Posttransplant B cell development and function in patients with B cell positive SCID caused by pathogenic variants in IL2RG and JAK3

Posttransplant B cell development and function in patients with B cell positive SCID caused by pathogenic variants in IL2RG and JAK3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Posttransplant B cell development and function in patients with B cell positive SCID caused by pathogenic variants in IL2RG and JAK3 Eva-Maria Jacobsen, Abdallah Khazaleh, Kerstin Felgentreff, Ingrid Furlan, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7329087/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2026 Read the published version in Journal of Clinical Immunology → Version 1 posted 11 You are reading this latest preprint version Abstract Genetic defects in IL2RG or JAK3 can cause the phenotype of severe combined immunodeficiency (SCID) with absent T- and non-functional B-lymphocytes (T-B+ SCID). B cell function and the need for immunoglobulin replacement after hematopoietic stem cell transplantation (HSCT) depends on the engraftment of donor B-lymphocytes. In a retrospective study we describe B-lymphocyte reconstitution after HSCT with the objective to identify B cell subpopulations as an early predictor for the maturation and function of B cells after HSCT. All patients included underwent HSCT in a single institution between 1980 and 2017. First, we studied B cell maturation in cryopreserved blood samples of 12 long-term surviving patients with B+ SCID (IL2RG-deficiency) after haploidentical HSCT and mixed B cell chimerism. Recipient and donor B cell subpopulations were identified by HLA-staining using flow cytometry. In a consecutive step we compared B cell subpopulations irrespective of chimerism between patients with or without post-transplant B cell function. Samples for this study had been obtained between day +90 to +250 after HSCT from 25 post-transplant long-term survivors with B-positive (9 with genetic variants in JAK3, 16 in IL2RG) SCID, 9/25 were dependent and 16/25 independent of Ig-substitution. We demonstrate that a proportion of less than 2% of donor B cells is sufficient for posttransplant B cell function and that a proportion of more than 4.7% of switched memory (IgM-) B cells in the memory B cell population (CD19+CD27+) between days +90 and +250 after HSCT correlates with normal B cell function and independence from immunoglobulin substitution. B cell positive SCID IL2RG JAK3 HSC-transplantation MZ-like B cells B-lymphocyte reconstitution immunoglobulin substitution Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Patients with B-lymphocyte positive SCID due to genetic variants in IL2RG or JAK3 need donor B cell engraftment to establish specific humoral immune functions after hematopoietic stem cell transplantation (HSCT). This is due to an intrinsic defect of recipient B cells to respond to IL-21 and to develop into mature class switched memory B cells and antibody producing plasma cells [ 1 ]. The common gamma chain (IL2RG, CD132) dimerizes with IL21A and is an indispensable subunit of a functional receptor for IL-21 [ 2 ] with effective signal transduction via JAK-3. Primary therapeutic objective in patients with SCID is the establishment of a T cell system as this prevents from life threatening infections and allows long term survival. The function of B-lymphocytes is dependent on T cell-help via CD40/CD40L and other coreceptor interactions in the germinal center as well as signaling via the IL-21 receptor in donor derived B cells [ 3 , 4 ]. Currently, HSCT is the only established therapy to cure patients with B-positive SCID [ 5 , 6 ]. Genetic correction of autologous stem cells by gene therapy is evaluated in several studies [ 7 , 8 ]. Before, during and early after cellular therapies, these patients are routinely substituted with immunoglobulins. The decision to stop immunoglobulin (Ig)-substitution after successful HSCT is usually based on parameters such as the cessation of GvHD-prophylaxis or -therapy, donor chimerism, level of T cell reconstitution, immunoglobulin levels and as gold standard specific antibody responses to Tetanus toxoid or polysaccharide antigens of H.influenzae or S.pneumoniae . This clinically important decision though is hampered by several aspects. Specific antigen responses and IgG-serum levels cannot be evaluated while patients are substituted. Testing for B cell chimerism can be complex as many patients have mixed chimerism after HSCT and therefore B cells have to be highly purified to avoid any signal of potentially contaminating donor T cells. The minimal level of donor-B-lymphocyte chimerism to provide robust B cell function is not known. For an evidence based clinical decision Ig-substitution would have to be interrupted for a period of several months, during which patients are potentially at risk to acquire serious infections if B cell function finally turns out to be absent. The objective of this study was to study B cell development in patients with B cell positive SCID and mixed B cell chimerism and to identify informative parameters which are independent from Ig-substitution and correlate with the development of a functional B cell system as an early indicator to stop immunoglobulin substitution post HSCT. 2. Patients and methods Blood samples and clinical data were studied from patients treated between 1980 and 2017 at the University Medical Centre Ulm, Department of Paediatrics. Written informed consent was obtained from patients and/or their legal guardians. Research study protocols were approved by the local review board of Ulm University (reference/review number 20122017) in accordance with the Declaration of Helsinki. Flow cytometry studies were performed on mononuclear cells (MNCs) isolated from peripheral blood of patients taken at routine follow up appointments after HSCT. MNCs were either directly stained after isolation from fresh blood by density gradient cell separation (Biocoll Separating Solution, Biochrom AG) or obtained from frozen samples. Flow cytometry was performed on a NAVIOS 3L10C (Beckman Coulter) cytometer after washing and incubation with antibodies as indicated below. Clinical Data (patient age, genetic variants, transplant data, serum Ig-levels, need for Ig-replacement) were retrospectively collected from patient files. 2.1. Chimerism studies based on HLA-antigen mismatches in flow cytometry Samples from 12 long-term surviving patients (cohort B) with IL2RG deficiency after haploidentical (mismatched related donor) HSCT 9/12 with myoablative conditioning, 3/12 without conditioning) resulting in mixed chimerism were analysed after a follow up of 1.5–34 years (see Table S1 B). All patients were independent of Ig-replacement. MNCs were incubated with unconjugated mouse HLA-antibodies against donor- and/or recipient HLA antigens followed by incubation with FITC-conjugated secondary goat-anti-mouse IgM- and IgG-antibodies respectively. B cell subpopulations were further characterized in a second step with fluorescence conjugated antibodies. Details on antibodies and fluorochromes are given in Table S2. In 6 out of 12 patients, antibodies, specific for recipient and donor HLA-antigens were available. In 4 patients, only anti-donor-type and in 2 patients, only anti-recipient-type HLA-antibodies could be tested. HLA positive and negative B cells were clearly distinguishable for gating. Positive and negative controls with HLA-typed cryopreserved buffy-coat MNCs were analysed in parallel with each experiment. Additional cell populations as monocytes, T and NK cells were characterized in the same experiment. As T cells are expected to originate completely from the donor in SCID-patients, these served as additional internal control. B cell phenotyping without HLA-staining led to equal results and distributions of subpopulations. If only one antibody was available, flow cytometric HLA results were confirmed once by an independent method (STR or XY-FISH, data not shown) after separation of populations by magnetic beads (EasySep™ Cell Separation, STEMCELL technologies, Vancouver, Canada). 2.2. B cell immunotyping by flow cytometry Samples from 25 long term surviving patients with B-positive SCID (cohort A) were included (Table S1 A). The selection of patients was based on the genotype and the availability of cryopreserved samples from the period of interest 90–250 days after HSCT. 7 patients of this cohort were also analysed for chimerism studies (annotated in Table S1 ). In 16 male patients, hemizygous variants in IL2RG were identified, in 9 patients, genetic variants affecting both alleles of JAK3 were detected. At their most recent presentation, patients had a median follow up of 11.1 years (0.4–33.1) years after HSCT. Conditioning was based on variable regimen in 15/25 (60%) patients and 10/25 (40%) had been transplanted without conditioning (Table S1 ). Grafts were donated by mismatched family donors (MMFD) in 18/25, matched sibling donors (MSD) in 6/25 and a matched family donor (MFD) in 1/25 cases. Definitions of B cell subpopulations and antibodies used for their identification by flow cytometry are given in Tables S3 and S2 respectively. A minimal cell population of 50 cells in the CD19 + CD27 + gate was considered necessary for the reliable analysis of subpopulations. Patient samples with less than 50 cells in this gate were excluded from the study. 2.3. Definition of patient groups According to the information taken from patient charts patients were either assigned to the immunoglobulin-dependent (IgDEP) or independent group (IgIND). All patients in the IgIND -group were demonstrated to have normal specific antibody responses after vaccination. 2.4. Statistical analysis Statistical analysis was performed by using IBM SPSS Statistics 24. A predictive cut-off for immune reconstitution was defined by using receiver operating characteristics (ROC) curves. 3. Results 3.1. HLA-chimerism analysis in flow cytometry: less than 2% of donor B cells are sufficient for posttransplant specific humoral immune function Samples of 12 patients were investigated in this part of the study originated from post-haplo-transplant long-term survivors with B-lymphocyte positive SCID caused by genetic variants in IL2RG . All patients were not substituted with immunoglobulins and had normal B cell function as determined by normal immunoglobulin values and normal response to vaccinations. The majority of patients (9/12) had been transplanted with and 3/12 without conditioning (for details see Table S1 , cohort B). As depicted in Fig. 1 , T cells completely originated from the donor, whereas donor chimerism for B cells, monocytes and NK cells were mixed. B cell chimerism revealed to be highly variable between patients and a proportion of less than 2% of donor derived B cells was sufficient to allow for normal B cell function. Monocyte donor chimerism was found at the detection limit for two patients. As expected, donor chimerism for NK cells is higher in comparison to monocytes in all but one patient as an indicator for a selective advantage of donor derived NK lymphocytes. 3.2. B cell maturation is deficient in autologous B cells As demonstrated for a representative sample in Fig. 2 , HLA staining allows for donor and recipient specific analysis of B cell subpopulations in patients with post-transplant mixed B cell chimerism. If donor and recipient CD19-positive B cells are set to 100% respectively, the relative composition of the B cell compartments can be compared between donor and recipient (colored bars in Fig. 2 and Fig. 3 ) and between patients. This analysis exemplifies the remaining post-transplant maturational defect of autologous B cells despite the presence of donor T cells in all patients tested (Fig. 1 ) and the normal maturational potential of donor B cells. Autologous switched memory B cells (CD19 + CD27 + IgM-) are hardly detectable whereas the majority (80–95%) of autologous B cells shows a naïve (CD19 + CD27-) phenotype with Marginal-Zone-like B cells as the dominant CD27 + population (Fig. 3 , Fig S2). This latter pattern is shared by B-lymphocyte positive SCID patients pretransplant or posttransplant with autologous reconstitution (Fig. 3 ). This finding is in contrast to the experience in patients with other SCID entities such as IL7RA or ADA (adenosine deaminase) deficiency in whom autologous B cells develop maturational capacity as soon as a donor derived T cell system is established (suppl. Figure 3). In the donor B cell compartment a negative correlation between the percentage of donor Bcells and the proportion of switched-memory B cells can be observed: the smaller the donor B cell compartment, the higher the percentage of switched-memory B cells (Fig. 3 d and Suppl. Figure 4). 3.3. Posttransplant immunoglobulin levels are of limited reliability as indicators of B cell function To check for potential B cell function after HSCT, the clinical routine in most institutions would include the close observation of patient IgA- and IgM-levels as these are not contained in pharmaceutical immunoglobulin substitution products and therefore indicate a production by mature plasma cells. In our cohort we were able to compare immunoglobulin levels at three periods post-HSCT: day 50, day 150, and between days 250 to 350. For IgA-levels, a significant difference between the IgIND and IgDEP group was only demonstrated for the latest period (Fig. 4 ) but not for earlier periods. Serum levels for IgM (suppl. Figure 5b) did not differ at any time. 3.4. The proportion of switched memory B cells in the CD27 + pool indicates the extend of B cell maturation and correlates with humoral specific immune function In order to transfer the robust finding that autologous cells are not able to mature to switched-memory B cells as observed in patients after haploidentical transplantation (cohort B, Table S1 ) to a more general and clinically more practicable approach we studied B cell subpopulations in cohort A irrespective of chimerism. Cohort A was -among other parameters- defined by the availability of cryopreserved samples taken from patients at routine follow up visits between 90 and 250 days post-HSCT. Patients in this cohort had been shown to have genetic variants in IL2RG and JAK3 and were transplanted with grafts donated by mismatched family donors, matched unrelated donors and matched sibling donors (see Table S1 ). B cell phenotyping was performed without any discrimination between donor and recipient cells. Available data on chimerism from the same period after HSCT are given in Table S1 . According to patient charts, 16 patients qualified for the IgIND and 9 patients for the IgDEP groups with a median follow up of 11.7 years (0.4–33.1 years) for the IgIND group and 8.3 years (1-24.6 years) for the IgDEP group after HSCT respectively. As expected, patients in the IgDEP group show a lack of CD27-positive B cells in comparison to the IgIND group but this population is not completely absent. As demonstrated in cohort B (Figs. 2 and 3 ), autologous B cells can mature to a CD27 + stage, which is represented almost completely by Marginal-Zone like B cells. The clearest difference between autologous and donor B cell maturation in cohort B was found in the percentage of switched-memory B cells (CD19 + CD27 + IgM-) in the CD27 + B cell pool. This population was absent or considerably reduced in all patients except one in the IgDEP group (Fig. 5 ). A ROC-analysis allowed to define a cut-off of 4.7% switched memory B cells in the CD27 + B cell pool to correctly assign a patient to the IgIND or IgDEP group with a specificity of 88.9% and a sensitivity of 93.8% (Fig S6). The two patients of the IgIND and the IgDEP group, who were found below this threshold in the IgIND and above this threshold in the IgDEP group, were contacted and reevaluated. Patient 346 (Tab. S1), who was substituted with immunoglobulins but had a proportion of 11.1% of switched memory B cells in the CD27 + B cell pool, was contacted and paused Ig-substitution for 4 months during summertime. He was challenged with inactivated vaccines (Tetanus, Diphtheria, Haemophilus and Pneumococci) and checked for specific antibodies. These turned out positive and he is off IVIG since. The proportion of switched memory B cells in patient 15 at the first analysis time point (d233 post HSCT) was still below the threshold of 4.7% in the CD27 + pool in spite of 14% donor B cells (Table S1 ). However, during 33 years of post HSCT follow up, he had no infectious history and was not substituted with immunoglobulins. His chimerism analysis at this late follow up time point (HLA-flow cytometry) revealed 4.6% of donor B cells with a proportion of 7.8% of switched memory B cells in the complete (donor and recipient) CD27 + B cell pool (not shown) and a proportion of 27% switched memory B cells in the donor CD27 + B cells (Fig. 3 d and S4a). IgG and IgM levels were found in the normal age adapted range but IgA was undetectable. Specific antibodies were positive for tetanus, pneumococci and rubella but negative for mumps and measles. 4. Discussion Without engraftment of donor derived B-lymphocytes, patients with SCID due to genetic variants in IL2RG and JAK3 will remain without specific humoral immune function and thus dependent on immunoglobulin substitution [ 1 ]. The underlying intrinsic defect of autologous B cells has been previously reported for this patient group by White et al [ 4 ] who demonstrated the inability of patient B cells, before and after HSCT, to perform a class switch and their inability to respond to IL-21 which was reported by Miggelbrink at al. [ 3 ]. With our approach to analyze CD19 + lymphocyte subpopulations and to label their donor or recipient origin by HLA-staining in flow cytometry, we provide further evidence for this primary defect in B cell maturation with an independent method. Beyond that, we can contribute an additional crucial clinical information: a very minor population of less than 2% of donor B cells in peripheral blood is sufficient to allow for normal specific immune functions “measured” retrospectively by an uneventful infectious history, normal Ig levels and the response to vaccinations with a median follow up of 14.3 (1.0-34.2) years. The formal weakness of this retrospective study turns to an advantage as this extensive follow up cannot be provided by a prospective approach and is a reliable clinical parameter to attribute patients in the cohort studied to the IgIND group. We moreover observed the correlation of a low percentage of donor B cells with a high proportion of class-switched memory cells within this donor B cell population. This negative correlation could be interpreted as an attempt to compensate for the lack of functional B cells by a supranormal percentage of maturation to switched memory B cells within the donor derived and thus functional B cell population. This of course raises further questions on the stability of such a system of exploitation. From the clinical perspective, these patients do not show any phenotype of immunodeficiency but this does not exclude the possibility of abnormal findings regarding BCR variability, clonal expansion or cellular signs of senescence or exhaustion, which will have to be further elucidated. The findings on B cells isolated from peripheral blood are not necessarily representative for the B cell system as a whole. Donor chimerism for B cell subpopulations in bone marrow and secondary lymphatic organs could vary considerably from those detected in CD19 positive cells in peripheral blood, but for obvious reasons this information will not be readily available. Even with these limitations, the information generated from blood samples remains essential for routine clinical decisions. Autologous B cells after HSCT have a relevant proportion of 2.6 to 14% of CD27 + cells, which almost all turn out not to be memory B cells but MZ-like B cells as they stain positive for IgM and IgD. To our surprise this has not been recognized and reported in the literature until present. Marginal zone-like B cells and their function have not been well characterized in humans yet and their function in mice is described as an important component for a T cell independent innate first wave of IgM-antibody-reaction of the immune system to bind with low affinity to highly conserved motifs of infectious agents. Beyond this, MZ-like B cells have also been shown to be able to interact with follicular T-helper cells and undergo class switch reaction and somatic hypermutation as follicular B cells do. In our studies we can clearly show, that CD19 + CD27 + IgM + IgD + cells can develop in the absence of signalling via the IL21-receptor but we strongly suspect that deep phenotyping and genetic studies (V(D)J-rearrangement and BCR-clonality) will be able to show major differences in this autologous B cell population in our cohort before HSCT (no T cell help, no IL-21 signalling), after HSCT (T cell help, no IL-21 signalling) and in comparison to donor derived MZ-like B cells (T cell help, normal IL-21 signalling) or samples from healthy controls. We aimed to practically exploit these findings for clinical use to help with the decision to stop immunoglobulin substitution in post HSCT patients with B-positive SCID. This decision can be clinically challenging- especially for patients with mixed chimerism. The mere presence of B cells is not sufficient to deduce B cell function. As reviewed by van der Maas et al [ 9 ], the reconstitution of non-switched Marginal-Zone like B cells and switched memory B cells proceeds slowly after HSCT and it can take up to two years or longer to reach age-matched normal levels. Gold standard and final proof for a functional B cell system is the detection of post-vaccination specific antibodies and Ig levels within the normal range in addition to an uneventful infectious history. For obvious reasons, IgG-levels and vaccination titers cannot be tested while patients are on Ig-substitution. IgM and IgA levels would be used by many centers as indicators for normal specific humoral immune function. For our cohort, we could demonstrate that there is no significant difference for IgM-levels between the IgIND and IgDEP groups and that IgA-levels become informative at a period after 250 days posttransplant. This parameter though remains with a major taint for clinical use as the absence of IgA in the age group tested here is within the normal range. The detection of donor derived B cells would be helpful but with the findings demonstrated above it becomes clear that a minor proportion donor derived B cells in the range of 2–5% cannot be reliably detected by other methods of chimerism analysis. Even after B cell enrichment or purification a percentage of less than 5% can be missed in STR analysis or XY-FISH. HLA-staining for flow cytometry is rarely established for routine chimerism analysis and is only feasible for patients transplanted from haploidentical donors. But with the experience of HLA-staining of B cell subpopulations we were able to clearly correlate the presence of CD19 + CD27 + IgM- B cells in peripheral blood as indicator for B cell function. The quantification of this population irrespective of its chimerism in cryopreserved posttransplant patient samples allowed to statistically define a threshold of 4.7% CD27 + IgM- (% of CD27 + CD19+) with the highest specificity and sensitivity to retrospectively attribute a patient to the IgIND group. The presence of this population of class-switched memory B cells was previously found to be informative for posttransplant B cell function for B cell positive SCID patients by Miggelbrink et al [ 3 ] - we can confirm this finding in a larger cohort and add the clinically important quantification and definition of a threshold together with a diagnostic window as early as 90–250 days post HSCT. This additional information now allows the use of this parameter for clinical decision making and the cessation of post HSCT Ig-substitution. An earlier time point would not be informative as the help of follicular T cells is necessary to allow germinal center reactions to induce the class switch of B cells. The reconstitution of T cells after HSCT cannot be observed before 2–3 months after HSCT and can be delayed by GvHD, immunosuppressive drugs or infections. The clinical use of the quantification of CD19 + CD27 + IgM- B cells is not confined to the cohort reported here but could be adapted to B cell positive SCID patients after autologous gene therapy or the growing group of patients after targeted B cell depleting therapies with monospecific (e.g. Rituximab) or bispecific (e.g. Blinatumomab) antibodies or CAR-T cells. Conclusion In this retrospective analysis on patients with B cell positive SCID we could demonstrate by HLA-staining that a very minor percentage of less than 2% of donor B cells in peripheral blood correlates with normal immune function. Between day 90–250 in post-transplant follow up a proportion of > 4.7% of CD19 + CD27 + IgM- within the CD27 + B cell pool is a sensitive and specific marker to indicate post HSCT B cell function in this cohort. Abbreviations ADA adenosine deaminase BCR B cell receptor HSCT hematopoetic stem cell transplantation Ig Immunoglobulin IgIND independent of immunglobulin substitution IgDEP dependent of immunglobulin substitution IL2RG interleukin 2 receptor gamma IL7RA interleukin 7 receptor alpha JAK3 janus kinase 3 MZ marginal zone SCID severe combined immunodeficiency Statements and Declarations Authorship contributions E-M.J. and A.K. performed the analysis of patient MNCs in Flow Cytometry and evaluated the data. W.F., A.S.S. K.F, I.F, K.W, M.S, U.P, K.S and M.H. were responsible for patient care and/ or provided clinical, genetic or laboratory data. M.H. wrote the main manuscript text, E.J. prepared figures 1-3 and A.K figures 4-5. All authors contributed to the discussion and the preparation of the draft and have read and approved the final version of the manuscript. Disclosure of potential conflicts of interest: The authors declare that they have no conflicts of (financial) interests with respect to the content of this manuscript. No funds, grants, or other support were received during the preparation of this manuscript. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the local Ethics Committee of Ulm University (reference/review number 20122017). Consent to participate: Written informed consent for participation was obtained from all patients and/or their legal guardians included in the study. Acknowledgements We would like to thank Carmen Blum, Andrea Hänsler, Gudrun Kirsch and Ulrike Tengler for excellent technical assistance. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Recher M, Berglund LJ, Avery DT, et al. IL-21 is the primary common gamma chain-binding cytokine required for human B-cell differentiation in vivo. Blood . 2011;118(26):6824-6835. Asao H, Okuyama C, Kumaki S, et al. Cutting edge: the common gamma-chain is an indispensable subunit of the IL-21 receptor complex. J Immunol . 2001;167(1):1-5. Miggelbrink AM, Logan BR, Buckley RH, et al. B-cell differentiation and IL-21 response in IL2RG/JAK3 SCID patients after hematopoietic stem cell transplantation. Blood . 2018;131(26):2967-2977. White H, Thrasher A, Veys P, Kinnon C, Gaspar HB. Intrinsic defects of B cell function in X-linked severe combined immunodeficiency. Eur J Immunol . 2000;30(3):732-737. Gennery AR, Slatter MA, Grandin L, et al. Transplantation of hematopoietic stem cells and long-term survival for primary immunodeficiencies in Europe: entering a new century, do we do better? J Allergy Clin Immunol . 2010;126(3):602-610 e601-611. Lankester AC, Neven B, Mahlaoui N, et al. Hematopoietic cell transplantation in severe combined immunodeficiency: The SCETIDE 2006-2014 European cohort. J Allergy Clin Immunol . 2021. Fischer A, Hacein-Bey-Abina S. Gene therapy for severe combined immunodeficiencies and beyond. J Exp Med . 2020;217(2). Fischer A, Hacein-Bey Abina S, Touzot F, Cavazzana M. Gene therapy for primary immunodeficiencies. Clin Genet . 2015;88(6):507-515. van der Maas NG, Berghuis D, van der Burg M, Lankester AC. B Cell Reconstitution and Influencing Factors After Hematopoietic Stem Cell Transplantation in Children. Front Immunol. 2019;10:782. doi:10.3389/fimmu.2019.00782 Additional Declarations No competing interests reported. Supplementary Files supplementarydataPosttransplantBcelldevelopmentJacobsenKhazalehetal.docx Cite Share Download PDF Status: Published Journal Publication published 21 Mar, 2026 Read the published version in Journal of Clinical Immunology → Version 1 posted Editorial decision: Revision requested 01 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviews received at journal 19 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers invited by journal 03 Sep, 2025 Editor assigned by journal 25 Aug, 2025 Submission checks completed at journal 25 Aug, 2025 First submitted to journal 08 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7329087","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512389226,"identity":"cb241e49-e092-4c7b-aa92-3de42f219800","order_by":0,"name":"Eva-Maria Jacobsen","email":"data:image/png;base64,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","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":true,"prefix":"","firstName":"Eva-Maria","middleName":"","lastName":"Jacobsen","suffix":""},{"id":512389227,"identity":"c12e0449-6f88-4f82-9320-64b8cc0beec1","order_by":1,"name":"Abdallah Khazaleh","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Abdallah","middleName":"","lastName":"Khazaleh","suffix":""},{"id":512389228,"identity":"e3f81170-86a4-4ac6-953b-61dc27533eba","order_by":2,"name":"Kerstin Felgentreff","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Kerstin","middleName":"","lastName":"Felgentreff","suffix":""},{"id":512389229,"identity":"bef3da66-225a-4562-ae75-fd95fc9099c1","order_by":3,"name":"Ingrid Furlan","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Ingrid","middleName":"","lastName":"Furlan","suffix":""},{"id":512389230,"identity":"2b0f2543-7312-4e7e-8a7d-6b10d7fc625a","order_by":4,"name":"Katharina Wustrau","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Katharina","middleName":"","lastName":"Wustrau","suffix":""},{"id":512389231,"identity":"e0d0f09e-a6f9-4450-a56e-34bdd4348d30","order_by":5,"name":"Mehtap Sirin","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Mehtap","middleName":"","lastName":"Sirin","suffix":""},{"id":512389232,"identity":"2caa9786-8d11-4083-b86d-901a6ae1a256","order_by":6,"name":"Ulrich Pannicke","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Ulrich","middleName":"","lastName":"Pannicke","suffix":""},{"id":512389233,"identity":"5edfa41a-be8e-40cc-ab5a-06fc8210a753","order_by":7,"name":"Klaus Schwarz","email":"","orcid":"","institution":"Institute for Transfusion Medicine, University of Ulm","correspondingAuthor":false,"prefix":"","firstName":"Klaus","middleName":"","lastName":"Schwarz","suffix":""},{"id":512389234,"identity":"8b1ed484-41f0-4c38-96e6-21dabb91b0cd","order_by":8,"name":"Klaus-Michael Debatin","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Klaus-Michael","middleName":"","lastName":"Debatin","suffix":""},{"id":512389235,"identity":"c025e0b1-632f-49c6-b14e-76ce74c5e731","order_by":9,"name":"Wilhelm Friedrich","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Wilhelm","middleName":"","lastName":"Friedrich","suffix":""},{"id":512389236,"identity":"1923e5ee-2f29-4278-ab4c-91c893f24743","order_by":10,"name":"Ansgar S. Schulz","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Ansgar","middleName":"S.","lastName":"Schulz","suffix":""},{"id":512389237,"identity":"f19d4421-e92a-4337-8f58-1553d6752a85","order_by":11,"name":"Manfred Hoenig","email":"","orcid":"","institution":"University Medical Center Ulm","correspondingAuthor":false,"prefix":"","firstName":"Manfred","middleName":"","lastName":"Hoenig","suffix":""}],"badges":[],"createdAt":"2025-08-08 16:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7329087/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7329087/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10875-026-02004-2","type":"published","date":"2026-03-21T15:59:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91071435,"identity":"65d8c13e-afe7-496b-9a7b-fd45f17faa15","added_by":"auto","created_at":"2025-09-11 10:52:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDonor chimerism in long term survivors with IL2RG-deficiency after haploidentical HSCT\u003c/strong\u003e Chimerism analysis of B cells was performed by flow-cytometric analysis after staining with donor and/or recipient-specific HLA-Antibodies and CD19 (B cells), CD3 (T cells), CD56/16 (NK cells) and CD14 (monocytes) as lineage markers. All patients are independent of immunoglobulin substitution. Patients who didn’t receive a conditioning regimen are shown with open circles. Dotted lines connect values of the same patients\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7329087/v1/c36a0b15c4a7f649ccd635ae.png"},{"id":91071442,"identity":"b2f9bb49-3f73-453e-a999-71808ff192a6","added_by":"auto","created_at":"2025-09-11 10:52:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":176565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eB cell maturation after HSCT is confined to donor B cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative example of donor and recipient B cell subpopulations after HLA haploidentical SCT in a patient with IL2RG-deficiency. Peripheral blood MNCs (UPN 454, 14 years post SCT) were initially stained with HLA-A2 specific antibodies (recipient) followed by staining with anti-CD19, anti-CD27, anti-IgM and anti IgD. \u003cstrong\u003ea.\u003c/strong\u003e Only donor B cells (green: CD19+ and HLA-A2 neg.) are able to switch to IgM-CD27+CD19+ B cells while recipient B cells (red: CD19+ and HLA-A2 pos.) remain IgM+. \u003cstrong\u003eb. \u003c/strong\u003eDonor CD27+IgM+ (non switched) B cells show a substantial proportion of IgD- (IgM only) B cells while most of the IgM+CD27+ autologous B cells are IgD+. \u003cstrong\u003ec. \u003c/strong\u003eIn donor B cells all stages of maturation, which are present in healthy controls, can be detected including switched memory B cells (red), IgM only memory B cells (green) and marginal-zone like B cells (orange). In recipient B cells mainly naïve B cells (blue) and a small proportion of marginal-zone like B cells are found. Definition of B cell subpopulations: IgD+CD27-: naïve; IgM+CD27+: non switched memory; IgM-/CD27+: switched memory (sw mem); IgM-/CD27-: atypical memory (atypical sw mem); IgM+IgD+ (gate IgM+CD27+): Marginal Zone-like (MZ-like); IgM+IgD- (gate IgM+CD27+): IgM only\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7329087/v1/7d4292cef10cabbe77df0ee0.png"},{"id":91071445,"identity":"dd3c5728-fcc2-4ec0-b2ce-c337f322bdb5","added_by":"auto","created_at":"2025-09-11 10:52:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of B cell subpopulations in B cells of IL2RG-deficient patients pre- and post-SCT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB cells in patients pre SCT (\u003cstrong\u003ea.\u003c/strong\u003e) and in patients without donor B cells post SCT (\u003cstrong\u003eb.\u003c/strong\u003e) consisted predominantly of naïve B cells and a small proportion of Marginal-Zone like B cells while in patients with complete donor B cell chimerism (\u003cstrong\u003ec.\u003c/strong\u003e) all maturation stages including substantial proportions of switched memory B cells and IgM only B cells could be detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003e The atypical distribution of B cell subpopulations in patients with mixed B cell chimerism with an accumulation at the stage of MZ-like B cells in the recipient B cell population (shown in detail in Fig 1 and suppl. Fig 1) was confirmed in 11 additional patients to patient UPN 454. B cells were analyzed with the same method of staining for donor (left) and recipient (right) specific HLA-alleles followed by staining for B cell subpopulations.\u003c/p\u003e\n\u003cp\u003eDefinition of B cell subpopulations: IgD+CD27-: naive; IgM+CD27+: non switched memory; IgM-/CD27+: switched memory (sw mem); IgM-/CD27-: atypical memory (atypical sw mem); IgM+IgD+ (gate IgM+CD27+): Marginal Zone-like (MZ-like); IgM+IgD- (gate IgM+CD27+): IgM only. The patients are listed according to their proportion of donor B cells (indicated in parentheses with the patient number)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7329087/v1/64fec988f90ce19bc332d8ac.png"},{"id":91073615,"identity":"d7ecd01b-a638-4ffe-98ac-f11192e234d1","added_by":"auto","created_at":"2025-09-11 11:00:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIgA-levels for patients with and without immunoglobulin substitution in different periods post-SCT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIgA-levels in patients who do not need Ig-substitution are significantly higher in the period after d+250 but show no difference in earlier periods\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7329087/v1/3a7466033be60106ab91a101.png"},{"id":91071444,"identity":"90ff6b71-2e73-482e-920b-509cf19a191e","added_by":"auto","created_at":"2025-09-11 10:52:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of proportions of B cell subpopulations for patients with and without the need for Ig-substitution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proportion of memory B cells in the B cell compartment shows considerable overlap between the cohorts but the proportion of switched memory B cells in the memory B cell compartment is lower in the IgDEP group and shows an overlap for two patients only. The cutoff of 4.7% CD27+IgM- of CD27+ is indicated by a horizontal line\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7329087/v1/367093a0822a11fd15190f3c.png"},{"id":105223776,"identity":"c09d30ff-f34c-4461-9fe8-782f6adf767f","added_by":"auto","created_at":"2026-03-23 16:10:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1193308,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7329087/v1/00b68b7a-8490-475a-a44a-cd4ca019a287.pdf"},{"id":91075466,"identity":"c8bcd334-bbe3-48f1-ba76-f4303e047d7b","added_by":"auto","created_at":"2025-09-11 11:08:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1508091,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydataPosttransplantBcelldevelopmentJacobsenKhazalehetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7329087/v1/32a8c01d34a8d207ee1c3bdb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Posttransplant B cell development and function in patients with B cell positive SCID caused by pathogenic variants in IL2RG and JAK3","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePatients with B-lymphocyte positive SCID due to genetic variants in IL2RG or JAK3 need donor B cell engraftment to establish specific humoral immune functions after hematopoietic stem cell transplantation (HSCT). This is due to an intrinsic defect of recipient B cells to respond to IL-21 and to develop into mature class switched memory B cells and antibody producing plasma cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The common gamma chain (IL2RG, CD132) dimerizes with IL21A and is an indispensable subunit of a functional receptor for IL-21 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] with effective signal transduction via JAK-3. Primary therapeutic objective in patients with SCID is the establishment of a T cell system as this prevents from life threatening infections and allows long term survival. The function of B-lymphocytes is dependent on T cell-help via CD40/CD40L and other coreceptor interactions in the germinal center as well as signaling via the IL-21 receptor in donor derived B cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Currently, HSCT is the only established therapy to cure patients with B-positive SCID [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Genetic correction of autologous stem cells by gene therapy is evaluated in several studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Before, during and early after cellular therapies, these patients are routinely substituted with immunoglobulins. The decision to stop immunoglobulin (Ig)-substitution after successful HSCT is usually based on parameters such as the cessation of GvHD-prophylaxis or -therapy, donor chimerism, level of T cell reconstitution, immunoglobulin levels and as gold standard specific antibody responses to Tetanus toxoid or polysaccharide antigens of \u003cem\u003eH.influenzae\u003c/em\u003e or \u003cem\u003eS.pneumoniae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis clinically important decision though is hampered by several aspects. Specific antigen responses and IgG-serum levels cannot be evaluated while patients are substituted. Testing for B cell chimerism can be complex as many patients have mixed chimerism after HSCT and therefore B cells have to be highly purified to avoid any signal of potentially contaminating donor T cells. The minimal level of donor-B-lymphocyte chimerism to provide robust B cell function is not known. For an evidence based clinical decision Ig-substitution would have to be interrupted for a period of several months, during which patients are potentially at risk to acquire serious infections if B cell function finally turns out to be absent.\u003c/p\u003e\u003cp\u003eThe objective of this study was to study B cell development in patients with B cell positive SCID and mixed B cell chimerism and to identify informative parameters which are independent from Ig-substitution and correlate with the development of a functional B cell system as an early indicator to stop immunoglobulin substitution post HSCT.\u003c/p\u003e"},{"header":"2. Patients and methods","content":"\u003cp\u003eBlood samples and clinical data were studied from patients treated between 1980 and 2017 at the University Medical Centre Ulm, Department of Paediatrics. Written informed consent was obtained from patients and/or their legal guardians. Research study protocols were approved by the local review board of Ulm University (reference/review number 20122017) in accordance with the Declaration of Helsinki.\u003c/p\u003e\u003cp\u003eFlow cytometry studies were performed on mononuclear cells (MNCs) isolated from peripheral blood of patients taken at routine follow up appointments after HSCT. MNCs were either directly stained after isolation from fresh blood by density gradient cell separation (Biocoll Separating Solution, Biochrom AG) or obtained from frozen samples. Flow cytometry was performed on a NAVIOS 3L10C (Beckman Coulter) cytometer after washing and incubation with antibodies as indicated below. Clinical Data (patient age, genetic variants, transplant data, serum Ig-levels, need for Ig-replacement) were retrospectively collected from patient files.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chimerism studies based on HLA-antigen mismatches in flow cytometry\u003c/h2\u003e\u003cp\u003eSamples from 12 long-term surviving patients (cohort B) with IL2RG deficiency after haploidentical (mismatched related donor) HSCT 9/12 with myoablative conditioning, 3/12 without conditioning) resulting in mixed chimerism were analysed after a follow up of 1.5\u0026ndash;34 years (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). All patients were independent of Ig-replacement.\u003c/p\u003e\u003cp\u003eMNCs were incubated with unconjugated mouse HLA-antibodies against donor- and/or recipient HLA antigens followed by incubation with FITC-conjugated secondary goat-anti-mouse IgM- and IgG-antibodies respectively. B cell subpopulations were further characterized in a second step with fluorescence conjugated antibodies. Details on antibodies and fluorochromes are given in Table S2. In 6 out of 12 patients, antibodies, specific for recipient and donor HLA-antigens were available. In 4 patients, only anti-donor-type and in 2 patients, only anti-recipient-type HLA-antibodies could be tested. HLA positive and negative B cells were clearly distinguishable for gating. Positive and negative controls with HLA-typed cryopreserved buffy-coat MNCs were analysed in parallel with each experiment. Additional cell populations as monocytes, T and NK cells were characterized in the same experiment. As T cells are expected to originate completely from the donor in SCID-patients, these served as additional internal control. B cell phenotyping without HLA-staining led to equal results and distributions of subpopulations. If only one antibody was available, flow cytometric HLA results were confirmed once by an independent method (STR or XY-FISH, data not shown) after separation of populations by magnetic beads (EasySep\u0026trade; Cell Separation, STEMCELL technologies, Vancouver, Canada).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. B cell immunotyping by flow cytometry\u003c/h2\u003e\u003cp\u003eSamples from 25 long term surviving patients with B-positive SCID (cohort A) were included (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The selection of patients was based on the genotype and the availability of cryopreserved samples from the period of interest 90\u0026ndash;250 days after HSCT. 7 patients of this cohort were also analysed for chimerism studies (annotated in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In 16 male patients, hemizygous variants in \u003cem\u003eIL2RG\u003c/em\u003e were identified, in 9 patients, genetic variants affecting both alleles of \u003cem\u003eJAK3\u003c/em\u003e were detected. At their most recent presentation, patients had a median follow up of 11.1 years (0.4\u0026ndash;33.1) years after HSCT. Conditioning was based on variable regimen in 15/25 (60%) patients and 10/25 (40%) had been transplanted without conditioning (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Grafts were donated by mismatched family donors (MMFD) in 18/25, matched sibling donors (MSD) in 6/25 and a matched family donor (MFD) in 1/25 cases. Definitions of B cell subpopulations and antibodies used for their identification by flow cytometry are given in Tables S3 and S2 respectively. A minimal cell population of 50 cells in the CD19\u0026thinsp;+\u0026thinsp;CD27\u0026thinsp;+\u0026thinsp;gate was considered necessary for the reliable analysis of subpopulations. Patient samples with less than 50 cells in this gate were excluded from the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Definition of patient groups\u003c/h2\u003e\u003cp\u003eAccording to the information taken from patient charts patients were either assigned to the immunoglobulin-dependent (IgDEP) or independent group (IgIND). All patients in the IgIND -group were demonstrated to have normal specific antibody responses after vaccination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed by using IBM SPSS Statistics 24. A predictive cut-off for immune reconstitution was defined by using receiver operating characteristics (ROC) curves.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003e3.1. HLA-chimerism analysis in flow cytometry: less than 2% of donor B cells are sufficient for posttransplant specific humoral immune function\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSamples of 12 patients were investigated in this part of the study originated from post-haplo-transplant long-term survivors with B-lymphocyte positive SCID caused by genetic variants in \u003cem\u003eIL2RG\u003c/em\u003e. All patients were not substituted with immunoglobulins and had normal B cell function as determined by normal immunoglobulin values and normal response to vaccinations. The majority of patients (9/12) had been transplanted with and 3/12 without conditioning (for details see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, cohort B). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, T cells completely originated from the donor, whereas donor chimerism for B cells, monocytes and NK cells were mixed. B cell chimerism revealed to be highly variable between patients and a proportion of less than 2% of donor derived B cells was sufficient to allow for normal B cell function. Monocyte donor chimerism was found at the detection limit for two patients. As expected, donor chimerism for NK cells is higher in comparison to monocytes in all but one patient as an indicator for a selective advantage of donor derived NK lymphocytes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.2. B cell maturation is deficient in autologous B cells\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eAs demonstrated for a representative sample in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, HLA staining allows for donor and recipient specific analysis of B cell subpopulations in patients with post-transplant mixed B cell chimerism. If donor and recipient CD19-positive B cells are set to 100% respectively, the relative composition of the B cell compartments can be compared between donor and recipient (colored bars in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and between patients. This analysis exemplifies the remaining post-transplant maturational defect of autologous B cells despite the presence of donor T cells in all patients tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the normal maturational potential of donor B cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAutologous switched memory B cells (CD19\u0026thinsp;+\u0026thinsp;CD27\u0026thinsp;+\u0026thinsp;IgM-) are hardly detectable whereas the majority (80\u0026ndash;95%) of autologous B cells shows a na\u0026iuml;ve (CD19\u0026thinsp;+\u0026thinsp;CD27-) phenotype with Marginal-Zone-like B cells as the dominant CD27\u0026thinsp;+\u0026thinsp;population (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig S2). This latter pattern is shared by B-lymphocyte positive SCID patients pretransplant or posttransplant with autologous reconstitution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This finding is in contrast to the experience in patients with other SCID entities such as IL7RA or ADA (adenosine deaminase) deficiency in whom autologous B cells develop maturational capacity as soon as a donor derived T cell system is established (suppl. Figure\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eIn the donor B cell compartment a negative correlation between the percentage of donor Bcells and the proportion of switched-memory B cells can be observed: the smaller the donor B cell compartment, the higher the percentage of switched-memory B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Suppl. Figure\u0026nbsp;4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Posttransplant immunoglobulin levels are of limited reliability as indicators of B cell function\u003c/h2\u003e\u003cp\u003eTo check for potential B cell function after HSCT, the clinical routine in most institutions would include the close observation of patient IgA- and IgM-levels as these are not contained in pharmaceutical immunoglobulin substitution products and therefore indicate a production by mature plasma cells. In our cohort we were able to compare immunoglobulin levels at three periods post-HSCT: day 50, day 150, and between days 250 to 350. For IgA-levels, a significant difference between the IgIND and IgDEP group was only demonstrated for the latest period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) but not for earlier periods. Serum levels for IgM (suppl. Figure\u0026nbsp;5b) did not differ at any time.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.4. The proportion of switched memory B cells in the CD27\u0026thinsp;+\u0026thinsp;pool indicates the extend of B cell maturation and correlates with humoral specific immune function\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn order to transfer the robust finding that autologous cells are not able to mature to switched-memory B cells as observed in patients after haploidentical transplantation (cohort B, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) to a more general and clinically more practicable approach we studied B cell subpopulations in cohort A irrespective of chimerism. Cohort A was -among other parameters- defined by the availability of cryopreserved samples taken from patients at routine follow up visits between 90 and 250 days post-HSCT. Patients in this cohort had been shown to have genetic variants in \u003cem\u003eIL2RG\u003c/em\u003e and \u003cem\u003eJAK3\u003c/em\u003e and were transplanted with grafts donated by mismatched family donors, matched unrelated donors and matched sibling donors (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). B cell phenotyping was performed without any discrimination between donor and recipient cells. Available data on chimerism from the same period after HSCT are given in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. According to patient charts, 16 patients qualified for the IgIND and 9 patients for the IgDEP groups with a median follow up of 11.7 years (0.4\u0026ndash;33.1 years) for the IgIND group and 8.3 years (1-24.6 years) for the IgDEP group after HSCT respectively. As expected, patients in the IgDEP group show a lack of CD27-positive B cells in comparison to the IgIND group but this population is not completely absent. As demonstrated in cohort B (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), autologous B cells can mature to a CD27\u0026thinsp;+\u0026thinsp;stage, which is represented almost completely by Marginal-Zone like B cells. The clearest difference between autologous and donor B cell maturation in cohort B was found in the percentage of switched-memory B cells (CD19\u0026thinsp;+\u0026thinsp;CD27\u0026thinsp;+\u0026thinsp;IgM-) in the CD27\u0026thinsp;+\u0026thinsp;B cell pool. This population was absent or considerably reduced in all patients except one in the IgDEP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA ROC-analysis allowed to define a cut-off of 4.7% switched memory B cells in the CD27\u0026thinsp;+\u0026thinsp;B cell pool to correctly assign a patient to the IgIND or IgDEP group with a specificity of 88.9% and a sensitivity of 93.8% (Fig S6).\u003c/p\u003e\u003cp\u003eThe two patients of the IgIND and the IgDEP group, who were found below this threshold in the IgIND and above this threshold in the IgDEP group, were contacted and reevaluated. Patient 346 (Tab. S1), who was substituted with immunoglobulins but had a proportion of 11.1% of switched memory B cells in the CD27\u0026thinsp;+\u0026thinsp;B cell pool, was contacted and paused Ig-substitution for 4 months during summertime. He was challenged with inactivated vaccines (Tetanus, Diphtheria, Haemophilus and Pneumococci) and checked for specific antibodies. These turned out positive and he is off IVIG since.\u003c/p\u003e\u003cp\u003eThe proportion of switched memory B cells in patient 15 at the first analysis time point (d233 post HSCT) was still below the threshold of 4.7% in the CD27\u0026thinsp;+\u0026thinsp;pool in spite of 14% donor B cells (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, during 33 years of post HSCT follow up, he had no infectious history and was not substituted with immunoglobulins. His chimerism analysis at this late follow up time point (HLA-flow cytometry) revealed 4.6% of donor B cells with a proportion of 7.8% of switched memory B cells in the complete (donor and recipient) CD27\u0026thinsp;+\u0026thinsp;B cell pool (not shown) and a proportion of 27% switched memory B cells in the donor CD27\u0026thinsp;+\u0026thinsp;B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and S4a). IgG and IgM levels were found in the normal age adapted range but IgA was undetectable. Specific antibodies were positive for tetanus, pneumococci and rubella but negative for mumps and measles.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWithout engraftment of donor derived B-lymphocytes, patients with SCID due to genetic variants in \u003cem\u003eIL2RG\u003c/em\u003e and \u003cem\u003eJAK3\u003c/em\u003e will remain without specific humoral immune function and thus dependent on immunoglobulin substitution [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The underlying intrinsic defect of autologous B cells has been previously reported for this patient group by White et al [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] who demonstrated the inability of patient B cells, before and after HSCT, to perform a class switch and their inability to respond to IL-21 which was reported by Miggelbrink at al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. With our approach to analyze CD19\u0026thinsp;+\u0026thinsp;lymphocyte subpopulations and to label their donor or recipient origin by HLA-staining in flow cytometry, we provide further evidence for this primary defect in B cell maturation with an independent method. Beyond that, we can contribute an additional crucial clinical information: a very minor population of less than 2% of donor B cells in peripheral blood is sufficient to allow for normal specific immune functions \u0026ldquo;measured\u0026rdquo; retrospectively by an uneventful infectious history, normal Ig levels and the response to vaccinations with a median follow up of 14.3 (1.0-34.2) years. The formal weakness of this retrospective study turns to an advantage as this extensive follow up cannot be provided by a prospective approach and is a reliable clinical parameter to attribute patients in the cohort studied to the IgIND group.\u003c/p\u003e\u003cp\u003eWe moreover observed the correlation of a low percentage of donor B cells with a high proportion of class-switched memory cells within this donor B cell population. This negative correlation could be interpreted as an attempt to compensate for the lack of functional B cells by a supranormal percentage of maturation to switched memory B cells within the donor derived and thus functional B cell population. This of course raises further questions on the stability of such a system of exploitation. From the clinical perspective, these patients do not show any phenotype of immunodeficiency but this does not exclude the possibility of abnormal findings regarding BCR variability, clonal expansion or cellular signs of senescence or exhaustion, which will have to be further elucidated.\u003c/p\u003e\u003cp\u003eThe findings on B cells isolated from peripheral blood are not necessarily representative for the B cell system as a whole. Donor chimerism for B cell subpopulations in bone marrow and secondary lymphatic organs could vary considerably from those detected in CD19 positive cells in peripheral blood, but for obvious reasons this information will not be readily available. Even with these limitations, the information generated from blood samples remains essential for routine clinical decisions.\u003c/p\u003e\u003cp\u003eAutologous B cells after HSCT have a relevant proportion of 2.6 to 14% of CD27\u0026thinsp;+\u0026thinsp;cells, which almost all turn out not to be memory B cells but MZ-like B cells as they stain positive for IgM and IgD. To our surprise this has not been recognized and reported in the literature until present. Marginal zone-like B cells and their function have not been well characterized in humans yet and their function in mice is described as an important component for a T cell independent innate first wave of IgM-antibody-reaction of the immune system to bind with low affinity to highly conserved motifs of infectious agents. Beyond this, MZ-like B cells have also been shown to be able to interact with follicular T-helper cells and undergo class switch reaction and somatic hypermutation as follicular B cells do. In our studies we can clearly show, that CD19\u0026thinsp;+\u0026thinsp;CD27\u0026thinsp;+\u0026thinsp;IgM\u0026thinsp;+\u0026thinsp;IgD\u0026thinsp;+\u0026thinsp;cells can develop in the absence of signalling via the IL21-receptor but we strongly suspect that deep phenotyping and genetic studies (V(D)J-rearrangement and BCR-clonality) will be able to show major differences in this autologous B cell population in our cohort before HSCT (no T cell help, no IL-21 signalling), after HSCT (T cell help, no IL-21 signalling) and in comparison to donor derived MZ-like B cells (T cell help, normal IL-21 signalling) or samples from healthy controls.\u003c/p\u003e\u003cp\u003eWe aimed to practically exploit these findings for clinical use to help with the decision to stop immunoglobulin substitution in post HSCT patients with B-positive SCID. This decision can be clinically challenging- especially for patients with mixed chimerism. The mere presence of B cells is not sufficient to deduce B cell function. As reviewed by van der Maas et al [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the reconstitution of non-switched Marginal-Zone like B cells and switched memory B cells proceeds slowly after HSCT and it can take up to two years or longer to reach age-matched normal levels. Gold standard and final proof for a functional B cell system is the detection of post-vaccination specific antibodies and Ig levels within the normal range in addition to an uneventful infectious history. For obvious reasons, IgG-levels and vaccination titers cannot be tested while patients are on Ig-substitution. IgM and IgA levels would be used by many centers as indicators for normal specific humoral immune function. For our cohort, we could demonstrate that there is no significant difference for IgM-levels between the IgIND and IgDEP groups and that IgA-levels become informative at a period after 250 days posttransplant. This parameter though remains with a major taint for clinical use as the absence of IgA in the age group tested here is within the normal range. The detection of donor derived B cells would be helpful but with the findings demonstrated above it becomes clear that a minor proportion donor derived B cells in the range of 2\u0026ndash;5% cannot be reliably detected by other methods of chimerism analysis. Even after B cell enrichment or purification a percentage of less than 5% can be missed in STR analysis or XY-FISH. HLA-staining for flow cytometry is rarely established for routine chimerism analysis and is only feasible for patients transplanted from haploidentical donors. But with the experience of HLA-staining of B cell subpopulations we were able to clearly correlate the presence of CD19\u0026thinsp;+\u0026thinsp;CD27\u0026thinsp;+\u0026thinsp;IgM- B cells in peripheral blood as indicator for B cell function. The quantification of this population irrespective of its chimerism in cryopreserved posttransplant patient samples allowed to statistically define a threshold of 4.7% CD27\u0026thinsp;+\u0026thinsp;IgM- (% of CD27\u0026thinsp;+\u0026thinsp;CD19+) with the highest specificity and sensitivity to retrospectively attribute a patient to the IgIND group.\u003c/p\u003e\u003cp\u003eThe presence of this population of class-switched memory B cells was previously found to be informative for posttransplant B cell function for B cell positive SCID patients by Miggelbrink et al [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] - we can confirm this finding in a larger cohort and add the clinically important quantification and definition of a threshold together with a diagnostic window as early as 90\u0026ndash;250 days post HSCT. This additional information now allows the use of this parameter for clinical decision making and the cessation of post HSCT Ig-substitution. An earlier time point would not be informative as the help of follicular T cells is necessary to allow germinal center reactions to induce the class switch of B cells. The reconstitution of T cells after HSCT cannot be observed before 2\u0026ndash;3 months after HSCT and can be delayed by GvHD, immunosuppressive drugs or infections.\u003c/p\u003e\u003cp\u003eThe clinical use of the quantification of CD19\u0026thinsp;+\u0026thinsp;CD27\u0026thinsp;+\u0026thinsp;IgM- B cells is not confined to the cohort reported here but could be adapted to B cell positive SCID patients after autologous gene therapy or the growing group of patients after targeted B cell depleting therapies with monospecific (e.g. Rituximab) or bispecific (e.g. Blinatumomab) antibodies or CAR-T cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this retrospective analysis on patients with B cell positive SCID we could demonstrate by HLA-staining that a very minor percentage of less than 2% of donor B cells in peripheral blood correlates with normal immune function. Between day 90\u0026ndash;250 in post-transplant follow up a proportion of \u0026gt;\u0026thinsp;4.7% of CD19\u0026thinsp;+\u0026thinsp;CD27\u0026thinsp;+\u0026thinsp;IgM- within the CD27\u0026thinsp;+\u0026thinsp;B cell pool is a sensitive and specific marker to indicate post HSCT B cell function in this cohort.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eADA\u0026nbsp; \u0026nbsp; \u0026nbsp;adenosine deaminase\u003c/p\u003e\n\u003cp\u003eBCR B cell receptor\u003c/p\u003e\n\u003cp\u003eHSCT \u0026nbsp; \u0026nbsp;hematopoetic stem cell transplantation\u003c/p\u003e\n\u003cp\u003eIg Immunoglobulin\u003c/p\u003e\n\u003cp\u003eIgIND\u0026nbsp; \u0026nbsp;independent of immunglobulin substitution\u003c/p\u003e\n\u003cp\u003eIgDEP\u0026nbsp; \u0026nbsp;dependent of immunglobulin substitution\u003c/p\u003e\n\u003cp\u003eIL2RG\u0026nbsp; \u0026nbsp;interleukin 2 receptor gamma\u003c/p\u003e\n\u003cp\u003eIL7RA\u0026nbsp; \u0026nbsp;interleukin 7 receptor alpha\u003c/p\u003e\n\u003cp\u003eJAK3\u0026nbsp; \u0026nbsp; \u0026nbsp;janus kinase 3\u003c/p\u003e\n\u003cp\u003eMZ\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;marginal zone\u003c/p\u003e\n\u003cp\u003eSCID\u0026nbsp; \u0026nbsp; \u0026nbsp;severe combined immunodeficiency\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE-M.J. and A.K. performed the analysis of patient MNCs in Flow Cytometry and evaluated the data. W.F., A.S.S. K.F, I.F, K.W, M.S, U.P, K.S and M.H. were responsible for patient care and/ or provided clinical, genetic or laboratory data. M.H. \u0026nbsp;wrote the main manuscript text, E.J. prepared figures 1-3 and A.K figures 4-5. All authors contributed to the discussion and the preparation of the draft and have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eDisclosure of potential conflicts of interest:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of (financial) interests with respect to the content of this manuscript. No funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEthics approval:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the local Ethics Committee of Ulm University (reference/review number 20122017).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConsent to participate:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for participation was obtained from all patients and/or their legal guardians included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Carmen Blum, Andrea H\u0026auml;nsler, Gudrun Kirsch and Ulrike Tengler for excellent technical assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRecher M, Berglund LJ, Avery DT, et al. IL-21 is the primary common gamma chain-binding cytokine required for human B-cell differentiation in vivo. \u003cem\u003eBlood\u003c/em\u003e. 2011;118(26):6824-6835.\u003c/li\u003e\n\u003cli\u003eAsao H, Okuyama C, Kumaki S, et al. Cutting edge: the common gamma-chain is an indispensable subunit of the IL-21 receptor complex. \u003cem\u003eJ Immunol\u003c/em\u003e. 2001;167(1):1-5.\u003c/li\u003e\n\u003cli\u003eMiggelbrink AM, Logan BR, Buckley RH, et al. B-cell differentiation and IL-21 response in IL2RG/JAK3 SCID patients after hematopoietic stem cell transplantation. \u003cem\u003eBlood\u003c/em\u003e. 2018;131(26):2967-2977.\u003c/li\u003e\n\u003cli\u003eWhite H, Thrasher A, Veys P, Kinnon C, Gaspar HB. Intrinsic defects of B cell function in X-linked severe combined immunodeficiency. \u003cem\u003eEur J Immunol\u003c/em\u003e. 2000;30(3):732-737.\u003c/li\u003e\n\u003cli\u003eGennery AR, Slatter MA, Grandin L, et al. Transplantation of hematopoietic stem cells and long-term survival for primary immunodeficiencies in Europe: entering a new century, do we do better? \u003cem\u003eJ Allergy Clin Immunol\u003c/em\u003e. 2010;126(3):602-610 e601-611.\u003c/li\u003e\n\u003cli\u003eLankester AC, Neven B, Mahlaoui N, et al. Hematopoietic cell transplantation in severe combined immunodeficiency: The SCETIDE 2006-2014 European cohort. \u003cem\u003eJ Allergy Clin Immunol\u003c/em\u003e. 2021.\u003c/li\u003e\n\u003cli\u003eFischer A, Hacein-Bey-Abina S. Gene therapy for severe combined immunodeficiencies and beyond. \u003cem\u003eJ Exp Med\u003c/em\u003e. 2020;217(2).\u003c/li\u003e\n\u003cli\u003eFischer A, Hacein-Bey Abina S, Touzot F, Cavazzana M. Gene therapy for primary immunodeficiencies. \u003cem\u003eClin Genet\u003c/em\u003e. 2015;88(6):507-515.\u003c/li\u003e\n\u003cli\u003evan der Maas NG, Berghuis D, van der Burg M, Lankester AC. B Cell Reconstitution and Influencing Factors After Hematopoietic Stem Cell Transplantation in Children. Front Immunol. 2019;10:782. doi:10.3389/fimmu.2019.00782\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"B cell positive SCID, IL2RG, JAK3, HSC-transplantation, MZ-like B cells, B-lymphocyte reconstitution, immunoglobulin substitution","lastPublishedDoi":"10.21203/rs.3.rs-7329087/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7329087/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenetic defects in \u003cem\u003eIL2RG \u003c/em\u003eor \u003cem\u003eJAK3 \u003c/em\u003ecan\u003cem\u003e \u003c/em\u003ecause the phenotype of severe combined immunodeficiency (SCID) with absent T- and non-functional B-lymphocytes (T-B+ SCID). B cell function and the need for immunoglobulin replacement after hematopoietic stem cell transplantation (HSCT) depends on the engraftment of donor B-lymphocytes. In a retrospective study we describe B-lymphocyte reconstitution after HSCT with the objective to identify B cell subpopulations as an early predictor for the maturation and function of B cells after HSCT. All patients included underwent HSCT in a single institution between 1980 and 2017. First, we studied B cell maturation in cryopreserved blood samples of 12 long-term surviving patients with B+ SCID (IL2RG-deficiency) after haploidentical HSCT and mixed B cell chimerism. Recipient and donor B cell subpopulations were identified by HLA-staining using flow cytometry. In a consecutive step we compared B cell subpopulations irrespective of chimerism between patients with or without post-transplant B cell function. Samples for this study had been obtained between day +90 to +250 after HSCT from 25 post-transplant long-term survivors with B-positive (9 with genetic variants in \u003cem\u003eJAK3, \u003c/em\u003e16 in \u003cem\u003eIL2RG)\u003c/em\u003e SCID, 9/25 were dependent and 16/25 independent of Ig-substitution.\u003c/p\u003e\n\u003cp\u003eWe demonstrate that a proportion of less than 2% of donor B cells is sufficient for posttransplant B cell function and that a proportion of more than 4.7% of switched memory (IgM-) B cells in the memory B cell population (CD19+CD27+) between days +90 and +250 after HSCT correlates with normal B cell function and independence from immunoglobulin substitution.\u003c/p\u003e","manuscriptTitle":"Posttransplant B cell development and function in patients with B cell positive SCID caused by pathogenic variants in IL2RG and JAK3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 10:51:59","doi":"10.21203/rs.3.rs-7329087/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-01T18:56:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T12:22:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T14:51:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T17:05:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166973595865092169173931130679951337295","date":"2025-09-09T09:05:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41002178904042436519939644601929354464","date":"2025-09-06T01:13:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172746890277213875122012130075499713767","date":"2025-09-05T18:04:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-03T17:17:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-26T02:51:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-26T02:50:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Clinical Immunology","date":"2025-08-08T16:34:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b4fc5f47-9ad5-44ca-869c-c8f2c61620d9","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:07:23+00:00","versionOfRecord":{"articleIdentity":"rs-7329087","link":"https://doi.org/10.1007/s10875-026-02004-2","journal":{"identity":"journal-of-clinical-immunology","isVorOnly":false,"title":"Journal of Clinical Immunology"},"publishedOn":"2026-03-21 15:59:12","publishedOnDateReadable":"March 21st, 2026"},"versionCreatedAt":"2025-09-11 10:51:59","video":"","vorDoi":"10.1007/s10875-026-02004-2","vorDoiUrl":"https://doi.org/10.1007/s10875-026-02004-2","workflowStages":[]},"version":"v1","identity":"rs-7329087","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7329087","identity":"rs-7329087","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}