wiskott  aldrich syndrome ( was ) is a monogenic x - linked primary immunodeficiency characterized by the classic triad of microthrombocytopenia , eczema and recurrent infections . multiple autoimmune manifestations and tumors are serious complications and the life expectancy of was patients is severely reduced , unless they are successfully cured by hematopoietic stem cell transplantation . was is caused by mutations in the was gene   impairing the  expression and/or function of the was protein ( wasp ) , a  hematopoietic - specific regulator of cytoskeletal reorganization also involved in signal transduction of cells . the absence or reduction of wasp compromises multiple processes of different immune cell types involved in innate and adaptive responses , resulting in a variable and progressive immunodeficiency . susceptibility of was patients to develop autoimmune diseases has been mainly attributed to the breakdown of self tolerance sustained by dysfunction of both natural t regulatory and effector t cells ( reviewed in ref . ) . however , b - cell intrinsic defects have been demonstrated to critically contribute to was - associated autoimmunity in was mouse model . in humans , the contribution of b - cell defects in the pathogenesis of was has been partially investigated . b cells from patients exhibit lower motility , migratory and adhesive capacities , likely due to defective f - actin nucleation . in contrast , despite the role of wasp in b - cell receptor ( bcr ) signaling , abnormalities in b - cell activation still remain controversial . a skewed distribution of serum immunoglobulin ( ig ) classes   and the inability to mount a proper antibody response , particularly to t - cell independent ( ti ) antigens , suggest defects in b - cell effector function . previous findings in was patients   show phenotypical b - cell perturbations in the periphery . in order to evaluate whether an abnormal b - cell development might generate a b - cell repertoire unable to unsure full protection against pathogens and tolerance against self - antigens , to this end , we have combined a detailed phenotypical analysis of b - cell maturation stages , from the bone marrow ( bm ) to the periphery , with a molecular study of ig repertoire and in  vivo b - cell maturation processes in a large cohort of was pediatric patients . our data show that wasp - deficiency affects critical stages of central and peripheral b - cell differentiation contributing to abnormalities in humoral immunity and b - cell tolerance in humans . the diagnoses were clinically defined and confirmed by genetic analysis . a description of all patients is reported in supplementary table  1 . human samples were obtained according to the code of ethics of the world medical association ( declaration of helsinki ) with the approval of the local medical ethical committees of the erasmus mc and the san raffaele scientific institute internal review board ( tiget02 ) . all results obtained from samples of was patients were compared to age and sex matched healthy donors ( hds ) . the composition of the precursor b - cell compartment was analyzed by flow cytometric immunophenotyping as described in the supplementary material . for the analysis of replication history and somatic hypermutation , four b - cell subsets were isolated from thawed peripheral blood mononuclear cells ( pbmcs ) using a  facs diva cell sorter ( bd biosciences ) . gating on cd19 cells ,  transitional ( cd27cd24cd38 ) , mature nave ( cd27igdcd24cd38 ) , natural effector ( cd27igd ) and memory ( cd27igd ) b - cell subsets were sorted with a purity of > 95% for all fractions . for intracytoplasmic detection of human wasp , cells were fixed and permeabilized using a cytofix / cytoperm kit ( bd pharmingen , oregon , usa ) . the anti - wasp antibody 503 ( a kind gift from prof h. d. ochs , seattle , wa , and l. d. notarangelo , boston , ma ) was used , followed by detection with pacific blue - labeled anti - rabbit igg secondary antibody ( invitrogen , san diego , usa ) . cd20 positive cells were purified from pbmcs of pediatric was patients and age - matched hds by immunomagnetic beads ( miltenyi biotec , germany ) or facs sorting . the purity of the isolated cells were analyzed by facs and ranged from 84% to 98% . after isolation , cells were left overnight at 37  c in culture medium composed of rpmi-1640 , 10% fbs , 2  mm glutamine , 100  iu / ml penicillin and 100  g / ml streptomycin ( lonza , basel , switzerland ) . in  vitro chemotaxis assay was performed using 5  m pore - size transwell inserts ( costar corporation , corning , ny , us ) in 24-well plates . filters were prewet 30  min at 37  c in presence of 600  l of medium supplemented with 250  ng / ml of recombinant human stromal cell - derived factor ( sdf)-1 ( cxcl12 ; peprotech , rocky hill , us ) . fifty thousand cd20 positive or negative cells were resuspended in 100  l of culture medium , seeded in the upper chamber and incubated at 37  c for 3  h. transmigrated cells , collected in the lower chamber , were counted for viable cells and stained with anti - cd19 , anti - cd24 , anti - cd38 , anti - cd27 and anti - cd3 ( bd biosciences ) for the phenotypical analysis by facs . migration frequency was estimated as the [ ( cell n at the lower chamber / the initial b cell input in the upper chamber )    100 ] . levels of b - cell activating factor ( baff ) were measured in duplicate in plasma samples of was patients and hds using a quantikine human baff / blys / tnfsf13b immunoassay kit ( r&d systems , minneapolis , usa ) . the assay was performed according to manufacturer 's instructions and the od was determined using a microplate reader set to 450  nm . ig and igh gene rearrangements were amplified and analyzed as described in the supplementary material . dna was extracted from sorted b - cell subsets with the genelute mammalian total dna miniprep kit ( sigma  aldrich ) and used to perform the ig-deleting recombination excision circles ( krec ) assay . the amounts of coding and signal joints of the ig-deleting rearrangement were measured by real - time quantitative pcr on an abi prism 7000 sequence detection system ( applied biosystems ) . ig restriction enzyme - based hot - spot mutation assay ( igrehma ) was performed on genomic dna isolated from sorted b - cell subsets . briefly , v3 - 20-j rearrangements were pcr - amplified and the pcr products ( 500  bp ) were first digested using the restriction enzyme fnu4hi and then kpni . the unmutated gene products can be visualized as 244 or 247 bp hex - coupled fragments and the mutated as 262 bp hex - coupled fragment . comparisons between proportions were calculated by using the chi - square test (  test ) ( with continuity correction ) . until now , the composition of the b - cell compartment in the bm of was patients has not been reported . to evaluate the effect of wasp - deficiency on b - cell development in the bm , we characterized five stages of b - cell differentiation   in three was patients . we observed a decreased frequency of immature b lymphocytes in was patients ( fig . this observation was further assessed in four additional patients , focusing on late stages of b - cell differentiation based on the expression of cd10 and cd20   ( gating strategy is shown in suppl . we confirmed a significant decrease in the frequency of immature b cells compared with age - matched hds ( fig . in addition , at the earliest differentiation stages , we observed a significant increase of small pre - b - ii cells in was patients ; while no statistical difference was detected in large pre - b - ii cell frequency ( fig . these findings might reflect a partial block in the bm b - cell differentiation between the small pre - b - ii and immature b - cell stages in was , or an early egress of immature b cells into the periphery . next we examined the distribution of b - cell subsets in the peripheral blood of was patients and age - matched hds . when available , we evaluated the absolute count of total b cells , which was found significantly reduced in patients less than three years old ( < 3y ) , while no differences were observed in the older was group ( fig . four b - cell subsets were identified according to the expression of cd24 , cd38 , igd and cd27 : transitional , mature nave , memory and plasmablasts ( fig . transitional b cells ( cd24cd38 ) are the most immature peripheral b - cell population that were significantly increased in both was age groups as frequency ( fig .  2c ) and absolute number ( suppl . the next maturation stage , represented by mature nave b cells ( cd27igdcd24cd38 ) , was significantly decreased only in was patients less than 3 years old both in frequency ( fig . finally , the frequency of plasmablasts ( cd24cd38 ) was significantly increased in the older group of patients ( fig . 2c ) , while the absolute number of both memory b cells ( cd27igdcd24cd38 ) and plasmablasts were significantly decreased in the younger group of was patients ( suppl . altogether , these findings demonstrate that was patients have a perturbed peripheral b - cell distribution . the overrepresentation of transitional b cells in the blood of was patients led us to hypothesize that an early egress of immature b cells from bm could occur . cxcr4 , the chemokine receptor for sdf-1 , has an important role in retaining b - cell precursors within the bm in order to prevent premature migration to the periphery [ 2224 ] . we tested the chemotactic response to sdf-1 of b lymphocytes of was patients and hds by transwell migration assay . as shown in fig .  3 , both transitional and mature peripheral b cells were less responsive to sdf-1 in  the absence of wasp ( fig . this defect was specific for b  lymphocytes since cd3 positive cells of was patients showed a normal migratory capacity to sdf-1 ( suppl . thus , we hypothesize that a diminished retention signal mediated by cxcr4/sdf-1 could explain the premature egress of immature b  cells from the bm leading to an increase of transitional b cells in periphery . to further characterize the peripheral b - cell distribution , we focused our analysis on b - cell subsets found expanded in autoimmune diseases and potentially autoreactive [ 2528 ] . we found a significant increase of cd19cd21cd35 subset in both was age groups ( fig . 4a ) , confirming data previously reported by park and  colleagues . we also analyzed the frequency of cd19cd21cd38 cells ( referred to as cd21 ) described to be expanded in autoimmune diseases and immunodeficiencies 
 . interestingly , we observed that the frequency of this unusual population was markedly increased in was patients , both in the younger and older groups ( fig . in addition , higher levels of soluble baff were found in the plasma of was patients as compared to pediatric hds ( fig . this finding was also associated with a decreased expression of its receptor , baffr , in transitional b cells of was patients ( fig . because of the role of baff in b - cell homeostasis and peripheral b - cell tolerance , high baff levels may affect the stringency of peripheral b - cell selection thus favoring the survival of b - cell subsets containing potentially autoreactive clones . in order to evaluate the effect of wasp - deficiency in an antigen dependent context , we analyzed in detail the memory b - cell compartment of was patients . we identified six distinct memory b - cell subsets   ( gating strategy is shown in suppl . igm - only  ) and cd27igg deriving from primary germinal center ( gc ) responses ; igg cd27 and iga cd27 switched memory b cells originating from secondary gc responses ; cd27igd ( named  natural effector  ) and cd27iga generated in a ti manner in the gastrointestinal tract or in the splenic marginal zone area , respectively . we did not observe differences in memory b - cell populations deriving from both primary and secondary gc responses between was patients and hds ( fig . a marked reduction in natural effector b cells was found in both age groups of was patients both as frequency ( fig . the frequency of cd27iga b cells was decreased in 312 years old patients compared with hds ( fig . , the absence of wasp mainly affects ti responses of b cells , whereas only mildly impairs the generation of early memory b cells in gc without influencing the class - switched memory b - cell compartment . we determined the in  vivo b - cell proliferation history by krec assay and in parallel shm by igrehma in sorted transitional , mature nave , natural effector and memory b cells . as expected , transitional b lymphocytes did not undergo cell divisions and mature nave b cells had a limited number of cell divisions in hds ( fig . both b - cell subsets were characterized by the absence of shm ( fig . 6b ) and we did not find differences in transitional and mature nave b cells of was subjects ( fig . in contrast , the memory compartment showed less proliferation in was memory b cells in association with a reduced shm level ( fig . interestingly , the defect in maturation was even more pronounced in natural effector was b lymphocytes . to study shm also in the ig heavy chain ( igh ) , we analyzed the frequency of mutated nucleotides in rearranged variable region of igh ( ighv ) genes by cloning and sequencing the most frequent ighv subgroups , the ighv3 and ighv4 gene families , of both - and -chain of constant regions ( c and c ) . the mutational frequency of was b cells was significantly lower for both c and c transcripts in all domains of v region ( fig . thus , the decreased in  vivo proliferation and shm levels of was memory b cells could be responsible of a lower protection against infections and persistence of pathogens that finally lead to autoimmunity . was b cells showed an increased frequency of ighv3 - 30 in both ig classes ( fig . 7a ) and the absence of ighv3 - 48 genes in c sequences ( suppl . 7a ) . in ighv4 transcripts , we noticed a dominant usage of the ighv4 - 34 genes in was c ( fig . moreover , the antibody repertoire of was b cells was devoid of ighv4 - 59 , a gene commonly used in b cells from hds . the distribution of d families was only slightly altered in was patients ( suppl . finally , we evaluated the csr process in total b cells by analyzing the frequency of ig subclasses in sequenced igh transcripts . a preferential usage of -chain c region 3 ( c3 ) and c1 , igh - proximal genes ( the igh locus is schematized in fig . 7d ) , accompanied by a reduction in the expression of distal c2 and c4 , were detected in was patients ( fig . the analysis of iga transcripts showed no significant difference in the subclass usage ( fig . 7e ) . in conclusion was patients show a skewed ig gene usage suggesting an altered selection of the antibody repertoire during b - cell development . we reported herein that wasp - deficiency affects many aspects of b - cell development , first in the bm and then in the periphery . indeed , precursor b - cell as well as memory b - cell development and selection appear affected by the absence of functional wasp . in the bm we have observed a decreased frequency of immature b cells , while in the periphery an overrepresentation of transitional b cells likely due to a reduced retention in the bm and/or a decreased migration to peripheral lymphoid tissues . the interaction of the chemokine receptor cxcr4 with its ligand sdf-1 is required for the retention of developing b cells in the bm . wasp is actively involved in cxcr4 signaling [ 3739 ] and was murine b cells show defective migratory response to sdf-1 . consistently , here we demonstrate in was patients that b cells migrate less to sdf-1. thus , was immature b cells could be unable to properly sense retention signals derived from cxcr4 in the bm leading to a premature release in the periphery . the increase of transitional b cells could also reflect an altered migration to the peripheral lymphoid organs resulting in a prolonged persistence in the circulation . consistently , our data allow us to exclude that the expansion of transitional b cells is due to homeostatic proliferation since we demonstrated the absence of replication history in this subset . transitional b cells are found expanded in immunodeficient conditions   or autoimmune diseases   and represent a reservoir of autoreactive b cells . the cohort of patients analyzed here did not show any overt sign of autoimmunity , probably due to their young age . however , the analysis of serum autoantibodies performed in four patients showed positivity for anti - nuclear antibodies in three patients . one of them also showed the presence of anti - platelet antibodies ( data not shown ) . these cells are enriched in anergic autoreactive clones   and expanded in systemic lupus erythematosus ( sle ) , rheumatoid arthritis and in common variable immunodeficiency groupia patients developing autoimmune syndromes . baff levels and signals through baffr coordinate the maintenance of the primary b - cell pool and the fate of self - reactive b cells . herein , we report for the first time increased baff levels and decreased baffr expression in was patients . elevated baff serum levels are often present in immunodeficiencies , autoimmune diseases   and viral infections   and may lower the thresholds for the survival of autoreactive b cell clones . the enrichment of cd21 b cells and the alterations in baff levels and expression of its receptor suggest that the mechanisms of b - cell selection could be altered in was patients . our data also suggest the presence of a defective selection of was b cells producing high - affinity antibodies . indeed , we noticed a restricted or null presence of ighv3 - 48 and ighv4 - 59 gene families and a preferential usage of ighv3 - 30 and ighv4 - 34 . in particular , ighv3 - 48 gene is selectively used against polysaccharide antigens   and its decrease in was could account for an inefficient antibody response . in contrast , ighv3 - 30 is highly represented among anti - platelet autoantibodies from patients with idiopathic thrombocytopenic purpura   and in sle patients . additionally , ighv4 - 34  encoded antibodies are intrinsically autoreactive when unmutated , as observed in our patients . this peculiar antibody repertoire reflects an altered selection of both protective and autoreactive ig gene families . the perturbation of b - cell homeostasis present in was patients supports the administration of anti - cd20 mab in the conditioning regimen of gene therapy to deplete b cells . in the memory b - cell compartment , the frequency of isotype - switched igg b cells we observed a normal frequency of memory b cells and of switched cd27igg and cd27iga b cells in our cohort of was patients . this suggests that t - cell dependent antigen response is induced normally in was patients . in contrast , the frequency and the shm level of ti memory b - cell subsets were reduced , likely due to their reduced in  vivo proliferation . the marked reduction in the natural effector b - cell subset , resembling marginal zone b cells in the spleen , mirrors the decreased number of splenic marginal zone b cells and the histological defects in the marginal zone area of the spleen already described in was mice   and patients . in addition , both the reduction in natural effector and igacd27 b cells , which are generated during ti responses , provide evidence of a defect in b - cell function independent from the cross - talk with t lymphocytes . the mutational status of both heavy and light ig chains is reduced in was in presence of a diminished rate of proliferation in the total memory b - cell pool . in addition , the analysis of ig gene selection showed a preferential use of igh - proximal genes ( ighg1 and ighg3 ) accompanied by reduced shm suggesting that was b cells have undergone less gc reactions . reduced igg2 switching in was patients could account for the poor response to ti antigens which is typical of the syndrome . the cytokine profile of was patients shows an impairment in the production of th1 cytokines   that could explain their reduced switching to igg2 subclass . moreover , the isotype - switching outcome is also influenced by two additional factors : cell division   and antibody affinity to antigens . the defective proliferation of memory and natural effector b cells and the reduced affinity maturation found in was might contribute to these alterations in class switching . our results in was patients show an early egress of immature b cells from bm leading to an overrepresentation of transitional b cells in the periphery . the memory compartment is characterized by a reduced maturation status that could affect the b - cell effector functions contributing to a lower clearance of pathogens and leading to chronic inflammation that can break tolerance . in conclusion , our results add novel immunological features of was b - cell phenotype that can complement the evaluation of the efficacy of various treatment approaches .
wiskott  aldrich syndrome protein ( wasp ) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the wiskott 
 aldrich syndrome ( was ) , a primary immunodeficiency with microthrombocytopenia , eczema and a higher susceptibility to develop tumors . 
 autoimmune manifestations , frequently observed in was patients , are associated with an increased risk of mortality and still represent an unsolved aspect of the disease . 
 b cells play a crucial role both in immune competence and self - tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity . we performed a phenotypical and molecular analysis of central and peripheral b - cell compartments in was pediatric patients . 
 we found a decreased proportion of immature b cells in the bone marrow correlating with an increased presence of transitional b cells in the periphery . 
 these results could be explained by the defective migratory response of was b cells to sdf-1 , essential for the retention of immature b cells in the bm . in the periphery 
 , we observed an unusual expansion of cd21low b - cell population and increased plasma baff levels that may contribute to the high susceptibility to develop autoimmune manifestations in was patients . 
 was memory b cells were characterized by a reduced in  vivo proliferation , decreased somatic hypermutation and preferential usage of ighv4 - 34 , an immunoglobulin gene commonly found in autoreactive b cells.in conclusion , our findings demonstrate that wasp - deficiency perturbs b - cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in was patients .