Hence, we conclude that ADA plays an essential role in the establishment of early B cell tolerance and the removal of developing autoreactive B cells in humans. Developing autoreactive B cells are normally removed at 2 distinct checkpoints, first in the bone marrow and then in the periphery. severely impaired in all ADA-SCID patients before HSC-GT, as illustrated by the prevalence of CD19+CD10+IgMhiCD27C immature new emigrant/transitional B cells (51%C75%, compared with 5%C20% of B cells in HDs) and the decreased frequencies of CD19+CD10CIgM+CD27C mature naive B cells (Figure ?(Figure1A).1A). HSC-GT resulted in improved B cell development in all ADA-SCID Sele patients, as illustrated by decreased new emigrant/transitional B cell and increased mature naive B cell frequencies (7%C17% before HSC-GT, compared with 20%C61% after). Patients 1 and 2 also showed increased production of memory B cells, as previously reported (3), whereas no difference was observed in patient 3, who was evaluated at a shorter time after HSC-GT (Figure NMS-P515 ?(Figure1B).1B). Hence, we concluded that HSC-GT improves the development of B cells in ADA-SCID patients by allowing the progression of new emigrant/transitional B cells into mature naive B cells. Open in a separate window Figure 1 HSC-GT rescues B cell development in ADA-SCID patients. (A) CD10 and IgM expression on gated CD19+CD27C naive B cells from an age-matched HD and 3 ADA-SCID patients (ADA1CADA3) before and after HSC-GT (preGT and postGT, respectively). (B) CD27 and IgM expression on gated CD19+ B cells from an age-matched HD and 3 ADA-SCID patients before and after HSC-GT. Impaired central B cell tolerance in ADA-SCID patients. ADA deficiency has been previously reported to interfere with TCR and BCR functions by altering the intracellular concentration of cyclic AMP (28, 29). To assess whether the central B cell tolerance checkpoint, which normally removes highly polyreactive and ANA-expressing developing B cells in the bone marrow, is affected by the absence of functional ADA, we cloned antibodies expressed by single new emigrant/transitional B cells from 3 ADA-SCID patients prior to HSC-GT (Table ?(Table1)1) and tested their reactivity by ELISA (15). The reactivities of antibodies expressed by new emigrant/transitional B cells from these ADA-SCID patients were compared with their counterparts in HDs (refs. 15, 24, 30, 31, Figure ?Figure2,2, and Supplemental Tables 1C5; supplemental material available online with this article; doi: 10.1172/JCI61788DS1). We found that polyreactive new emigrant/transitional B cells were significantly increased in all 3 ADA-SCID patients compared with HDs (25%C40% of clones, compared with 5%C11%; Figure ?Figure2,2, A and B, and refs. 15, 30, 32, 33). Using indirect immunofluorescence assays with HEp-2 cellCcoated slides, we found that the proportion of ANA-expressing clones in new emigrant/transitional B cells from the 3 ADA-SCID patients (representing 20%, 18%, and 27%) was also significantly increased compared with HDs (Figure ?(Figure2C).2C). These ANAs displayed Ig heavy chain (IgH) complementarity determining regions 3 (CDR3) that contained the highest number of positively charged aas, such as arginines, previously shown to favor anti-DNA autoreactivity (Supplemental Figure 1A, Supplemental Tables 3C6, and refs. 15, 24, 30, 31). ANAs expressed by ADA-SCID B NMS-P515 cells showed a large diversity of anti-nuclear staining patterns and could be divided into those that reacted or not with the condensed chromatin material in mitotic cells (Figure ?(Figure2,2, C and D). Chromatin-nonreactive ANAs accounted for 14%C18% of new emigrant (ne) B cells of ADA-SCID patients; in contrast, chromatin-reactive ANAs represented 4%C14% of these cells (Figure ?(Figure2E).2E). The chromatin-nonreactive patterns included speckled patterns, such as neADA2-2 and neADA3-26, that may recognize nuclear proteins, anti-mitotic spindle clones (neADA1-33), and, most often, nucleolar patterns potentially associated with anti-RNA polymerase I complex antibodies (neADA1-9, neADA1-45, neADA2-209, neADA2-225, neADA3-2, and neADA3-6; Figure ?Figure2D2D and ref. 34). Most of the chromatin-reactive recombinant antibodies displayed diverse nucleolar-like clumpy staining, potentially associated with fibrillarin recognition patterns (neADA2-30, neADA3-38, and neADA3-46, Figure ?Figure2D2D and ref. 34). The reactivity of recombinant antibodies from ADA-SCID patients was further analyzed by indirect immunofluorescence on (Figure ?(Figure2,2, E and F). Antibody recognition of the kinetoplast of and were often ANA-reactive clones recognizing chromosomal material (Figure ?(Figure2F2F and Supplemental Table 6). Similarly to IRAK-4C and MyD88-deficient patients, who also fail to counterselect ANA-expressing and kinetoplast-reactive clones (24), we found that ANAs and kinetoplast-reactive antibodies expressed by new NMS-P515 emigrant/transitional B cells from ADA-SCID patients used chains (Figure ?(Figure2,2, D and F, and Supplemental Table 6), which NMS-P515 indicates that editing using chains was.