| Data source |
GEO: GSE158102
|
| Description |
Three healthy rainbow trout were killed by benzocaine overdose. To isolate peripheral blood leukocytes (PBLs) blood was drawn with a heparinized needle from the caudal vein and diluted 10 times with of Leibovitz medium. The resulting cell suspension was placed onto 51% Percoll density cushions collecting the interface cells after centrifugation. Leukocytes were incubated with anti-trout MHC-II APC and YO-PRO in FACS staining buffer. MHC-II+ leukocytes from lymphoid gate were isolated by sorting. A total of 2,500 cells (700 cells/?L) from each fish were loaded to chips from the Chromium Single Cell 5' Gel Beads Kit (10x Genomics, CA, USA) and subjected to the Chromium Controller Instrument (10x Genomics) to generate single-cell gel beads in emulsion following manufacturer's instructions. Libraries for sequencing were constructed using Chromium Single Cell 5' Library Construction Kit following manufacturer's instructions. Libraries were sequenced using a NextSeq instrument (Illumina) with 150PE chemistry. |
| Key word |
[1]channel catfish; gene-expression; one antibody; exclusion; isotypes; igt; [2]teleost; b cells; single cell transcriptomics; immunoglobulins; immune markers; transcription factors; long non-coding rnas; toll-like receptor; t-cell; differential expression; regulatory factor 4; cc-chemokine; teleost fish; roles; identification; proliferation |
| Publication |
[1] Perdiguero P, Morel E, D P, Tafalla C. Individual B cells transcribe multiple rearranged immunoglobulin light chains in teleost fish. iScience 2021 Jun 25;24(6):102615. PMID: 34142062. [2]Perdiguero P, Morel E, Tafalla C. Diversity of Rainbow Trout Blood B Cells Revealed by Single Cell RNA Sequencing. Biology (Basel) 2021 Jun 9;10(6). PMID: 34207643 |
| Abstract |
[1]B cells express a unique antibody protein which comprises two pairs of immunoglobulin (Ig) heavy (H) and light (L) chains. In addition to an invariable constant (C) region, IgH and IgL chains encompass a variable (V) region mediating antigen binding. This unique region stems from Ig V(D)J gene recombination, which generates diversity by assembling these gene segments into VHDJH and VLJL genes. To ensure that one B cell only expresses one antibody, VHDJH rearrangement occurs only in one IgH locus (allelic exclusion), whereas VLJL rearrangement only in either the k or locus (isotype exclusion). However, teleosts express multiple IgLs encoded by distinct CL genes. Using single-cell transcriptomics, we have demonstrated the transcription of distinct rearranged VLJLCL genes in single rainbow trout B cells. Our results highlight the laxity of isotype exclusion in teleosts and strongly suggest that fish B cells can produce antibodies of different specificities. [2]Single-cell sequencing technologies capable of providing us with immune information from dozens to thousands of individual cells simultaneously have revolutionized the field of immunology these past years. However, to date, most of these novel technologies have not been broadly applied to non-model organisms such as teleost fish. In this study, we used the 10X Genomics single cell RNA sequencing technology and used it to analyze for the first time in teleost fish the transcriptional pattern of single B cells from peripheral blood. The analysis of the data obtained in rainbow trout revealed ten distinct cell clusters that seem to be associated with different subsets and/or maturation/differentiation stages of circulating B cells. The potential characteristics and functions of these different B cell subpopulations are discussed on the basis of their transcriptomic profile. The results obtained provide us with valuable information to understand the biology of teleost B cells and offer us a repertoire of potential markers that could be used in the future to differentiate trout B cell subsets. |
