchy mouth, lips, and/or pharynx II. Abdominal discomfort and/or diarrhea III. Nausea or vomiting IV. Severe skin itching or hives, acute or angioedema V. Rhinitis and/or conjunctivitis and/or respiratory compromise VI. Dizziness (feeling loss of consciousness) VII. Syncope (loss of consciousness) VIII. Desaturation with respiratory compromise F, Female; M, male.