In-depth blood immune profiling of Good syndrome patients.

Introduction: Good syndrome (GS) is a rare adult-onset immunodeficiency first described in 1954. It is characterized by the coexistence of a thymoma and hypogammaglobulinemia, associated with an increased susceptibility to infections and autoimmunity. The classification and management of GS has been long hampered by the lack of data about the underlying immune alterations, a controversy existing on whether it is a unique diagnostic entity vs. a subtype of Common Variable Immune Deficiency (CVID). Methods: Here, we used high-sensitive flow cytometry to investigate the distribution of up to 70 different immune cell populations in blood of GS patients (n=9) compared to age-matched CVID patients (n=55) and healthy donors (n=61). Results: All 9 GS patients displayed reduced B-cell counts -down to undetectable levels (<0.1 cells/µL) in 8/9 cases-, together with decreased numbers of total CD4+ T-cells, NK-cells, neutrophils, and basophils vs. age-matched healthy donors. In contrast, they showed expanded TCRγδ+ T-cells (p ≤ 0.05). Except for a deeper B-cell defect, the pattern of immune cell alteration in blood was similar in GS and (age-matched) CVID patients. In depth analysis of CD4+ T-cells revealed significantly decreased blood counts of naïve, central memory (CM) and transitional memory (TM) TCD4+ cells and their functional compartments of T follicular helper (TFH), regulatory T cells (Tregs), T helper (Th)2, Th17, Th22, Th1/Th17 and Th1/Th2 cells. In addition, GS patients also showed decreased NK-cell, neutrophil, basophil, classical monocyte and of both CD1c+ and CD141+ myeloid dendritic cell counts in blood, in parallel to an expansion of total and terminal effector TCRγδ+ T-cells. Interestingly, those GS patients who developed hypogammaglobulinemia several years after the thymoma presented with an immunological and clinical phenotype which more closely resembled a combined immune humoral and cellular defect, with poorer response to immunoglobulin replacement therapy, as compared to those in whom the thymoma and hypogammaglobulinemia were simultaneously detected. Discussion: Our findings provide a more accurate definition of the immune cell defects of GS patients and contribute to a better discrimination among GS patients between those with a pure B-cell defect vs. those suffering from a combined immunodeficiency with important consequences on the diagnosis and management of the disease.

Automated EuroFlow approach for standardized in-depth dissection of human circulating B-cells and plasma cells.

Background: Multiparameter flow cytometry (FC) immunophenotyping is a key tool for detailed identification and characterization of human blood leucocytes, including B-lymphocytes and plasma cells (PC). However, currently used conventional data analysis strategies require extensive expertise, are time consuming, and show limited reproducibility. Objective: Here, we designed, constructed and validated an automated database-guided gating and identification (AGI) approach for fast and standardized in-depth dissection of B-lymphocyte and PC populations in human blood. Methods: For this purpose, 213 FC standard (FCS) datafiles corresponding to umbilical cord and peripheral blood samples from healthy and patient volunteers, stained with the 14-color 18-antibody EuroFlow BIgH-IMM panel, were used. Results: The BIgH-IMM antibody panel allowed identification of 117 different B-lymphocyte and PC subsets. Samples from 36 healthy donors were stained and 14 of the datafiles that fulfilled strict inclusion criteria were analysed by an expert flow cytometrist to build the EuroFlow BIgH-IMM database. Data contained in the datafiles was then merged into a reference database that was uploaded in the Infinicyt software (Cytognos, Salamanca, Spain). Subsequently, we compared the results of manual gating (MG) with the performance of two classification algorithms -hierarchical algorithm vs two-step algorithm- for AGI of the cell populations present in 5 randomly selected FCS datafiles. The hierarchical AGI algorithm showed higher correlation values vs conventional MG (r2 of 0.94 vs. 0.88 for the two-step AGI algorithm) and was further validated in a set of 177 FCS datafiles against conventional expert-based MG. For virtually all identifiable cell populations a highly significant correlation was observed between the two approaches (r2>0.81 for 79% of all B-cell populations identified), with a significantly lower median time of analysis per sample (6 vs. 40 min, p=0.001) for the AGI tool vs. MG, respectively and both intra-sample (median CV of 1.7% vs. 10.4% by MG, p<0.001) and inter-expert (median CV of 3.9% vs. 17.3% by MG by 2 experts, p<0.001) variability. Conclusion: Our results show that compared to conventional FC data analysis strategies, the here proposed AGI tool is a faster, more robust, reproducible, and standardized approach for in-depth analysis of B-lymphocyte and PC subsets circulating in human blood.

Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

Improving Properties of Podophyllic Aldehyde-Derived Cyclolignans: Design, Synthesis and Evaluation of Novel Lignohydroquinones, Dual-Selective Hybrids against Colorectal Cancer Cells.

New lignohydroquinone conjugates (L-HQs) were designed and synthesized using the hybridization strategy, and evaluated as cytotoxics against several cancer cell lines. The L-HQs were obtained from the natural product podophyllotoxin and some semisynthetic terpenylnaphthohydroquinones, prepared from natural terpenoids. Both entities of the conjugates were connected through different aliphatic or aromatic linkers. Among the evaluated hybrids, the L-HQ with the aromatic spacer clearly displayed the in vitro dual cytotoxic effect derived from each starting component, retaining the selectivity and showing a high cytotoxicity at short (24 h) and long (72 h) incubation times (4.12 and 0.0450 µM, respectively) against colorectal cancer cells. In addition, the cell cycle blockade observed by flow cytometry studies, molecular dynamics, and tubulin interaction studies demonstrated the interest of this kind of hybrids, which docked adequately into the colchicine binding site of tubulin despite their large size. These results prove the validity of the hybridization strategy and encourage further research on non-lactonic cyclolignans.

The Euroflow PID Orientation Tube in the diagnostic workup of primary immunodeficiency: Daily practice performance in a tertiary university hospital.

Introduction: Multiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can identify different lymphoid-PIDs with high sensitivity, clinical validation in a broad spectrum of patients with suspicion of PID is missing. In this study, we investigated the diagnostic performance of PIDOT, as part of the EuroFlow diagnostic screening algorithm for lymphoid-PID, in a daily practice at a tertiary reference center for PID. Methods: PIDOT was tested in 887 consecutive patients suspicious of PID at the Ghent University Hospital, Belgium. Patients were classified into distinct subgroups of lymphoid-PID vs. non-PID disease controls (non-PID DCs), according to the IUIS and ESID criteria. For the clinical validation of PIDOT, comprehensive characterization of the lymphoid defects was performed, together with the identification of the most discriminative cell subsets to distinguish lymphoid-PID from non-PID DCs. Next, a decision-tree algorithm was designed to guide subsequent FCM analyses. Results: The mean number of lymphoid defects detected by PIDOT in blood was 2.87 times higher in lymphoid-PID patients vs. non-PID DCs (p < 0.001), resulting in an overall sensitivity and specificity of 87% and 62% to detect severe combined immunodeficiency (SCID), combined immunodeficiency with associated or syndromic features (CID), immune dysregulation disorder (ID), and common variable immunodeficiency (CVID). The most discriminative populations were total memory and switched memory B cells, total T cells, TCD4+cells, and naive TCD4+cells, together with serum immunoglobulin levels. Based on these findings, a decision-tree algorithm was designed to guide further FCM analyses, which resulted in an overall sensitivity and specificity for all lymphoid-PIDs of 86% and 82%, respectively. Conclusion: Altogether, our findings confirm that PIDOT is a powerful tool for the diagnostic screening of lymphoid-PID, particularly to discriminate (S)CID, ID, and CVID patients from other patients suspicious of PID. The combination of PIDOT and serum immunoglobulin levels provides an efficient guide for further immunophenotypic FCM analyses, complementary to functional and genetic assays, for accurate PID diagnostics.

Age and Primary Vaccination Background Influence the Plasma Cell Response to Pertussis Booster Vaccination.

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole-cell to acellular pertussis vaccines, although other factors may also contribute. Here, we applied high-dimensional flow cytometry to investigate changes in B cells in individuals of different ages and distinct priming backgrounds upon administration of an acellular pertussis booster vaccine. Participants were divided over four age cohorts. We compared longitudinal kinetics within each cohort and between the different cohorts. Changes in the B-cell compartment were correlated to numbers of vaccine-specific B- and plasma cells and serum Ig levels. Expansion and maturation of plasma cells 7 days postvaccination was the most prominent cellular change in all age groups and was most pronounced for more mature IgG1+ plasma cells. Plasma cell responses were stronger in individuals primed with whole-cell vaccine than in individuals primed with acellular vaccine. Moreover, IgG1+ and IgA1+ plasma cell expansion correlated with FHA-, Prn-, or PT- specific serum IgG or IgA levels. Our study indicates plasma cells as a potential early cellular marker of an immune response and contributes to understanding differences in immune responses between age groups and primary vaccination backgrounds.

Highly Sensitive Flow Cytometry Allows Monitoring of Changes in Circulating Immune Cells in Blood After Tdap Booster Vaccination.

Antigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In these models, in-depth knowledge of cellular processes associated with protective versus poor responses may bring added value. We applied high-throughput multicolor flow cytometry to track over-time changes in circulating immune cells in 10 individuals following pertussis booster vaccination (Tdap, Boostrix®, GlaxoSmithKline). Next, we applied correlation network analysis to extensively investigate how changes in individual cell populations correlate with each other and with Ag-specific Ig levels. We further determined the most informative cell subsets and analysis time points for future studies. Expansion and maturation of total IgG1 plasma cells, which peaked at day 7 post-vaccination, was the most prominent cellular change. Although these cells preceded the increase in Ag-specific serum Ig levels, they did not correlate with the increase of Ig levels. In contrast, strong correlation was observed between Ag-specific IgGs and maximum expansion of total IgG1 and IgA1 memory B cells at days 7 to 28. Changes in circulating T cells were limited, implying the need for a more sensitive approach. Early changes in innate immune cells, i.e. expansion of neutrophils, and expansion and maturation of monocytes up to day 5, most likely reflected their responses to local damage and adjuvant. Here we show that simultaneous monitoring of multiple circulating immune subsets in blood by flow cytometry is feasible. B cells seem to be the best candidates for vaccine monitoring.

Dynamic Intracellular Metabolic Cell Signaling Profiles During Ag-Dependent B-Cell Differentiation.

Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells undergoing antigen-dependent maturation using a label-free LC-MS/MS approach applied on 5 purified B-cell subpopulations (naive, centroblasts, centrocytes, memory and plasma B-cells) from human tonsils (data are available via ProteomeXchange with identifier PXD006191). Our results revealed that the actual differences among these B-cell subpopulations are a combination of expression of a few maturation stage-specific proteins within each B-cell subset and maturation-associated changes in relative protein expression levels, which are related with metabolic regulation. The considerable overlap of the proteome of the 5 studied B-cell subsets strengthens the key role of the regulation of the stoichiometry of molecules associated with metabolic regulation and programming, among other signaling cascades (such as antigen recognition and presentation and cell survival) crucial for the transition between each B-cell maturation stage.

Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating.

Background: Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods: FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results: The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I. Conclusion: Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles.

A Novel Cytotoxic Conjugate Derived from the Natural Product Podophyllotoxin as a Direct-Target Protein Dual Inhibitor.

Natural products are the ideal basis for the design of novel efficient molecular entities. Podophyllotoxin, a naturally occurring cyclolignan, is an example of natural product which displays a high versatility from a biological activity point of view. Based on its unique chemical structure, different derivatives have been synthesized presenting the original antitumoral properties associated with the compound, i.e., the tubulin polymerization inhibition and arising anti-topoisomerase II activity from structural modifications on the cyclolignan skeleton. In this report, we present a novel conjugate or hybrid which chemically combines both biological activities in one single molecule. Chemical design has been planned based in our lead compound, podophyllic aldehyde, as an inhibitor of tubulin polymerization, and in etoposide, an approved antitumoral drug targeting topoisomerase II. The cytotoxicity and selectivity of the novel synthetized hybrid has been evaluated in several cell lines of different solid tumors. In addition, these dual functional effects of the novel compound have been also evaluated by molecular docking approaches.

Detection of Circulating Tumor Plasma Cells in Monoclonal Gammopathies: Methods, Pathogenic Role, and Clinical Implications.

Cancer dissemination and distant metastasis most frequently require the release of tumor cells into the blood circulation, both in solid tumors and most hematological malignancies, including plasma cell neoplasms. However, detection of blood circulating tumor cells in solid tumors and some hematological malignancies, such as the majority of mature/peripheral B-cell lymphomas and monoclonal gammopathies, has long been a challenge due to their very low frequency. In recent years, the availability of highly-sensitive and standardized methods for the detection of circulating tumor plasma cells (CTPC) in monoclonal gammopathies, e.g., next-generation flow cytometry (NGF), demonstrated the systematic presence of CTPC in blood in virtually every smoldering (SMM) and symptomatic multiple myeloma (MM) patient studied at diagnosis, and in the majority of patients with newly-diagnosed monoclonal gammopathies of undetermined significance (MGUS). These methods set the basis for further detailed characterization of CTPC vs. their bone marrow counterpart in monoclonal gammopathies, to investigate their role in the biology of the disease, and to confirm their strong impact on patient outcome when measured both at diagnosis and after initiating therapy. Here, we review the currently available techniques for the detection of CTPC, and determine their biological features, physiopathological role and clinical significance in patients diagnosed with distinct diagnostic categories of plasma cell neoplasms.

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children.

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube.

CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models-monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM), and common variable immunodeficiency (CVID). Overall, 145 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design-testing-evaluation-redesign, combined with in vitro functional studies, gene expression profiling, and multicentric evaluation of manual vs. automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0-89 years) were used to establish reference values, and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL (n = 8), SM (n = 7), and CVID (n = 10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T cells, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, Th2, and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4 tube allows fast, automated, and reproducible identification of ≥89 subsets of CD4+ blood T cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.

Dissection of the Pre-Germinal Center B-Cell Maturation Pathway in Common Variable Immunodeficiency Based on Standardized Flow Cytometric EuroFlow Tools.

Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients. Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD). Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21-CD24- (6.1 vs 0.74 cells/µl, p<0.0001) and CD21-CD24++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD+ (21 vs 32 cells/µl, p=0.03) and IgMD- (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%). Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.

Monozygotic Twins Concordant for Common Variable Immunodeficiency: Strikingly Similar Clinical and Immune Profile Associated With a Polygenic Burden.

Monozygotic twins provide a unique opportunity to better understand complex genetic diseases and the relative contribution of heritable factors in shaping the immune system throughout life. Common Variable Immunodeficiency Disorders (CVID) are primary antibody defects displaying wide phenotypic and genetic heterogeneity, with monogenic transmission accounting for only a minority of the cases. Here, we report a pair of monozygotic twins concordant for CVID without a family history of primary immunodeficiency. They featured a remarkably similar profile of clinical manifestations and immunological alterations at diagnosis (established at age 37) and along the subsequent 15 years of follow-up. Interestingly, whole-exome sequencing failed to identify a monogenic cause for CVID, but unraveled a combination of heterozygous variants, with a predicted deleterious impact. These variants were found in genes involved in relevant immunological pathways, such as JUN, PTPRC, TLR1, ICAM1, and JAK3. The potential for combinatorial effects translating into the observed disease phenotype is inferred from their roles in immune pathways, namely in T and B cell activation. The combination of these genetic variants is also likely to impose a significant constraint on environmental influences, resulting in a similar immunological phenotype in both twins, despite exposure to different living conditions. Overall, these cases stress the importance of integrating NGS data with clinical and immunological phenotypes at the single-cell level, as provided by multi-dimensional flow-cytometry, in order to understand the complex genetic landscape underlying the vast majority of patients with CVID, as well as those with other immunodeficiencies.

Delineating Human B Cell Precursor Development With Genetically Identified PID Cases as a Model.

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.

Complete Multilineage CD4 Expression Defect Associated With Warts Due to an Inherited Homozygous CD4 Gene Mutation.

Idiopathic T-CD4 lymphocytopenia (ICL) is a rare and heterogeneous syndrome characterized by opportunistic infections due to reduced CD4 T-lymphocytes (<300 cells/µl or <20% T-cells) in the absence of HIV infection and other primary causes of lymphopenia. Molecular testing of ICL has revealed defects in genes not specific to CD4 T-cells, with pleiotropic effects on other cell types. Here we report for the first time an absolute CD4 lymphocytopenia (<0.01 CD4+ T-cells/µl) due to an autosomal recessive CD4 gene mutation that completely abrogates CD4 protein expression on the surface membrane of T-cells, monocytes, and dendritic cells. A 45-year-old female born to consanguineous parents consulted because of exuberant, relapsing, and treatment-refractory warts on her hands and feet since the age of 10 years, in the absence of other recurrent infections or symptoms. Serological studies were negative for severe infections, including HIV 1/2, HTLV-1, and syphilis, but positive for CMV and EBV. Blood analysis showed the absence of CD4+ T-cells (<0.01%) with repeatedly increased counts of B-cells, naïve CD8+ T-lymphocytes, and particularly, CD4/CD8 double-negative (DN) TCRαß+ TCRγδ- T-cells (30% of T-cells; 400 cells/µl). Flow cytometric staining of CD4 using monoclonal antibodies directed against five different epitopes, located in two different domains of the protein, confirmed no cell surface membrane or intracytoplasmic expression of CD4 on T-cells, monocytes, and dendritic cells but normal soluble CD4 plasma levels. DN T-cells showed a phenotypic and functional profile similar to normal CD4+ T-cells as regards expression of maturation markers, T-helper and T-regulatory chemokine receptors, TCRvß repertoire, and in vitro cytokine production against polyclonal and antigen-specific stimuli. Sequencing of the CD4 gene revealed a homozygous (splicing) mutation affecting the last bp on intron 7-8, leading to deletion of the juxtamembrane and intracellular domains of the protein and complete abrogation of CD4 expression on the cell membrane. These findings support previous studies in CD4 KO mice suggesting that surrogate DN helper and regulatory T-cells capable of supporting antigen-specific immune responses are produced in the absence of CD4 signaling and point out the need for better understanding the role of CD4 on thymic selection and the immune response.

CD Maps-Dynamic Profiling of CD1-CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets.

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

Immunophenotypic dissection of normal hematopoiesis.

Flow cytometry immunophenotyping is essential for diagnosis, classification and monitoring of clonal hematopoietic diseases, particularly of hematological malignancies and primary immunodeficiencies. Optimal use of immunophenotyping for these purposes requires detailed knowledge about the phenotypic patterns of normal hematopoietic cells. In the past few decades, flow cytometry has benefited from technological developments allowing simultaneous analysis of multiple antigen stainings with ≥3-35 distinct fluorochrome-conjugated antibodies for increasingly higher numbers of cells. These advances have contributed to expand our knowledge about the phenotypic differentiation profiles of normal hematopoietic cells, from uncommitted CD34+ precursors in the bone marrow (BM) and peripheral blood (PB), to the several hundreds of populations of circulating myeloid and (B and T) lymphoid cells identified so far. Detailed dissection of the normal phenotypic profiles of hematopoietic cells has settled the basis for identification of aberrant phenotypes on leukemia and lymphoma cells. Thus, it has contributed to: i) more sensitive identification of leukemia/lymphoma cells (especially when represented at low frequencies in a sample), and ii) more accurate classification of hematological malignancies. In this manuscript, we review the major phenotypic features of hematopoietic cells, from the more immature BM CD34+ precursors committed to the myeloid and lymphoid lineages toward mature hematopoietic cells circulating in PB (e.g. neutrophils, monocytes, basophils, eosinophils, dendritic cells, erythroid cells, and B- and T-cells) and those homing to other tissues (e.g. plasma cells, mast cells).