Abstract
We investigated how the equine fetus prepares its pre-immune humoral repertoire for an imminent exposure to pathogens in the neonatal period, particularly how the primary hematopoietic organs are equipped to support B cell hematopoiesis and immunoglobulin (Ig) diversity. We demonstrated that the liver and the bone marrow at approximately 100 days of gestation (DG) are active sites of hematopoiesis based on the expression of signature messenger RNA (mRNA) (c-KIT, CD34, IL7R, CXCL12, IRF8, PU.1, PAX5, NOTCH1, GATA1, CEBPA) and protein markers (CD34, CD19, IgM, CD3, CD4, CD5, CD8, CD11b, CD172A) of hematopoietic development and leukocyte differentiation molecules, respectively. To verify Ig diversity achieved during the production of B cells, V(D)J segments were sequenced in primary lymphoid organs of the equine fetus and adult horse, revealing that similar heavy chain VDJ segments and CDR3 lengths were most frequently used independent of life stage. In contrast, different lambda light chain segments were predominant in equine fetal compared to adult stage, and surprisingly, the fetus had less restricted use of variable gene segments to construct the lambda chain. Fetal Igs also contained elements of sequence diversity, albeit to a smaller degree than that of the adult horse. Our data suggest that the B cells produced in the liver and bone marrow of the equine fetus generate a wide repertoire of pre-immune Igs for protection, and the more diverse use of different lambda variable gene segments in fetal life may provide the neonate an opportunity to respond to a wider range of antigens at birth.
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Abbreviations
- Ig:
-
Immunoglobulin
- DG:
-
Days of gestation
- HSC:
-
Hematopoietic stem cell
- mRNA:
-
Messenger RNA
- RT-PCR:
-
Reverse transcription polymerase chain reaction
- CD:
-
Cluster of differentiation
- CDR:
-
Complementary determining region
- AGM:
-
Aorta-gonad-mesonephros
- YS:
-
Yolk sac
- IGHV:
-
Ig heavy chain variable gene
- IGHD:
-
Ig heavy chain diversity gene
- IGHJ:
-
Ig heavy chain joining gene
- IGLV:
-
Ig lambda light chain variable gene
- IGLJ:
-
Ig lambda light chain joining gene
- IGLC:
-
Ig lambda light chain constant gene
- IGKV:
-
Ig kappa light chain variable gene
- IGKJ:
-
Ig kappa light chain joining gene
- IGKC:
-
Ig kappa light chain constant gene
- TdT:
-
Terminal deoxynucleotidyl transferase
- N-nucleotide:
-
Non-template nucleotide
- P-nucleotide:
-
Palindromic nucleotide
- PBS:
-
Phosphate-buffered solution
- cDNA:
-
Complementary DNA
- dNTPs:
-
The four deoxynucleotide triphosphates
- M-MuLV:
-
Moloney murine leukemia virus
- CLP:
-
Common lymphoid progenitor
- TBS:
-
Tris-buffered saline
- RACE:
-
Rapid amplification of cDNA ends
- MHC:
-
Major histocompatibility complex
- AEC:
-
3-Amino-9-ethylcarbazole
- FITC:
-
Fluorescein isothiocyanate
- LB broth:
-
Luria-Bertani broth
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Acknowledgments
We would like to thank Steven C. Miller and Mary Beth Matychak for their technical support. This research was partially funded by the NIH Director’s New Innovator Award 1 DP2 OD007216.
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Battista, J.M., Tallmadge, R.L., Stokol, T. et al. Hematopoiesis in the equine fetal liver suggests immune preparedness. Immunogenetics 66, 635–649 (2014). https://doi.org/10.1007/s00251-014-0799-9
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DOI: https://doi.org/10.1007/s00251-014-0799-9