Unraveling the Repertoire in Wiskott–Aldrich Syndrome

Human immunology is entering the next frontier. With the fast developing technology, we can today sequence the whole genome from an individual in a relatively short time. It is now possible to decipher pathological mechanisms in immunological diseases, including primary immunodeficiencies, with high specificity. A particularly interesting aspect to study is the development and maintenance of the immune repertoire diversity and its consequences for disease progression. Until recently, a major difficulty in analysis of peripheral blood cells has been to sequence the locus encoding the T cell receptor (TCR) and the B cell receptor (BCR) in each cell. These receptors are assembled from a large array of V, (D), and J gene segments in a process that inserts and deletes nucleotides in the V(D)J junctions (Figures 1A,B). The antigenic specificity of the BCR and TCR is determined by the complementaritydetermining regions (CDR) 1–3, where the CDR3 covers the junctions between the V, (D), and J segments and is the most variable part of the receptor (Figures 1A,B). To add to the complexity, B cells undergo further gene diversification in the peripheral germinal centers by class switch recombination and induction of somatic hypermutations. Mutations in the Wiskott–Aldrich syndrome protein (WASp) cause the severe immunodeficiency disease Wiskott– Aldrich syndrome (WAS) (1, 2). WAS has been associated with numerous cellular defects and is termed a cell-trafficking disease of the immune system. WAS−/− B cells are hyperactive and induce an autoreactive response, whereas WAS−/− T cells are hyporesponsive and WAS−/− T regulatory cells fail to suppress effector T cells (1, 2). Based on the important role of WASp for peripheral function, it has been somehow surprising that B and T cell development is intact as evident in normal progression through maturation stages in the bone marrow and thymus, respectively (2, 3). The role of WASp in creating a diverse BCR and TCR repertoire has until recently remained unknown. Since a skewed and oligoclonal BCR and TCR repertoire is linked to autoimmunity (2), a number of laboratories have now addressed the immune receptor repertoire in WAS patients. Two recent studies show that B cells from WAS patients have a decreased BCR repertoire, altered V gene usage, and decreased somatic hypermutation (4, 5). In T cells, Wada et al. showed already in 2005 that the TCRVβ repertoire was skewed in WAS patients older than 15 years, while younger WAS patients showed no repertoire skewing (6). This year, Braun et al. and Wu et al. showed that also young WAS patients often had a skewed TCRVβ repertoire (7, 8). O’Connell and colleagues (9) have in the present investigation examined the BCR and TCR repertoire in WAS patients using next generation sequencing [NGS, see recent reviews in Ref. (10, 11)]. Using this technique, the authors collected a vast amount of data that allowed them to analyze the diversity of the receptor repertoire, including V(D)J segment usage, CDR3 size distribution, clonal expansions, and for BCRs; class switch recombination and frequency of somatic hypermutations. In B cells from WAS patients, the repertoire diversity tended to be lower than in controls, and the usage of some V heavy chain (VH) gene segments was skewed. However, WAS B cells had normal rate of somatic hypermutation. In T cells of WAS patients, clonal expansions were present in the memory CD4 T cells and both in naïve and memory CD8 T cells. The usage of TCRVβ gene segments tended to be skewed to a higher extent in WAS patient CD8 T cells than in WAS patient CD4 T cells. These results provide valuable information regarding the development and maintenance of the immune repertoire in WAS and importantly, describes alterations mainly in CD8 T cells. Interestingly, one out of the three patients in the study by O’Connell et al. showed increased VH4–34 expression that recognizes self-antigens (12). Two other recent studies have examined the BCR diversification in WAS patient B cells and also found overrepresentation of the VH4– 34 gene (4, 5). This suggests that autoreactive B cells are expanded in WAS patients, and the current study indicates that these B cells may be present even before clinical signs of autoimmune disease. Simon et al. and Castiello et al. describe decreased somatic hypermutation in WAS patients