Aberrant early hematopoietic progenitor formation marks the onset of hematopoietic defects in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder that often presents at infancy. Progress has been made in revealing causal mutated genes (SBDS and others), ribosome defects, and hematopoietic aberrations in SDS. However, the mechanism underlying the hematopoietic failure remained unknown, and treatment options are limited. Herein, we investigated the onset of SDS embryonic hematopoietic impairments. We generated SDS and control human-derived induced pluripotent stem cells (iPSCs). SDS iPSCs recapitulated the SDS hematological phenotype. Detailed stepwise evaluation of definitive hematopoiesis revealed defects that started at the early emerging hematopoietic progenitor (EHP) stage after mesoderm and hemogenic endothelium were normally induced. Hematopoietic potential of EHPs was markedly reduced, and the introduction of SBDS in SDS iPSCs improved colony formation. Transcriptome analysis revealed reduced expression of ribosome and oxidative phosphorylation-related genes in undifferentiated and differentiated iPSCs. However, certain pathways (e.g., DNA replication) and genes (e.g., CHCHD2) were exclusively or more severely dysregulated in EHPs compared with earlier and later stages. To our knowledge, this study offers for the first time an insight into the embryonic onset of human hematopoietic defects in an inherited bone marrow failure syndrome and reveals cellular and molecular aberrations at critical stages of hematopoietic development toward EHPs.

Gut Immunomodulation with Vedolizumab prior to Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients with Inflammatory Bowel Disease.

Inborn errors of immunity (IEI) are often associated with inflammatory bowel disease (IBD). IEI can be corrected by allogeneic hematopoietic stem cell transplantation (HSCT); however, peritransplantation intestinal inflammation may increase the risk of gut graft-versus-host disease (GVHD). Vedolizumab inhibits the homing of lymphocytes to the intestine and may attenuate gut GVHD, yet its role in preventing GVHD in pediatric patients with IEI-associated IBD has not been studied. Here we describe a cohort of pediatric patients with IEI-associated IBD treated with vedolizumab before and during allogeneic HSCT. The study involved a retrospective chart review of pediatric patients with IEI-associated IBD treated with vedolizumab at 6 weeks, 4 weeks, and 1 week before undergoing HSCT. The conditioning regimen consisted of treosulfan, fludarabine, and cyclophosphamide with rabbit antithymocyte globulin, and GVHD prophylaxis included tacrolimus and steroids. Eleven patients (6 females) with a median age of 5 years (range, 0.4 to 14 years) with diverse IEI were included. IBD symptoms were characterized by abdominal pain, loose stools, and blood in stools. Four patients had developed a perianal fistula, and 1 patient had a rectal prolapse. One patient had both a gastrostomy tube and a jejunal tube in situ. Treatment of IBD before HSCT included steroids in 11 patients, anakinra in 2, infliximab in 4, sulfasalazine in 2, mesalazine in 2, and vedolizumab. IBD symptoms were considered controlled in the absence of abdominal pain, loose stools, or blood in stools. Graft sources for HSCT were unrelated donor cord in 5 patients (2 with a 5/8 HLA match, 2 with a 7/8 match, and 1 with a 6/8 match), peripheral blood stem cells in 5 patients (2 haploidentical, 1 with a 9/10 HLA match, and 2 with a 10/10 match), and bone marrow in 1 patient (10/10 matched sibling donor). The median number of vedolizumab infusions was 4 (range, 3 to 12) before HSCT and 1 (range, 1 to 3) after HSCT, and all were reported to be uneventful. All patients had engrafted. Acute GVHD occurred in 4 patients and was limited to grade I skin GVHD only. Chronic GVHD occurred in 1 patient and again was limited to the skin. There was no gut GVHD. Three patients experienced cytomegalovirus viremia, and 2 patients had Epstein-Barr virus viremia. At the time of this report, all patients were alive with no evidence of IBD at a median follow-up of 15 months (range, 3 to 39 months). Administration of vedolizumab pre- and post-HSCT in pediatric patients with IEI-associated IBD is well tolerated and associated with a low rate of gut GVHD. These findings provide a platform for the prospective study and use of vedolizumab for GVHD prophylaxis in pediatric patients with known intestinal inflammation as a pre-HSCT comorbidity.

Allogeneic hematopoietic cell transplantation is effective for p47phox chronic granulomatous disease: A Primary Immune Deficiency Treatment Consortium study.

BACKGROUND: P47phox (neutrophil cytosolic factor-1) deficiency is the most common cause of autosomal recessive chronic granulomatous disease (CGD) and is considered to be associated with a milder clinical phenotype. Allogeneic hematopoietic cell transplantation (HCT) for p47phox CGD is not well-described. OBJECTIVES: We sought to study HCT for p47phox CGD in North America. METHODS: Thirty patients with p47phox CGD who received allogeneic HCT at Primary Immune Deficiency Treatment Consortium centers since 1995 were included. RESULTS: Residual oxidative activity was present in 66.7% of patients. In the year before HCT, there were 0.38 CGD-related infections per person-years. Inflammatory diseases, predominantly of the lungs and bowel, occurred in 36.7% of the patients. The median age at HCT was 9.1 years (range 1.5-23.6 years). Most HCTs (90%) were performed after using reduced intensity/toxicity conditioning. HCT sources were HLA-matched (40%) and -mismatched (10%) related donors or HLA-matched (36.7%) and -mismatched (13.3%) unrelated donors. CGD-related infections after HCT decreased significantly to 0.06 per person-years (P = .038). The frequency of inflammatory bowel disease and the use of steroids also decreased. The cumulative incidence of graft failure and second HCT was 17.9%. The 2-year overall and event-free survival were 92.3% and 82.1%, respectively, while at 5 years they were 85.7% and 77.0%, respectively. In the surviving patients evaluated, ≥95% donor myeloid chimerism at 1 and 2 years after HCT was 93.8% and 87.5%, respectively. CONCLUSIONS: Patients with p47phox CGD suffer from a significant disease burden that can be effectively alleviated by HCT. Similar to other forms of CGD, HCT should be considered for patients with p47phox CGD.

Intestinal microbiome and metabolome signatures in patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments. OBJECTIVE: We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD. METHODS: We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium. RESULTS: We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD. CONCLUSION: Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets.

Genotype, oxidase status, and preceding infection or autoinflammation do not affect allogeneic HCT outcomes for CGD.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening infections and inflammatory conditions. Hematopoietic cell transplantation (HCT) is the definitive treatment for CGD, but questions remain regarding patient selection and impact of active disease on transplant outcomes. We performed a multi-institutional retrospective and prospective study of 391 patients with CGD treated either conventionally (non-HCT) enrolled from 2004 to 2018 or with HCT from 1996 to 2018. Median follow-up after HCT was 3.7 years with a 3-year overall survival of 82% and event-free survival of 69%. In a multivariate analysis, a Lansky/Karnofsky score <90 and use of HLA-mismatched donors negatively affected survival. Age, genotype, and oxidase status did not affect outcomes. Before HCT, patients had higher infection density, higher frequency of noninfectious lung and liver diseases, and more steroid use than conventionally treated patients; however, these issues did not adversely affect HCT survival. Presence of pre-HCT inflammatory conditions was associated with chronic graft-versus-host disease. Graft failure or receipt of a second HCT occurred in 17.6% of the patients and was associated with melphalan-based conditioning and/or early mixed chimerism. At 3 to 5 years after HCT, patients had improved growth and nutrition, resolved infections and inflammatory disease, and lower rates of antimicrobial prophylaxis or corticosteroid use compared with both their baseline and those of conventionally treated patients. HCT leads to durable resolution of CGD symptoms and lowers the burden of the disease. Patients with active infection or inflammation are candidates for transplants; HCT should be considered before the development of comorbidities that could affect performance status. This trial was registered at www.clinicaltrials.gov as #NCT02082353.

Updated Management Guidelines for Adenosine Deaminase Deficiency.

Inherited defects in the adenosine deaminase (ADA) gene typically cause severe combined immunodeficiency. In addition to infections, ADA-deficient patients can present with neurodevelopmental, behavioral, hearing, skeletal, lung, heart, skin, kidney, urogenital, and liver abnormalities. Some patients also suffer from autoimmunity and malignancies. In recent years, there have been remarkable advances in the management of ADA deficiency. Most ADA-deficient patients can be identified by newborn screening for severe combined immunodeficiency, which facilitates early diagnosis and treatment of asymptomatic infants. Most patients benefit from enzyme replacement therapy (ERT). Allogeneic hematopoietic cell transplantation from an HLA-matched sibling donor or HLA-matched family member donor with no conditioning is currently the preferable treatment. When matched sibling donor or matched family member donor is not available, autologous ADA gene therapy with nonmyeloablative conditioning and ERT withdrawal, which is reported in recent studies to result in 100% overall survival and 90% to 95% engraftment, should be pursued. If gene therapy is not immediately available, ERT can be continued for a few years, although its excessive cost might be prohibitive. The recent improved outcome of hematopoietic cell transplantation using HLA-mismatched family-related donors or HLA-matched unrelated donors, after reduced-intensity conditioning, suggests that such procedures might also be considered rather than continuing ERT for prolonged periods. Long-term follow-up will further assist in determining the optimal treatment approach for ADA-deficient patients.

Long-Term Immune Reconstitution in ADA-Deficient Patients Treated With Elapegademase: A Real-World Experience.

BACKGROUND: ADAGEN, a bovine-based enzyme replacement therapy (ERT), has been used to treat adenosine deaminase severe combined immunodeficiency (ADA-SCID). In 2018, ADAGEN was replaced by REVCOVI (elapegademase), a modified bovine recombinant protein. OBJECTIVE: To determine the real-life long-term benefits of REVCOVI in ADA-SCID. METHODS: Data on ERT, infectious and noninfectious complications, and metabolic and immune evaluations were collected from 17 patients with ADA-SCID treated for 6 months or more with REVCOVI. RESULTS: Eleven patients had previously received ADAGEN for 16 to 324 months, whereas 6 patients were ERT-naive. REVCOVI was administered twice weekly at 0.4 mg/kg/wk in ERT-naive patients, whereas patients transitioning to REVCOVI from ADAGEN typically continued at the same frequency and equivalent dosing as ADAGEN, resulting in a significantly lower (P = .007) total REVCOVI dose in the transitioning group. REVCOVI treatment in the ERT-naive group led to the resolution of many clinical and laboratory complications of ADA deficiency, whereas there were no new adverse effects among the transitioning patients. REVCOVI treatment increased plasma ADA activity and decreased dAXP (which included deoxyadenosine mono-, di-, and tri phosphate) among most patients, effects that persisted throughout the 7- to 37-month treatment periods, except in 2 patients with incomplete adherence. Among some patients, after 0.5 to 6 months, injection frequency was reduced to once a week, while maintaining adequate metabolic profiles. All ERT-naive infants treated with REVCOVI demonstrated an increase in the number of CD4+ T and CD19+ B cells, although these counts remained stable but lower than normal in most transitioning patients. CONCLUSIONS: REVCOVI is effective for the management of ADA-SCID.

Platelet Activating Factor (PAF): A Mediator of Inflammation.

Platelet-activating factor (PAF) is a phospholipid-derived mediator with an established role in multiple inflammatory states. PAF is synthesized and secreted by multiple cell types and is then rapidly hydrolyzed and degraded to an inactive metabolite, lyso-PAF, by the enzyme PAF acetylhydrolase. In addition to its role in platelet aggregation and activation, PAF contributes to allergic and nonallergic inflammatory diseases such as anaphylaxis, sepsis, cardiovascular disease, neurological disease, and malignancy as demonstrated in multiple animal models and, increasingly, in human disease states. Recent research has demonstrated the importance of the PAF pathway in multiple conditions including the prediction of severe pediatric anaphylaxis, effects on blood-brain barrier permeability, effects on reproduction, ocular diseases, and further understanding of its role in cardiovascular risk. Investigation of PAF as both a biomarker and a therapeutic target continues because of the need for directed management of inflammation. Collectively, studies have shown that therapies focused on the PAF pathway have the potential to provide targeted and effective treatments for multiple inflammatory conditions.

Disease Progression of WHIM Syndrome in an International Cohort of 66 Pediatric and Adult Patients.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS) is a combined immunodeficiency caused by gain-of-function mutations in the C-X-C chemokine receptor type 4 (CXCR4) gene. We characterize a unique international cohort of 66 patients, including 57 (86%) cases previously unreported, with variable clinical phenotypes. Of 17 distinct CXCR4 genetic variants within our cohort, 11 were novel pathogenic variants affecting 15 individuals (23%). All variants affect the same CXCR4 region and impair CXCR4 internalization resulting in hyperactive signaling. The median age of diagnosis in our cohort (5.5 years) indicates WHIM syndrome can commonly present in childhood, although some patients are not diagnosed until adulthood. The prevalence and mean age of recognition and/or onset of clinical manifestations within our cohort were infections 88%/1.6 years, neutropenia 98%/3.8 years, lymphopenia 88%/5.0 years, and warts 40%/12.1 years. However, we report greater prevalence and variety of autoimmune complications of WHIM syndrome (21.2%) than reported previously. Patients with versus without family history of WHIM syndrome were diagnosed earlier (22%, average age 1.3 years versus 78%, average age 5 years, respectively). Patients with a family history of WHIM syndrome also received earlier treatment, experienced less hospitalization, and had less end-organ damage. This observation reinforces previous reports that early treatment for WHIM syndrome improves outcomes. Only one patient died; death was attributed to complications of hematopoietic stem cell transplantation. The variable expressivity of WHIM syndrome in pediatric patients delays their diagnosis and therapy. Early-onset bacterial infections with severe neutropenia and/or lymphopenia should prompt genetic testing for WHIM syndrome, even in the absence of warts.

Purine nucleoside phosphorylase deficiency induces p53-mediated intrinsic apoptosis in human induced pluripotent stem cell-derived neurons.

Purine nucleoside phosphorylase (PNP) is an important enzyme in the purine degradation and salvage pathway. PNP deficiency results in marked T lineage lymphopenia and severe immunodeficiency. Additionally, PNP-deficient patients and mice suffer from diverse non-infectious neurological abnormalities of unknown etiology. To further investigate the cause for these neurologic abnormalities, induced pluripotent stem cells (iPSC) from two PNP-deficient patients were differentiated into neurons. The iPSC-derived PNP-deficient neurons had significantly reduced soma and nuclei volumes. The PNP-deficient neurons demonstrated increased spontaneous and staurosporine-induced apoptosis, measured by cleaved caspase-3 expression, together with decreased mitochondrial membrane potential and increased cleaved caspase-9 expression, indicative of enhanced intrinsic apoptosis. Greater expression of tumor protein p53 was also observed in these neurons, and inhibition of p53 using pifithrin-α prevented the apoptosis. Importantly, treatment of the iPSC-derived PNP-deficient neurons with exogenous PNP enzyme alleviated the apoptosis. Inhibition of ribonucleotide reductase (RNR) in iPSC derived from PNP-proficient neurons with hydroxyurea or with nicotinamide and trichostatin A increased the intrinsic neuronal apoptosis, implicating RNR dysfunction as the potential mechanism for the damage caused by PNP deficiency. The findings presented here establish a potential mechanism for the neurological defects observed in PNP-deficient patients and reinforce the critical role that PNP has for neuronal viability.

A Sherlock Approach to a Kindred With a Variable Immunohematologic Phenotype.

Given the ubiquity of leukopenia and sinopulmonary infections in childhood, differentiating patients with inborn errors of immunity (IEI) from otherwise healthy patients can be challenging. The diagnostic complexity is further exacerbated in disorders with wide phenotypic variability such as warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. However, using a Sherlock approach with careful attention to details in the patient's medical history and physical examination coupled with a comprehensive family history can heighten the index of suspicion for underlying IEI. Subsequent iterative and deductive reasoning incorporating results from laboratory interrogation, response (or lack thereof) to standard therapy, and emergence of new symptoms can further aid in a timely diagnosis of IEI. Herein, we detail a WHIM syndrome kindred with marked phenotype variability, identified after the presentation of a child with intermittent neutropenia and sinopulmonary infections. The complexity of this kindred highlights the utility of an interspecialty, collaborative Sherlock approach to diagnosis, and care. In addition, the genetic underpinnings, diagnostic approaches, clinical features, supportive care options, and management of WHIM syndrome are reviewed.

Monitoring patients with uncomplicated common variable immunodeficiency: a systematic review.

BACKGROUND: Non-infectious complications have become a major cause of morbidity and mortality in patients with Common Variable Immunodeficiency (CVID). The monitoring of patients with CVID prior to the development of non-infectious complications is not well defined. OBJECTIVE: Our objectives were to systematically review the current literature on the monitoring of CVID patients without non-infectious complications and to develop recommendations for such monitoring. METHODS: MEDLINE and EMBASE were searched from January 1st, 2000 to March 25th, 2021. Studies on any aspects of CVID monitoring were included. Studies that included only children, on monitoring CVID patients with existing non-infectious complications, or in the format of case reports were excluded. RESULTS: Nine studies on CVID monitoring, including 3 cohort studies, 3 experts' opinions, 2 consensus statements and a single guideline report were identified. These studies revealed that clinical assessment and bloodwork were preformed every 6 to 12 months in asymptomatic patients. Some centers performed computerized tomography scan of the chest every 2-5 years to identify chronic lung disease, although the majority did chest imaging in accordance with clinical indications. Pulmonary function tests were done annually at most centers. Most studies did not address the role of abdominal imaging to screen for liver diseases or endoscopy to screen for gastric cancer in asymptomatic patients with uncomplicated CVID. CONCLUSIONS: There is paucity of evidence-based information to guide the routine monitoring of CVID patients without non-infectious complications. Prospective studies are needed to determine the best monitoring practices in this group of patients.

The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development.

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.

Homozygous duplication identified by whole genome sequencing causes LRBA deficiency.

In more than one-third of primary immunodeficiency (PID) patients, extensive genetic analysis including whole-exome sequencing (WES) fails to identify the genetic defect. Whole-genome sequencing (WGS) is able to detect variants missed by other genomics platforms, enabling the molecular diagnosis of otherwise unresolved cases. Here, we report two siblings, offspring of consanguineous parents, who experienced similar severe events encompassing early onset of colitis, lymphoproliferation, and hypogammaglobulinemia, typical of lipopolysaccharide-responsive and beige-like anchor (LRBA) or cytotoxic T lymphocyte antigen 4 (CTLA4) deficiencies. Gene-panel sequencing, comparative genomic hybridization (CGH) array, and WES failed to reveal a genetic aberration in relevant genes. WGS of these patients detected a 12.3 kb homozygous tandem duplication that was absent in control cohorts and is predicted to disrupt the reading frame of the LRBA gene. The variant was validated by PCR and Sanger sequencing, demonstrating the presence of the junction between the reference and the tandem-duplicated sequence. Droplet digital PCR (ddPCR) further confirmed the copy number in the unaffected parents (CN = 3, heterozygous) and affected siblings (CN = 4, homozygous), confirming the expected segregation pattern. In cases of suspected inherited immunodeficiency, WGS may reveal a mutation when other methods such as microarray and WES analysis failed to detect an aberration.

SARS-CoV-2-Reactive Mucosal B Cells in the Upper Respiratory Tract of Uninfected Individuals.

SARS-CoV-2 is a respiratory pathogen that can cause severe disease in at-risk populations but results in asymptomatic infections or a mild course of disease in the majority of cases. We report the identification of SARS-CoV-2-reactive B cells in human tonsillar tissue obtained from children who were negative for coronavirus disease 2019 prior to the pandemic and the generation of mAbs recognizing the SARS-CoV-2 Spike protein from these B cells. These Abs showed reduced binding to Spike proteins of SARS-CoV-2 variants and did not recognize Spike proteins of endemic coronaviruses, but subsets reacted with commensal microbiota and exhibited SARS-CoV-2-neutralizing potential. Our study demonstrates pre-existing SARS-CoV-2-reactive Abs in various B cell populations in the upper respiratory tract lymphoid tissue that may lead to the rapid engagement of the pathogen and contribute to prevent manifestations of symptomatic or severe disease.

Progressive decline of T and B cell numbers and function in a patient with CDC42 deficiency.

Single allele mutations in the Cell Division Control protein 42 homolog (CDC42) gene were recently shown to cause Takenouchi-Kosaki syndrome with diverse manifestations. These include persistent mild thrombocytopenia with large platelet size, severe developmental delay, growth retardation, facial dysmorphism, and other neurodevelopmental and hematological anomalies. CDC42 deficiency might also cause myelofibrosis, myeloproliferation, and severe autoinflammation. CDC42 closely interacts with the Wiskott-Aldrich Syndrome Protein, but little is still known about the immune abnormalities associated with CDC42 deficiency. Detailed immune evaluations were performed in a patient diagnosed with a CDC42 Tyr64Cys mutation. The 19-year-old female suffered from recurrent pneumonia, otitis media, and bacteremia, which resolved at 10 years of age, concordant with the initiation of amoxicillin prophylaxis. In addition, the patient had frequent viral upper respiratory tract infections, which resolved without need for medical interventions. Immune evaluations demonstrated decreased immunoglobulin levels, inability to maintain antibody responses, progressive decline in the number of CD19+ B cells, and decreased switched memory B cells. There was also a decrease in CD4+ and CD8+ T cells, markedly reduced naïve T cells, and intermittent depressed proliferation of T cells to stimulation. Natural killer cells' number and functions were normal. However, no opportunistic infections were observed, nor was there evidence for autoinflammation. CDC42 deficiency might also be associated with decline in T and B cell function. Therefore, immunity in patients with CDC42 defects should be closely monitored, particularly among those with frequent infections or systemic autoinflammation.

Partial Purine Nucleoside Phosphorylase Deficiency Helps Determine Minimal Activity Required for Immune and Neurological Development.

Introduction: Complete or near complete absence of the purine nucleoside phosphorylase (PNP) enzyme causes a profound T cell immunodeficiency and neurological abnormalities that are often lethal in infancy and early childhood. We hypothesized that patients with partial PNP deficiency, characterized by a late and mild phenotype due to residual PNP enzyme, would provide important information about the minimal PNP activity needed for normal development. Methods: Three siblings with a homozygous PNP gene mutation (c.769C>G, p.His257Asp) resulting in partial PNP deficiency were investigated. PNP activity was semi-quantitively assayed by the conversion of [14C]inosine in hemolysates, mononuclear cells, and lymphoblastoid B cells. PNP protein expression was determined by Western Blotting in lymphoblastoid B cells. DNA repair was quantified by measuring viability of lymphoblastoid B cells following ionizing irradiation. Results: A 21-year-old female was referred for recurrent sino-pulmonary infections while her older male siblings, aged 25- and 28- years, did not suffer from significant infections. Two of the siblings had moderately reduced numbers of T, B, and NK cells, while the other had near normal lymphocyte subset numbers. T cell proliferations were normal in the two siblings tested. Hypogammaglobulinemia was noted in two siblings, including one that required immunoglobulin replacement. All siblings had typical (normal) neurological development. PNP activity in various cells from two patients were 8-11% of the normal level. All siblings had normal blood uric acid and increased PNP substrates in the urine. PNP protein expression in cells from the two patients examined was similar to that observed in cells from healthy controls. The survival of lymphoblastoid B cells from 2 partial PNP-deficient patients after irradiation was similar to that of PNP-proficient cells and markedly higher than the survival of cells from a patient with absent PNP activity or a patient with ataxia telangiectasia. Conclusions: Patients with partial PNP deficiency can present in the third decade of life with mild-moderate immune abnormalities and typical development. Near-normal immunity might be achieved with relatively low PNP activity.

Detection of Human CD38 Using Variable Lymphocyte Receptor (VLR) Tetramers.

CD38 is a multifunctional cell surface receptor expressed on multiple cell lineages of hematopoietic origin with high levels of expression on human plasma cells. Previously, we isolated the monoclonal variable lymphocyte receptor B (VLRB) MM3 antibody from the evolutionarily distant sea lamprey, which recognized the CD38 ectoenzyme exclusively on human plasma cells in a manner that correlated with CD38 enzymatic activity. The plasma cell-specific binding of VLRB MM3 contrasts with the broad pattern of expression of CD38-determined conventional antibodies specific for this antigen. In an effort to facilitate the application of this unique reagent in combination with conventional antibody panels, we explored a strategy to generate VLRB MM3 tetramers. The resulting reagent maintained the threshold-based recognition of CD38. Increased sensitivity achieved with VLRB MM3 tetramers also showed preferential recognition of germinal center centroblasts over centrocytes. VLRB MM3 tetramers thus provided a unique and versatile single-step staining reagent for the detection of human CD38 that is readily incorporated into multi-color flow cytometry panels.