THE IGE REPERTOIRE IN HEALTHY, NON-ALLERGIC INDIVIDUALS: IMPLICATIONS FOR EFFICIENT ALLERGEN DESENSITATION IMMUNOTHERAPY
(Abstract release date: 05/19/16)
EHA Library. Koning M. 06/09/16; 132671; E1122
Dr. Marvyn Koning
Contributions
Contributions
Abstract
Abstract: E1122
Type: Eposter Presentation
Background
Type I allergic disease is a hypersensitivity reaction mediated by allergen-specific IgE. Allergen desensitation immunotherapy consists of serial injections of increasing doses of the allergen. A possible mechanism for the restriction of the severity of type I allergy by desensitation is the induction of allergen-specific neutralizing antibodies of non-IgE isotype. It is unclear whether allergen-specific IgG clones in desensitised individuals have been recruited form the naive repertoire or from pre-existing clones from which the IgE clones originated. The largest reported IgE repertoire from a single person comprises 32 immunoglobulin sequences.
Aims
To characterise the IgE repertoire in healthy donors in-depth by an unbiased approach.
Methods
Peripheral blood CD19+ cells from six healthy donors were isolated by magnetic bead separation and divided into aliquots of 2x106 cells. For each donor, mRNA was amplified from 5 aliquots by ARTISAN PCR, an anchored RT-PCR primed on constant immunoglobulin regions and employing a template-switching reverse transcriptase. ARTISAN PCR was performed in parallel to obtain IgE, IgG, and IgM libraries. PacBio sequencing of the resulting 15 amplicon libraries per donor yielded a median of 284 (range: 100-743) full-length sequences/aliquot and 27500 sequences in total.
Results
A median of 261 (range: 175-358) unique IgE sequences were obtained per donor. 9.1-61.8% of individual IgE sequences were represented in >2 aliquots of the same donor. In contrast, 1.4-7.8% IgM and 5.7-16.3% IgG sequences, respectively, were represented in multiple aliquots. 29 unique VDJ sequences were identified in both IgM and IgG compartments. All IgE sequences were strictly restricted to their isotype. Somatic mutation loads per sequence differed significantly between IgE (median: 5.6%), IgM (0.3%), and IgG (8.3%). Intraclonal sequence diversity in IgE clones was predominantly due to silent framework region mutations. IGHV3-11, IGHV3-9, and the IGHV3 family were significantly overrepresented in IgE compared to IgM/IgG. IGHV3-30, IGHV3-33, IGHV4-59, and IGHV2, 4, 5, and 6 families were underrepresented.
Conclusion
These data provide a reference for investigations of the IgE repertoire in allergy and parasitology. In healthy donors, the peripheral IgE repertoire is less diverse than IgM/IgG, displays a strong IGHV bias, and carries an intermediate mutation load without evidence for ongoing affinity maturation. IgE and IgG/IgM repertoires apparently do not overlap. The lack of overlap between IgE sequences and expanded IgG clones, as well as the higher mutation load of the latter, suggest that most IgE sequences have undergone class switch recombination directly from IgM, rather than from class-switched expanded IgG clones as intermediates. These findings suggest that desensitisation strategies should focus on recruiting novel allergen-specific cells from the naïve B-cell compartment, rather than attempting to activate and expand existing IgG-expressing B cells that belong to the same B-cell clone and have the identical antigen specificity as the allergy-causing B-cells that have undergone alternative or subsequent class switching to IgE.
Session topic: E-poster
Keyword(s): B cell subsets, B lymphocyte, IgH rearrangment
Type: Eposter Presentation
Background
Type I allergic disease is a hypersensitivity reaction mediated by allergen-specific IgE. Allergen desensitation immunotherapy consists of serial injections of increasing doses of the allergen. A possible mechanism for the restriction of the severity of type I allergy by desensitation is the induction of allergen-specific neutralizing antibodies of non-IgE isotype. It is unclear whether allergen-specific IgG clones in desensitised individuals have been recruited form the naive repertoire or from pre-existing clones from which the IgE clones originated. The largest reported IgE repertoire from a single person comprises 32 immunoglobulin sequences.
Aims
To characterise the IgE repertoire in healthy donors in-depth by an unbiased approach.
Methods
Peripheral blood CD19+ cells from six healthy donors were isolated by magnetic bead separation and divided into aliquots of 2x106 cells. For each donor, mRNA was amplified from 5 aliquots by ARTISAN PCR, an anchored RT-PCR primed on constant immunoglobulin regions and employing a template-switching reverse transcriptase. ARTISAN PCR was performed in parallel to obtain IgE, IgG, and IgM libraries. PacBio sequencing of the resulting 15 amplicon libraries per donor yielded a median of 284 (range: 100-743) full-length sequences/aliquot and 27500 sequences in total.
Results
A median of 261 (range: 175-358) unique IgE sequences were obtained per donor. 9.1-61.8% of individual IgE sequences were represented in >2 aliquots of the same donor. In contrast, 1.4-7.8% IgM and 5.7-16.3% IgG sequences, respectively, were represented in multiple aliquots. 29 unique VDJ sequences were identified in both IgM and IgG compartments. All IgE sequences were strictly restricted to their isotype. Somatic mutation loads per sequence differed significantly between IgE (median: 5.6%), IgM (0.3%), and IgG (8.3%). Intraclonal sequence diversity in IgE clones was predominantly due to silent framework region mutations. IGHV3-11, IGHV3-9, and the IGHV3 family were significantly overrepresented in IgE compared to IgM/IgG. IGHV3-30, IGHV3-33, IGHV4-59, and IGHV2, 4, 5, and 6 families were underrepresented.
Conclusion
These data provide a reference for investigations of the IgE repertoire in allergy and parasitology. In healthy donors, the peripheral IgE repertoire is less diverse than IgM/IgG, displays a strong IGHV bias, and carries an intermediate mutation load without evidence for ongoing affinity maturation. IgE and IgG/IgM repertoires apparently do not overlap. The lack of overlap between IgE sequences and expanded IgG clones, as well as the higher mutation load of the latter, suggest that most IgE sequences have undergone class switch recombination directly from IgM, rather than from class-switched expanded IgG clones as intermediates. These findings suggest that desensitisation strategies should focus on recruiting novel allergen-specific cells from the naïve B-cell compartment, rather than attempting to activate and expand existing IgG-expressing B cells that belong to the same B-cell clone and have the identical antigen specificity as the allergy-causing B-cells that have undergone alternative or subsequent class switching to IgE.
Session topic: E-poster
Keyword(s): B cell subsets, B lymphocyte, IgH rearrangment
Abstract: E1122
Type: Eposter Presentation
Background
Type I allergic disease is a hypersensitivity reaction mediated by allergen-specific IgE. Allergen desensitation immunotherapy consists of serial injections of increasing doses of the allergen. A possible mechanism for the restriction of the severity of type I allergy by desensitation is the induction of allergen-specific neutralizing antibodies of non-IgE isotype. It is unclear whether allergen-specific IgG clones in desensitised individuals have been recruited form the naive repertoire or from pre-existing clones from which the IgE clones originated. The largest reported IgE repertoire from a single person comprises 32 immunoglobulin sequences.
Aims
To characterise the IgE repertoire in healthy donors in-depth by an unbiased approach.
Methods
Peripheral blood CD19+ cells from six healthy donors were isolated by magnetic bead separation and divided into aliquots of 2x106 cells. For each donor, mRNA was amplified from 5 aliquots by ARTISAN PCR, an anchored RT-PCR primed on constant immunoglobulin regions and employing a template-switching reverse transcriptase. ARTISAN PCR was performed in parallel to obtain IgE, IgG, and IgM libraries. PacBio sequencing of the resulting 15 amplicon libraries per donor yielded a median of 284 (range: 100-743) full-length sequences/aliquot and 27500 sequences in total.
Results
A median of 261 (range: 175-358) unique IgE sequences were obtained per donor. 9.1-61.8% of individual IgE sequences were represented in >2 aliquots of the same donor. In contrast, 1.4-7.8% IgM and 5.7-16.3% IgG sequences, respectively, were represented in multiple aliquots. 29 unique VDJ sequences were identified in both IgM and IgG compartments. All IgE sequences were strictly restricted to their isotype. Somatic mutation loads per sequence differed significantly between IgE (median: 5.6%), IgM (0.3%), and IgG (8.3%). Intraclonal sequence diversity in IgE clones was predominantly due to silent framework region mutations. IGHV3-11, IGHV3-9, and the IGHV3 family were significantly overrepresented in IgE compared to IgM/IgG. IGHV3-30, IGHV3-33, IGHV4-59, and IGHV2, 4, 5, and 6 families were underrepresented.
Conclusion
These data provide a reference for investigations of the IgE repertoire in allergy and parasitology. In healthy donors, the peripheral IgE repertoire is less diverse than IgM/IgG, displays a strong IGHV bias, and carries an intermediate mutation load without evidence for ongoing affinity maturation. IgE and IgG/IgM repertoires apparently do not overlap. The lack of overlap between IgE sequences and expanded IgG clones, as well as the higher mutation load of the latter, suggest that most IgE sequences have undergone class switch recombination directly from IgM, rather than from class-switched expanded IgG clones as intermediates. These findings suggest that desensitisation strategies should focus on recruiting novel allergen-specific cells from the naïve B-cell compartment, rather than attempting to activate and expand existing IgG-expressing B cells that belong to the same B-cell clone and have the identical antigen specificity as the allergy-causing B-cells that have undergone alternative or subsequent class switching to IgE.
Session topic: E-poster
Keyword(s): B cell subsets, B lymphocyte, IgH rearrangment
Type: Eposter Presentation
Background
Type I allergic disease is a hypersensitivity reaction mediated by allergen-specific IgE. Allergen desensitation immunotherapy consists of serial injections of increasing doses of the allergen. A possible mechanism for the restriction of the severity of type I allergy by desensitation is the induction of allergen-specific neutralizing antibodies of non-IgE isotype. It is unclear whether allergen-specific IgG clones in desensitised individuals have been recruited form the naive repertoire or from pre-existing clones from which the IgE clones originated. The largest reported IgE repertoire from a single person comprises 32 immunoglobulin sequences.
Aims
To characterise the IgE repertoire in healthy donors in-depth by an unbiased approach.
Methods
Peripheral blood CD19+ cells from six healthy donors were isolated by magnetic bead separation and divided into aliquots of 2x106 cells. For each donor, mRNA was amplified from 5 aliquots by ARTISAN PCR, an anchored RT-PCR primed on constant immunoglobulin regions and employing a template-switching reverse transcriptase. ARTISAN PCR was performed in parallel to obtain IgE, IgG, and IgM libraries. PacBio sequencing of the resulting 15 amplicon libraries per donor yielded a median of 284 (range: 100-743) full-length sequences/aliquot and 27500 sequences in total.
Results
A median of 261 (range: 175-358) unique IgE sequences were obtained per donor. 9.1-61.8% of individual IgE sequences were represented in >2 aliquots of the same donor. In contrast, 1.4-7.8% IgM and 5.7-16.3% IgG sequences, respectively, were represented in multiple aliquots. 29 unique VDJ sequences were identified in both IgM and IgG compartments. All IgE sequences were strictly restricted to their isotype. Somatic mutation loads per sequence differed significantly between IgE (median: 5.6%), IgM (0.3%), and IgG (8.3%). Intraclonal sequence diversity in IgE clones was predominantly due to silent framework region mutations. IGHV3-11, IGHV3-9, and the IGHV3 family were significantly overrepresented in IgE compared to IgM/IgG. IGHV3-30, IGHV3-33, IGHV4-59, and IGHV2, 4, 5, and 6 families were underrepresented.
Conclusion
These data provide a reference for investigations of the IgE repertoire in allergy and parasitology. In healthy donors, the peripheral IgE repertoire is less diverse than IgM/IgG, displays a strong IGHV bias, and carries an intermediate mutation load without evidence for ongoing affinity maturation. IgE and IgG/IgM repertoires apparently do not overlap. The lack of overlap between IgE sequences and expanded IgG clones, as well as the higher mutation load of the latter, suggest that most IgE sequences have undergone class switch recombination directly from IgM, rather than from class-switched expanded IgG clones as intermediates. These findings suggest that desensitisation strategies should focus on recruiting novel allergen-specific cells from the naïve B-cell compartment, rather than attempting to activate and expand existing IgG-expressing B cells that belong to the same B-cell clone and have the identical antigen specificity as the allergy-causing B-cells that have undergone alternative or subsequent class switching to IgE.
Session topic: E-poster
Keyword(s): B cell subsets, B lymphocyte, IgH rearrangment
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