Genetic background and immunological status influence B cell repertoire diversity in mice

International audience; The relationship between the immune repertoire and the physiopathological status of individuals is essential to apprehend the genesis and the evolution of numerous pathologies.

Nevertheless, the methodological approaches to understand these complex interactions are challenging.

We performed a study evaluating the diversity harbored by different immune repertoires as a function of their physiopathological status.

In this study, we base our analysis on a murine scFv library previously described and representing four different immune repertoires: i) healthy and naïve, ii) healthy and immunized, iii) autoimmune prone and naïve, and iv) autoimmune prone and immunized.

This library, 2.6 × 10 9 in size, is submitted to high throughput sequencing (Next Generation Sequencing, NGS) in order to analyze the gene subgroups encoding for immunoglobulins.

A comparative study of the distribution of immunoglobulin gene subgroups present in the four libraries has revealed shifts in the B cell repertoire originating from differences in genetic background and immunological status of mice.

The adaptive immune system is capable of producing antibodies against a large number of immunogens.

This vast diversity of immunoglobulin sequences is not provided by the limited number of genes present in the genome, but by rearrangements of the germline at specific loci.

In the case of B cell receptors, rearrangement of variable (V), diversity (D), and joining (J) gene segments in V-Domain creates a combinatorial diversity for the immu-noglobulin heavy chain (IGH), whereas rearrangement of V and J gene segments provides a similar diversity for the lambda or kappa light chains (IGL/IGK) 1 (Fig. 1).

Additionally, at the junctions of V-D and D-J segments, a process of random deletion and addition of nucleotides creates an immense junctional diversity.

Finally, somatic hypermutations focused on Complementary Determining Regions (CDR) supplement the mechanisms of immu-noglobulin maturation, expanding still further the diversity and leading to affine and specific antibodies.

Studies have shown that this vast diversity, as well as other characteristics of the immune repertoire, can be influenced by factors such as immunization 2,3 or pathology, notably autoimmune diseases 4-6.

Generation of antibody libraries is a crucial step in the attempt to study in vivo immune repertoires 7,8.

Care needs to be taken to ensure the coverage of a large antibody sequence diversity in order to mimic the natural B cell repertoire as close as possible.

Recently, we have described an original strategy allowing to improve the library construction process and increase its diversity 9.

This strategy is based on a technological optimization relying on Rolling Circle Amplification (RCA), combined with a newly designed set of oligonucleotide primers based on a thorough analysis of the IMGT/LIGM-DB database 10.

In the present study, we have used this strategy to generate libraries form two murine inbred strains were used, namely Balb/C (healthy) and SJL/J (susceptible to autoimmune disease), together representing 4 different IgG immune repertoires: i) healthy and naïve (NB for naïve Balb/C), ii) healthy and immunized (IB for immunized Balb/C), iii) autoimmune prone and naïve (NS for naïve SJL/J), and iv) autoimmune prone and immunized (IS for immunized SJL/J) 11.

We have decidedly chosen to