In this research study, Spaceflight is known to affect immune cell populations. Splenic B cell numbers decrease during spaceflight and in ground-based physiological models. Although antibody isotype changes have been assessed during and after space flight, an extensive characterization of the impact of spaceflight on antibody composition has not been conducted in mice. Next Generation Sequencing and bioinformatic tools are now available to assess antibody repertoires.
For B cell development and specificity, there are many heavy chain (IgH) and kappa light chain (Igκ) gene segments that are used to produce the Ig (antibody) receptor population repertoire. This antibody repertoire is quite large and the possible specificities can theoretically exceed the number of actual antibody molecules in the host.
In antibodies, the antigen binding region is formed by six complementarities determining regions (CDRs) that loop out from the V region backbone formed by two beta-pleated sheets. The germline V gene segment repertoire is necessary for host responses to pathogens and CDR1 and CDR2 are completely encoded for by variable (V region) gene segments
Spaceflight presents unique difficulties in the collection, preparation and preservation of cells and tissues. Normal preparation methods such as the creation of single-cell suspensions are difficult and normal tissue preservations methods such as the use of liquid nitrogen are unavailable.
To determine the acceptability of whole tissue preparations compared to more traditional single cell suspensions, we examined the differences in Ig sequences obtained from both treatment groups. We were concerned that tissue isolation methods may introduce artifacts into the data since many studies specifically focus on single cell suspensions; often sorted, to isolate B cells specifically.
Our data indicate that comparable results were obtained from both the tissue and the cells treatment groups. There were strong correlations in V-gene usage and the CDR3 sequences identities were very similar.
In conclusion, our goals for this project were to examine the breadth of the antibody repertoire gathering information about V, D, J, and constant region usage and CDR3 composition and to lay the foundation for future studies that will examine the immune response to vaccination during space flight.
They have determined that whole tissue preparations as will be available from the ISS will yield similar results when examining the antibody repertoire. By performing a size selection to isolate likely antibody sequences provided the highest number of Ig reads. A novel workflow using multiple mapping methods.
To characterize NGS data for Ig repertoire data was developed and genome and reference mapping methods were validated using publically available datasets. This novel workflow can be used for future studies on the antibody repertoire regardless of whether they are ISS- or ground-based.