Induced B Cell Development in Adult Mice

We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed

High-Throughput Single-Cell Analysis of B Cell Receptor Usage among Autoantigen-Specific Plasma Cells in Celiac Disease

Characterization of Ag-specific BCR repertoires is essential for understanding disease mechanisms involving humoral immunity. This is optimally done by interrogation of paired H chain V region (VH) and L chain V region (VL) sequences of individual and Ag-specific B cells. By applying single-cell high-throughput sequencing on gut lesion plasma cells (PCs), we have analyzed the transglutaminase 2 (TG2)-specific VH:VL autoantibody repertoire of celiac disease (CD) patients. Autoantibodies against TG2 are a hallmark of CD, and anti-TG2 IgA-producing gut PCs accumulate in patients upon gluten ingestion. Altogether, we analyzed paired VH and VL sequences of 1482 TG2-specific and 1421 non–TG2-specific gut PCs from 10 CD patients. Among TG2-specific PCs, we observed a striking bias in IGHV and IGKV/IGLV gene usage, as well as pairing preferences with a particular presence of the IGHV5-51:IGKV1-5 pair. Selective and biased VH:VL pairing was particularly evident among expanded clones. In general, TG2-specific PCs had lower numbers of mutations both in VH and VL genes than in non–TG2-specific PCs. TG2-specific PCs using IGHV5-51 had particularly few mutations. Importantly, VL segments paired with IGHV5-51 displayed proportionally low mutation numbers, suggesting that the low mutation rate among IGHV5-51 PCs is dictated by the BCR specificity. Finally, we observed selective amino acid changes in VH and VL and striking CDR3 length and J segment selection among TG2-specific IGHV5-51:IGKV1-5 pairs. Hence this study reveals features of a disease- and Ag-specific autoantibody repertoire with preferred VH:VL usage and pairings, limited mutations, clonal dominance, and selection of particular CDR3 sequences. © 2017 American Association of Immunologist

Liquid Biopsies: Emerging role and clinical applications in solid tumours

Late detection and lack of precision diagnostics are the major challenges in cancer prevention and management. Biomarker discovery in specific cancers, especially at the pre-invasive stage, is vital for early diagnosis, positive treatment response, and good disease prognosis. Traditional diagnostic measures require invasive procedures such as tissue excision using a needle, an endoscope, and/or surgical resection which can be unsafe, expensive, and painful. Additionally, the presence of comorbid conditions in individuals might render them ineligible for undertaking a tissue biopsy, and in some cases, it is difficult to access tumours depending on the site of occurrence. In this context, liquid biopsies are being explored for their clinical significance in solid malignancies management. These non-invasive or minimally invasive methods are being developed primarily for identification of biomarkers for early diagnosis and targeted therapeutics. In this review, we have summarised the use and importance of liquid biopsy as significant tool in diagnosis, prognosis prediction, and therapeutic development. We have also discussed the challenges that are encountered and future perspective

PEC and splenic B1 cell derived IgA VH sequences display high frequencies of nucleotide exchanges due to somatic hypermutation.

<p>Frequency of mutations observed in the IgM or IgA VH region sequences derived from PEC and splenic B1 cells cultured for 4 days with the indicated combinations of IgA CSR inducing factors. (A) Each colored sector represents a particular number of mutations found at a particular frequency. Total number of sequences is shown in the middle. (B) Number of replacement and silent mutations amongst IgM and IgA VH sequences derived from indicated B cell populations. Sequences were analyzed using SEQUENCHER™ Version 4.1 for Macintosh software (Gene Codes Corporation). Assignment of VH chain was done using VBASE2 database (<a href="http://www.vbase2.org/" target="_blank">http://www.vbase2.org/</a>). Only functional sequences were included for the mutation analysis. Bars represent mean ± SD.</p

TGF-β in combination with RA synergistically induces IgA switching by PEC B cells.

<p>(A) Results of IgA and IgM ELISPOT assays performed in triplicates on sort purified PEC B cell subpopulations after 4 days of treatment with the indicated combination of stimulatory factors in culture. Surface IgA⁺ cells were excluded by sorting from B1a (IgA^-CD19^hiCD5⁺CD43⁺Mac-1⁺), B1b (IgA^-CD19^hiCD5^-CD43⁺Mac-1⁺) and B2 (IgA^-CD19^loCD5^-CD43^-Mac-1^-) B cell subpopulations. (B) Amount of secretory Igs determined by ELISA in the supernatant of B cells cultured for 4 days under the indicated stimulatory conditions. Data show the results of one of two independent experiments. Bars represent mean ± SD.</p

RA and TGF-β treated PEC B1 cells migrate to spleen and gut and produce serum and intestinal secretory IgA.

<p>IgA^- PEC B1a and B1b cells sorted in a fashion similar to previous experiments were cultured under various IgA CSR inducing conditions for 3 days and transferred intraperitoneally (16000 live cells/mouse) into lymphopenic Rag1^-/- recipients. (A) Unstimulated splenocytes and 2 days LPS (25 µg/ml) stimulated PECs of the recipient mice were analyzed for the presence of IgA and IgM producing cells by ELISPOT 2 weeks after adoptive B cell transfer. (B) Levels of secretory Igs in the serum and gut lavage of recipient mice were determined by ELISA. Per group, 2-3 mice were used as recipients. Cells pooled from the recipients belonging to the same group were used for ELISPOT assay and it was done in triplicates. Data show the results of one of two independent experiments. Bars represent mean ± SD.</p

Induced B Cell Development in Adult Mice.

We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed

RA treatment leads to up-regulation of gut homing molecules on the surface of cultured PEC B cells.

<p>Expression of surface α4β7 and CCR9 on IgA^- PEC (A) or splenic (B) B cells sorted in fashion similar to previous experiments was checked by flow cytometry after four days of culture under various combinations of stimulatory conditions. Using the same cultures (as in A and B), expression of surface IgA and IgM on PEC (C) and splenic (D) B cells was checked by flow cytometry. Data show the results of one of two independent experiments.</p