ena-STUDY-LUND UNIVERSITY-11-01-2017-07:42:34:022-142
Antibody heavy chain variable domains encoded in transcriptomes of bone marrow and peripheral blood of allergic subjects.
Bone marrow and peripheral blood was collected (2*10 ml of each) from subjects suffering from allergic rhinitis out of season of exposure to most seasonal allergens. RNA was purified, reversed transcribed into cDNA and amplified by PCR using the 5' Biomed2 primer set annealing to FR1 and 3' isotype (IgA, IgD, IgG, and IgM)-specific primers. Sequencing was performed using Illumina MiSeq with a 2x301 setup in High Output mode. Bcl to Fastq conversion was performed using bcl2Fastq v1.8.3 from the CASAVA software suite. The quality scale is Sanger / phred33 / Illumina 1.8+.For downstream analysis raw sequences were processed by the pRESTO pipeline, divided into sets encoding different isotypes and analyzed using IMGT HighV-QUEST as outlined in Levin et al (2017) (PubMed ID 27521279).Main conclusions: IgE repertoires of BM samples are more diverse and more reproducible in their composition than those of commonly sized PB samples. Samples from BM should consequently be preferred over those from PB in future studies of IgE and its sequence-based biomarkers. Studies of allergen-specific antibodies of isotypes other than IgE must not a priori be used to define the character of IgE because the degree of overlap between these repertoires is very small. Furthermore, we propose that there is a window of opportunity for NGS to assist in the assessment of successful SIT, if such treatment is able to induce a high degree of clonal overlap between IgE and other isotypes, and the IgG4 subclass in particular. Finally, we envisage that BM-encoded repertoires, through their larger diversity, may also be an excellent source of IgE diversity for the development of human monoclonal IgE through cellular cloning or combinatorial library approaches, antibodies that will enhance our understanding of IgE-allergen interactions and their outcomes.
Bone marrow and peripheral blood was collected (2*10 ml of each) from subjects suffering from allergic rhinitis out of season of exposure to most seasonal allergens. RNA was purified, reversed transcribed into cDNA and amplified by PCR using the 5' Biomed2 primer set annealing to FR1 and 3' isotype (IgA, IgD, IgG, and IgM)-specific primers. Sequencing was performed using Illumina MiSeq with a 2x301 setup in High Output mode. Bcl to Fastq conversion was performed using bcl2Fastq v1.8.3 from the CASAVA software suite. The quality scale is Sanger / phred33 / Illumina 1.8+.For downstream analysis raw sequences were processed by the pRESTO pipeline, divided into sets encoding different isotypes and analyzed using IMGT HighV-QUEST as outlined in Levin et al (2017) (PubMed ID 27521279).Main conclusions: IgE repertoires of BM samples are more diverse and more reproducible in their composition than those of commonly sized PB samples. Samples from BM should consequently be preferred over those from PB in future studies of IgE and its sequence-based biomarkers. Studies of allergen-specific antibodies of isotypes other than IgE must not a priori be used to define the character of IgE because the degree of overlap between these repertoires is very small. Furthermore, we propose that there is a window of opportunity for NGS to assist in the assessment of successful SIT, if such treatment is able to induce a high degree of clonal overlap between IgE and other isotypes, and the IgG4 subclass in particular. Finally, we envisage that BM-encoded repertoires, through their larger diversity, may also be an excellent source of IgE diversity for the development of human monoclonal IgE through cellular cloning or combinatorial library approaches, antibodies that will enhance our understanding of IgE-allergen interactions and their outcomes.