Human IgE repertoires

Background and aim

Approach

 

To further the understanding of allergic reactions we have sequenced genes encoding human gE as it is found in allergic individuals (i.e. in allergy-encoding transcriptomes). We ahave lso (by phage display technology) isolated sequences encoding allergen-specific IgE and characterized such proteins in order to define the diversity of recognition of allergens as it occurs in human allergy.

 

The human IgE-encoding transcriptome

     

• We demonstrated that the IgE-encoding transcriptome was highly diverse with respect to V gene usage and showed evidence of somatic hypermutation similar to that found in repertoires encoding e.g. IgG (Andréasson et al., 2006).
• We showed that despite its diversity the size of the IgE-encoding transcriptomes in these two allergic individuals was highly restricted, i.e. just a few clonotypes populated the entire population of IgE-poducing B cells. Each set of related clones had, however, been diversified and routes taken in the evolution of these clonotypes could be followed (Figure 1) (Andréasson et al., 2006). 
• We demonstrated diversity and somatic hypermutation in IgE repertoires in sinus mucosa of patients diagnosed with non-allergic (as assessed by conventional diagnostic tests) sinusitis involving fungal infection and eosinophilic mucus (Levin et al., 2011).

Figure 1. Evolution paths taken by genes encoding IgE with an origin in three different germline genes. The number of mutations differing the various members of these clonotypes derived from different germline genes are shown next to the arrows. (Andréasson et al., 2006)

 

• It was shown that very similar rearrangements populated the IgE-encoding transcriptome even in different individuals. Thus, it is likely that for certain epitopes as found on allergens, there are only few ways in which a selectable antigen-binding site can be created based on the available repertoire (Figure 2) (Andréasson et al., 2006). This notion was further explored in a subsequent study (Persson et al., 2008) that demonstrated such similar antibodies targeting the important grass-pollenallergen Phl p 2 (see below).

 

Figure 2. Two similar rearrangements identified in two different individuals (A3 and A4). These rearrangements, that due to their similarity shared a common specificity, originated from the same VH and JH genes but used different D-genes. Despite the differences in D gene usage, the rearrangement resulted in almost identical CDRH3 sequences. Several mutations were also shared or very similar (stars) in-between the different clonotypes as they evolve in vivo, further supporting the notion that they evolved their affinity for the same antigen. (Andréasson et al., 2006)

 

 

Allergen-specific repertoires in the IgE population

 

In order to address the composition of allergen-specifi recognition of IgE, we cloned genes encoding heavy chain variable domains as found in the IgE with genes encoding human kappa and lambda light chain sequences. Combinatorial antibody libraries have been made and allergen-specific antibody fragments were selected by phage display technology.

1. The antibody repertoire targeting the major timothy allergen Phl p 5 has been characterized (Andréasson et al., 2006; Persson et al., 2007).

• Antibody fragments specific for the important thimoty allergen Phl p 5 were selected from our combinatorial libraries. Their sequences demonstarted that they were derived from a diverse set of VH genes. derived from the IGHV1, 3 and 5 subgroups. Thus the response against a single allergen may be complex in geneic terms.
• We demonstrated that specific antibodies against Phl p 5 targeted a range of epitopes on this allergen. At least five such epitopes, three of which are located on a 110 aa long fragment of Phl p 5, were identified.
• We were able to put one specific clone, scFv 4.2, into one of the clonotype familes identified above Thus evolutionary paths, as it occurs in vivo, taken by an allergen-specific human clone targeting an biologically relevant, known antigen have been established (Figure 3). Thus by using the toolbox available to us on the IgE-encoding transcriptome, it is possible to define evolutionary paths taken during human immune responses.

Figure 3. Evolution of a clonotype derived from the IGHV5-51 germline gene that has been shown to encode a known specficity, namely Phl p 5. This particular clonotype amounted to approximately 9% of all transcript in the IgE-encoding transcriptome found in peripheral blood B cells of an allergic individual. The numbers next to the arrows indicate the number of nucleotide substitutions that differ the various members in this proposed evolution pathway. (Andréasson et al., 2006)

2. To further assess the make-up of the IgE-encoding transcripome, we selected specific binders for additional timothy pollen allergens, namely Phl p 1, Phl p 2, Phl p 4, Phl p 6 and Phl p 11 (Persson et al., 2007).

• We concluded that the response against timothy allergens is diverse and involves genes with an origin in all major VH subgroups (IGHV1, 3, 4 and 5)).
• We defined part of the limited set of antigens that are involved in mounting an IgE response as the 5 investigated timothy allergens accounted for not less than 25% of the Ig-encoding transcriptome of this individual.
• IgE antibodies developed by this phage display approach have recently been shown to be biologically active and to mediate biological activity in the form of antibody-enhanced antigen presentation by dendritic cells that induces a TH2-type immune response (Lundberg et al., 2008).

Altogether, we have developed a unique panel of human IgE specificities targeting six different groups of grass pollen allergens, a probe resource for future assessment of the recognition of allergens by the human humoral immune system. Collectively they represent the first extensive set of an Anti-Allergome Resouce, i.e. sets of IgE representing the human antibody reponse that can interact with the much more extensively characterized Allergome, the set of allergenic proteins that induce type I hypersensitivity reactions by activating effector cells of the immune system that carry IgE.

  

A common idiotype in IgE repertoires

 

We recently isolated human antibody fragments against the timothy allergen Phl p 2 (Persson et al., 2007) from an antibody library with an origin in the IgE-encoding transcriptome. Interestingly, the sequence of this scFv was very similar to a set of other antibody fragments specific for the same allergen but derived from a library developed from transcripts from another subject (Persson et al., 2008). Furthermore, some transcripts encoding IgE VH in other individuals allergic to grass pollen allergens were very similar in sequence, too. It thus appear that a common sequence motif is involved in the development of specific antibodies targeting a major epitope in the allergen Phl p 2. These findings extend the observation of existence of restricted antibody repertoires (previously defined in e.g. the response against haptens, viral and bacterial antigens, xenoantigens and autoantigens) also to human antibody repertoires targting allergens.

 

General conclusion

 

We have demonstrated that molecular library technology can be used to unravel molecular details of IgE as it occurs in allergic disease. It can also be used to develop unique research reagents targeting allergens. We envisage that such knowledge and reagents will help us understand allergic disease and to improve treatment, diagnosis and prevention of such diseases in the future.

 

References

• Andréasson U, Flicker S, Lindstedt M, Valenta R, Greiff L, Korsgren M, Borrebaeck CAK and Ohlin M (2006) The human IgE-encoding transcriptome to assess antibody repertoires and repertoire evolution. J Mol Biol 362, 212-227. (Abstract)
• Persson H, Karbalaei Sadegh M, Greiff L and Ohlin M (2007) Delineating the specificity of an IgE-encoding transcriptome. J Allergy Clin Immunol 120, 1186-1192. (Abstract) (see comment in the journal's Editors' choice section)
• Persson H (2007) Generation and evolution of human antibody repertoires. (doctoral thesis, Lund University) (Abstract)
• Persson H, Flicker S, Karbalaei Sadegh M, Valenta R, Greiff L and Ohlin M (2008) A common idiotype in IgE and its relation to recognition of the grass pollen allergen Phl p 2. Mol Immunol 45, 2715-2720. (Abstract)
• Persson J, Augustsson P, Laurell T and Ohlin M (2008) Acoustic microfluidic chip technology to facilitate automation of phage display selection. FEBS J 275, 5657-5666. (Abstract) (journal cover)
• Lundberg K, Lindstedt M, Larsson K, Dexlin L, Wingren C, Ohlin M, Greiff L and Borrebaeck CAK (2008) Augmented Phl p 5-specific Th2 response after exposure of dendritic cells to allergen in complex with specific IgE compared to IgG1 and IgG4. Clin Immunol 128, 358-365. (Abstract)
• Levin M, Tan LW, Baker L, Wormald PJ, Greiff L, Ohlin M (2011) Diversity of IgE-encoding transcripts in sinus mucosa of subjects diagnosed with non-allergic fungal eosinophilic sinusitis. Clin Exp Allergy 41, 811-820. (Abstract)
• Levin M, Ohlin M (2013) Inconclusive evidence for or against positive antigen selection in the shaping of human IgE repertoires - a call for new approaches. Int Arch Allergy Immunol 161, 122-126. (Abstract)

    

This project has been supported by grants from

   

• Alfred Österlund's Foundation
• the Swedish Research Council
• the Konsul Th C Bergh's Foundation
• the Swedish Asthma- and Allergy Association's Research Foundation

  

Contact

 

Dr. Mats Ohlin
Dept. of Immunotechnology
Lund University
Medicon Village (Building 406)
S-223 81 LUND
Sweden
telephone: +46-46-222 4322

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