E-Mail karger@karger.com

Original Paper

Int Arch Allergy Immunol 2014;163:292–296 DOI: 10.1159/000360733

Characterization of T Cell Epitopes in Bovine α-Lactalbumin

Laura A.P.M. Meulenbroek a, b Constance F. den Hartog Jager a

Ans F.M. Lebens a André C. Knulst a Carla A.F.M. Bruijnzeel-Koomen a

Johan Garssen b, c Léon M.J. Knippels b, c Els van Hoffen a

a Department of Dermatology/Allergology, University Medical Center Utrecht, and b Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht , and c Department of Immunology, Nutricia Research, Utrecht , The Netherlands

Introduction

Cow’s milk allergy (CMA) is a common food allergy, affecting 0.3–3.5% of the young children and 0.1–0.3% of the adults [1, 2] . One of the proteins in cow’s milk that may induce an allergic response is α-lactalbumin (α-LAC).

α-LAC is a small protein of 123 amino acids (AA) with a molecular weight of 14.2 kDa. It belongs to the whey fraction and is present in relatively high concentrations in the milk of all mammalian species [3] . The proteins of the different species show a strong homology, for exam-ple, about 74% of the AA in bovine and human α-LAC are identical [4, 5] . α-LAC has an important role in the bio-synthesis of lactose by acting as a coenzyme in the lactose synthase complex. In addition, it is rich in essential AA, such as tryptophan, leucine and lysine, and therefore im-portant in newborn nutrition [3, 6] .

Despite the strong homology between human and bo-vine α-LAC, bovine α-LAC has been described as one of the major allergens in cow’s milk [7] . However, the per-centage of CMA patients that show IgE reactivity to α-LAC is variable in different study populations. Two studies describe that 75–80% of the patients recognize the protein [5, 7] , whereas others observed α-LAC-specific IgE in only 30–35% of the patients [8, 9] . Depending on the population tested, these percentages were either low-er or similar to the percentages observed for β-lactoglobu-

Key Words

Cow’s milk allergy · α-Lactalbumin · T cell epitopes

Abstract

Background: Recent studies have indicated that peptides containing T cell epitopes may be used for immunotherapy. While for several cow’s milk allergens the T cell epitopes have been described, the T cell epitopes in the major aller-gen α-lactalbumin (α-LAC) are unknown. Therefore, the aim of this study was to determine the T cell epitopes in α-LAC. Methods: Nineteen synthetic peptides spanning α-LAC were obtained. Cow’s milk-specific T cell lines (TCLs) of 46 subjects were generated and tested for their specificity for α-LAC. The lines responding to α-LAC were subsequently tested to de-termine their activation in response to the peptides. Results: More than half of the TCLs generated did not respond to α-LAC or lost their responsiveness during subsequent exper-iments, which indicates that α-LAC has low immunogenicity. Only 8 TCLs recognized 1 or more peptides. The recognition of the peptides was diverse and no major epitopes could be defined. Conclusion: The immunogenicity of α-LAC is very low compared to other major allergens in cow’s milk. More-over, there seems to be no dominant epitope present in the protein. Therefore, it seems unlikely that peptides of this protein can be used for immunotherapy.

© 2014 S. Karger AG, Basel

Received: August 20, 2013 Accepted after revision: February 17, 2014 Published online: April 26, 2014

Correspondence to: Dr. Laura A.P.M. Meulenbroek Department of Dermatology/Allergology (G02.124) University Medical Center Utrecht PO Box 85.500, NL–3508 GA Utrecht (The Netherlands) E-Mail laurameulenbroek   @   home.nl

© 2014 S. Karger AG, Basel1018–2438/14/1634–0292$39.50/0

www.karger.com/iaa

Dow

nloa

ded

by:

Uni

vers

ität Z

üric

h, Z

entr

albi

blio

thek

Zür

ich

13

0.60

.206

.43

- 7/

15/2

014

10:2

4:56

AM

T Cell Epitopes in α-Lactalbumin Int Arch Allergy Immunol 2014;163:292–296 DOI: 10.1159/000360733

293

lin and casein, the other major allergens in cow’s milk [7–9] . Interestingly, in a recent study, we investigated the basophil response of adult CMA patients to the major al-lergens [10] . Whereas in general the IgE levels for α-LAC were lower than for the other major allergens, the re-sponse in the basophil activation test was similar, suggest-ing that α-LAC is a potent allergen. According to IgE epi-tope studies, conformational epitopes play a major role in the IgE recognition of α-LAC [11] .

In addition to IgE, T cells also play an important role in food allergy by stimulating B cells to switch to IgE pro-duction. On the other hand, they are also important for tolerance induction. Previous studies indicated that im-munotherapy with peptides containing T cell epitopes re-duced the allergic response to intact protein [12] . While for other major allergens in cow’s milk T cell epitopes have been described [13–15] , the T cell epitopes in α-LAC are still unknown. In this study, the proliferative and cy-tokine responses of cow’s milk-specific short- and long-term T cell lines (TCLs) to synthetic peptides of α-LAC were determined to identify the T cell epitopes.

Materials and Methods

Peptides Nineteen sequential synthetic peptides spanning α-LAC (se-

quence of the mature protein, i.e. AA 20–142, as described in Gen-Bank No. AAA30615.1) were obtained from JPT Peptide Tech-nologies (Berlin, Germany). The peptides were 18 AA long with a 12-AA overlap ( fig. 1 ). They were dissolved at a concentration of 1 mg/ml in 10% dimethylsulfoxide (Sigma Aldrich, Louis, Mo., USA) in PBS, aliquoted and stored at –80   °   C.

Generation of TCLs Cow’s milk-specific short-term TCLs were generated from 14

adult CMA patients. For the long-term TCLs, PBMCs of 14 chil-dren with CMA, 11 atopic children without CMA and 7 healthy

controls were used (see online supplementary table  1, www.karger.com/doi/10.1159/000360733). The diagnosis of CMA was based on a suggestive history, positive cow’s milk-specific IgE lev-els, positive skin prick test and/or a positive, double-blind, place-bo-controlled food challenge. Typical symptoms in CMA children were erythema and urticaria, while adult CMA patients experi-enced symptoms varying from oral allergy symptoms to respira-tory and/or cardiovascular symptoms [9, 16, 17] . Ethical approval for this study was obtained from the Ethics Committee of the Uni-versity Medical Center Utrecht.

The TCLs were generated as described previously [16, 18] . In short, PBMCs were incubated for 7 days with cow’s milk protein (CMP). For the short-term TCLs, medium was refreshed and IL-2 was added to the culture on day 7. Four days later, the cells were stimulated with a mixture of irradiated allogeneic PBMCs, irradi-ated allogeneic EBV-transformed B cells, PHA and IL-2. On day 21, cells were tested for specificity. The long-term TCLs were maintained in medium containing both IL-2 and IL-4. Every 14 days, the cells were restimulated with autologous EBV-trans-formed B cells pre-incubated with CMP. At the time of restimula-tion, cells were tested for their specificity.

T Cell Specificity To test the specificity of T cells, their response to the major al-

lergens and peptides of α-LAC was determined as described previ-ously [14, 18] . In short, short-term TCLs were incubated with ir-radiated autologous PBMCs and the allergens/peptides of interest. After 48 h, supernatant was collected and tritium-labeled thymi-dine was added to determine proliferation. The following day, cells were harvested and thymidine uptake was measured with a betaplate counter. Cytokine concentrations (IL-13, IFNγ and IL-10) in the supernatant were determined with ELISA. For long-term TCLs, irradiated EBV-transformed B cells were pre-incubat-ed overnight with the allergens/peptides of interest. The next day, the long-term TCLs were added and incubated for another 24 h. Subsequently, thymidine uptake was determined as described above. To determine the degree of proliferation, the stimulation index (SI), i.e. the ratio between the proliferation of allergen/pep-tide-stimulated cells and non-stimulated cells, was calculated. SI ≥ 2 was considered positive. The major allergens, namely CMP [a mixture of whey (Lactalis, Laval, France) and casein (FrieslandCampina Domo, Amersfoort, The Netherlands) in a 1: 1 ratio], casein, whey, β-lactoglobulin and α-LAC (both from

Fig. 1. AA sequence of α-LAC (GenBank No. AAA30615.1; AA 20–142) used for the production of the synthetic peptides. The peptides were 18 AA long and had a 12-AA overlap as indicated in the figure.

Dow

nloa

ded

by:

Uni

vers

ität Z

üric

h, Z

entr

albi

blio

thek

Zür

ich

13

0.60

.206

.43

- 7/

15/2

014

10:2

4:56

AM

Meulenbroek    et al.

Int Arch Allergy Immunol 2014;163:292–296 DOI: 10.1159/000360733

294

NIZO Food Research BV, Ede, The Netherlands) were tested in a concentration of 50 μg/ml. Due to lipopolysaccharide contamina-tion, β-lactoglobulin and α-LAC could not be used for the short-term TCLs. The peptides (10 μg/ml) were first tested in mixtures of 2–3 peptides, after which positive peptides were tested sepa-rately.

Results

Of the 32 long-term TCLs that were generated, only 15 TCLs responded to α-LAC (online suppl. table 1). In ad-dition, 7 of these 15 lines lost their response to the protein during the subsequent experiments. Of the remaining α-LAC-specific TCLs, only 4, 2 from children with CMA (TCL 26 and 30) and 2 from atopic children without CMA (TCL 12 and 13), responded to the peptides. The 14 short-term TCLs from CMA adults all responded to whey. However, of these 14 TCLs, only 4 TCLs (38, 40, 41 and 43) responded to the α-LAC peptides.

The T cell response to the peptides was diverse ( ta-ble 1 ). The 8 TCLs recognized 10 different peptides, which covered almost the complete protein. Three TCLs (13, 30 and 38) recognized 2 consecutive peptides, suggesting that the overlapping part contains the epitope. Four pep-tides (AA 19–36, 25–42, 31–48 and 43–60) were able to induce a proliferative response in 2 TCLs.

The cytokine response of the short-term TCLs reflect-ed their degree of activation. For example, TCL 40 showed a strong proliferative and cytokine response after stimu-lation with the peptides. Both IL-13 and IFNγ were found in the supernatant of this TCL. In contrast, high IL-13 levels were measured in the supernatant of TCL 41 after stimulation with the major allergens but not with the pep-tides. This correlated with the proliferation of this TCL, which was much stronger in response to the major aller-gens than to the peptides (SI of 31 vs. 3.5).

Table 1. Peptide recognition by human TCLs

TCL 12 13 26 30 38 40 41 43

SI SI SI SI SI IL-13 IFNγ IL-10 SI IL-13 IFNγ IL-10 SI IL-13 IFNγ IL-10 SI IL-13 IFNγ IL-10

CMP 14.8 32.7 3.4 108.8 20.8 1.2 193.3 21.8 9.9 8.3 6.0 0Casein 14.7 32.7 3.8 152.3 57.9 6.3 215.0 32.9 16.4 11.5 12.0 0Whey 28.1 19.9 0 223.3 34.3 9.2 143.1 16.0 6.3 7.9 14.7 0α-LAC ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDβ-LG ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND1 – 187 – 2413 – 3019 – 36 2.0 3.5 0.725 – 4231 – 4837 – 5443 – 6049 – 66 0 2.8 ND55 – 72 0 0 ND61 – 7867 – 8473 – 9079 – 9685 – 102 93.3 95.0 091 – 10897 – 114 12.1 0.8 17101 – 120104 – 123 0 1.3 0

The numbers in the left column indicate the AA of the peptides. SI is an indication for the degree of proliferation, which is determined by measuring thymidine uptake. The cytokine concentrations are in pg/ml. β-LG = β-Lactoglobulin; ND = not determined; light grey = 2 < SI < 5; grey = 5 < SI < 20; black = SI > 20.

Dow

nloa

ded

by:

Uni

vers

ität Z

üric

h, Z

entr

albi

blio

thek

Zür

ich

13

0.60

.206

.43

- 7/

15/2

014

10:2

4:56

AM

T Cell Epitopes in α-Lactalbumin Int Arch Allergy Immunol 2014;163:292–296 DOI: 10.1159/000360733

295

Discussion

In this study, short- and long-term TCLs of in total 46 subjects were generated to investigate the T cell epitopes of α-LAC. The initial specificity tests of the TCLs already indicated that α-LAC has low immunogenicity. More than half of the long-term TCLs did not respond to α-LAC or lost their responsiveness during the subsequent exper-iments. In addition, most of the lines that did respond showed a lower SI in response to α-LAC compared to the other major allergens ( table 1 ; online suppl. table 1). A similar effect had been seen in a previous study that in-vestigated the primary lymphocyte response of CMA pa-tients [8] . A possible explanation for the low immunoge-nicity of α-LAC may be the high homology (74%) be-tween bovine and human α-LAC [4, 5] , because the body usually does not respond to ‘self’ proteins.

Only 8 TCLs responded to 1 or more peptides. The peptides that were recognized were diverse and no dis-crimination between major and minor epitopes could be made. This may suggest that there is no dominant epitope present in the protein. In 2 of the short-term TCLs (38 and 41), the proliferative and cytokine responses induced by the peptides were relatively low when compared with the response to whey. Because CMP was used for the gen-eration of the TCLs, it is possible that these TCLs not only recognize α-LAC but also other CMPs. This hypothesis was supported by the fact that TCL 38 and 41 also recog-

nized peptides of β-lactoglobulin, while TCL 39 and 40 did not [unpubl. data]. In addition, by using a mixture of proteins, there is a risk that responses to weakly immuno-genic proteins are lost due to the dominance of stronger immunogenic proteins. This may explain why only a small number of TCLs responded to α-LAC.

Several studies in both humans and mice have indi-cated that peptides may be used for immunotherapy [12, 19, 20] . Because peptides are too small to cross-link IgE on basophils and mast cells but are capable of activating T cells, they may induce tolerance without provoking side effects, as is seen with conventional immunotherapy. However, because of the low immunogenicity of α-LAC, it is questionable whether peptides of this protein are able to induce tolerance.

In conclusion, α-LAC has low immunogenicity com-pared to the other major allergens in cow’s milk. The TCLs that do respond to α-LAC recognize diverse epi-topes, which suggests that there is no dominant epitope present in the protein. Therefore, it seems unlikely that peptides of this protein can be used for immunotherapy.

Acknowledgments

This work was conducted at the Department of Dermatology/Allergology of the University Medical Center Utrecht. Current af-filiation of E.v.H.: NIZO Food Research BV, Ede, The Netherlands.

References

1 Venter C, Arshad SH: Epidemiology of food allergy. Pediatr Clin North Am 2011; 58: 327–349.

2 Rona RJ, Keil T, Summers C, Gislason D, Zuidmeer L, Sodergren E, Sigurdardottir ST, Lindner T, Goldhahn K, Dahlstrom J, Mc-Bride D, Madsen C: The prevalence of food allergy: a meta-analysis. J Allergy Clin Immu-nol 2007; 120: 638–646.

3 Barbana C, Sanchez L, Perez MD: Bioactivity of alpha-lactalbumin related to its interaction with fatty acids: a review. Crit Rev Food Sci Nutr 2011; 51: 783–794.

4 Fiocchi A, Schunemann HJ, Brozek J, Restani P, Beyer K, Troncone R, Martelli A, Terrac-ciano L, Bahna SL, Rance F, Ebisawa M, Heine RG, Assa’ad A, Sampson H, Verduci E, Bouygue GR, Baena-Cagnani C, Canonica W, Lockey RF: Diagnosis and Rationale for Ac-tion Against Cow’s Milk Allergy (DRACMA): a summary report. J Allergy Clin Immunol 2010; 126: 1119–1128.

5 Hochwallner H, Schulmeister U, Swoboda I, Focke-Tejkl M, Civaj V, Balic N, Nystrand M, Harlin A, Thalhamer J, Scheiblhofer S, Keller W, Pavkov T, Zafred D, Niggemann B, Quirce S, Mari A, Pauli G, Ebner C, Papadopoulos NG, Herz U, van Tol EA, Valenta R, Spitzau-er S: Visualization of clustered IgE epitopes on alpha-lactalbumin. J Allergy Clin Immu-nol 2010; 125: 1279–1285.

6 Heine WE, Klein PD, Reeds PJ: The impor-tance of alpha-lactalbumin in infant nutri-tion. J Nutr 1991; 121: 277–283.

7 Restani P, Ballabio C, Di Lorenzo C, Tripodi S, Fiocchi A: Molecular aspects of milk aller-gens and their role in clinical events. Anal Bioanal Chem 2009; 395: 47–56.

8 Shek LP, Bardina L, Castro R, Sampson HA, Beyer K: Humoral and cellular responses to cow milk proteins in patients with milk-in-duced IgE-mediated and non-IgE-mediated disorders. Allergy 2005; 60: 912–919.

9 Lam HY, van Hoffen E, Michelsen A, Guikers K, van der Tas CH, Bruijnzeel-Koomen CA, Knulst AC: Cow’s milk allergy in adults is rare but severe: both casein and whey proteins are involved. Clin Exp Allergy 2008; 38: 995–1002.

10 Meulenbroek LA, Oliveira S, den Hartog Jag-er CF, Klemans RJ, Lebens AF, van Baalen T, Knulst AC, Bruijnzeel-Koomen CA, Garssen J, Knippels LM, van Hoffen E: The degree of whey hydrolysis does not uniformly affect in vitro basophil and T cell responses of cow’s milk allergic patients. Clin Exp Allergy 2013, DOI: 10.1111/cea.12254.

11 Jarvinen KM, Chatchatee P, Bardina L, Beyer K, Sampson HA: IgE and IgG binding epi-topes on alpha-lactalbumin and beta-lacto-globulin in cow’s milk allergy. Int Arch Al-lergy Immunol 2001; 126: 111–118.

12 Oldfield WL, Larche M, Kay AB: Effect of T-cell peptides derived from Fel d 1 on allergic reactions and cytokine production in patients sensitive to cats: a randomised controlled tri-al. Lancet 2002; 360: 47–53.

Dow

nloa

ded

by:

Uni

vers

ität Z

üric

h, Z

entr

albi

blio

thek

Zür

ich

13

0.60

.206

.43

- 7/

15/2

014

10:2

4:56

AM

Meulenbroek    et al.

Int Arch Allergy Immunol 2014;163:292–296 DOI: 10.1159/000360733

296

13 Inoue R, Matsushita S, Kaneko H, Shinoda S, Sakaguchi H, Nishimura Y, Kondo N: Identi-fication of beta-lactoglobulin-derived pep-tides and class II HLA molecules recognized by T cells from patients with milk allergy. Clin Exp Allergy 2001; 31: 1126–1134.

14 Ruiter B, Tregoat V, M’rabet L, Garssen J, Bruijnzeel-Koomen CA, Knol EF, Hoffen E: Characterization of T cell epitopes in alphas1-casein in cow’s milk allergic, atopic and non-atopic children. Clin Exp Allergy 2006; 36: 303–310.

15 Meulenbroek LA, van Esch BC, Hofman GA, den Hartog Jager CF, Nauta AJ, Willemsen LE, Bruijnzeel-Koomen CA, Garssen J, van Hoffen E, Knippels LM: Oral treatment with beta-lactoglobulin peptides prevents clinical symptoms in a mouse model for cow’s milk allergy. Pediatr Allergy Immunol 2013; 24: 656–664.

16 Schade RP, Van Ieperen-Van Dijk AG, Van Reijsen FC, Versluis C, Kimpen JL, Knol EF, Bruijnzeel-Koomen CA, Van Hoffen E: Dif-ferences in antigen-specific T-cell responses between infants with atopic dermatitis with and without cow’s milk allergy: relevance of TH2 cytokines. J Allergy Clin Immunol 2000; 106: 1155–1162.

17 Tiemessen MM, Van Ieperen-Van Dijk AG, Bruijnzeel-Koomen CA, Garssen J, Knol EF, Van Hoffen E: Cow’s milk-specific T-cell re-activity of children with and without persis-tent cow’s milk allergy: key role for IL-10. J Allergy Clin Immunol 2004; 113: 932–939.

18 Flinterman AE, Pasmans SG, den Hartog Jag-er CF, Hoekstra MO, Bruijnzeel-Koomen CA, Knol EF, van Hoffen E: T cell responses to ma-jor peanut allergens in children with and without peanut allergy. Clin Exp Allergy 2010; 40: 590–597.

19 Campbell JD, Buckland KF, McMillan SJ, Kearley J, Oldfield WL, Stern LJ, Gronlund H, van Hage M, Reynolds CJ, Boyton RJ, Cob-bold SP, Kay AB, Altmann DM, Lloyd CM, Larche M: Peptide immunotherapy in allergic asthma generates IL-10-dependent immuno-logical tolerance associated with linked epi-tope suppression. J Exp Med 2009; 206: 1535–1547.

20 Rupa P, Mine Y: Oral immunotherapy with immunodominant T-cell epitope peptides al-leviates allergic reactions in a Balb/c mouse model of egg allergy. Allergy 2012; 67: 74–82.

Dow

nloa

ded

by:

Uni

vers

ität Z

üric

h, Z

entr

albi

blio

thek

Zür

ich

13

0.60

.206

.43

- 7/

15/2

014

10:2

4:56

AM