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Background/aimNeuroblastoma is a common childhood cancer with poor survival for children with high-risk disease, and ongoing research to improve outcomes is needed. Patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM) are reliable models for oncologic research; however, they are resource-intensive, expensive, and require significant expertise to develop and maintain. We developed an orthotopic xenograft murine model of neuroblastoma that utilizes cryopreserved banks of human neuroblastoma cell lines, requires minimal equipment, and is easily reproducible.Materials and methodsThe neuroblastoma cell line NB1643 was obtained from the Children's Oncology Group (COG) Childhood Cancer Repository. Nod-SCID-gamma (NSG) mice underwent orthotopic injection of 2x106 NB1643 cells suspended in 10 μl of collagen hydrogel directly into the adrenal gland via an open retroperitoneal surgical approach. Mice were monitored by ultrasound and in vivo imaging system (IVIS) until the tumor reached the volume of the ipsilateral kidney. Tumor identity was confirmed by necropsy and histologic analysis.ResultsA total of 55 mice underwent surgery. Eight died due to anesthetic or surgical complications. 39/47 (78%) survivors grew primary adrenal tumors. Average anesthesia time was 30 min. Ultrasound and IVIS successfully characterized tumor growth in all mice. Average time to target tumor size was 5 weeks (range=3-9). Gross pathologic and histologic analysis confirmed adrenal tumors consistent with neuroblastoma in all mice with adrenal masses.ConclusionA cell-derived orthotopic xenograft murine model can be successfully used to create an in vivo model of neuroblastoma. This model can be utilized in environments where PDX or GEMM models are not feasible.

Purpose: The measurement of T-cell receptor excision circle (TREC) is used for newborn screening (NBS) in dried blood spot (DBS) samples from Guthrie card for severe combined immunodeficiency (SCID). Here, we report the results of first newborn screening pilot program for SCID conducted in Türkiye. Methods: The study was carried out together with Ankara University School of Medicine and The Ministry of Health, Public Health General Directorate, Pediatric and Adolescent Health Department. TREC measurements were performed in randomly selected Guthrie card samples obtained from 20253 babies born between October 2018 and October 2020. The TREC analyses were performed together with beta Actin (β-Actin) via RT-PCR (Real Time Polymerase Chain Reaction). Results: TRECs found to be normal (≥15 copies/µl) in 98,6% of the newborns (n: 19975) but low (<15 copies/µl) in 1.4% (n:278) at the initial analyses. TRECs were retested in 278 suspected infants and found to be normal in 160 (0.8%) while low in 118 (0.58%). New DBS were obtained from the babies with low TRECs (new sample test). TRECs were normal in 108 (0.53%) of the new sample tests and low in 10 (0.049%). Two among 10 babies who had abnormal (undetectable) TRECs were diagnosed as SCID; ADA (P1) and RAG1 (P2) defects were confirmed respectively. They both received curative treatments [gene therapy (P1) and HSCT (P2)]. The remaining 6 of 8 newborns with abnormal TRECs were found normal after clinical and laboratory immune work-up, while medical records of other two revealed early postnatal death due to extreme prematurity. Conclusion: In the light of this study the incidence of SCID was detected at least 1/10000 live births in Türkiye. This study shows the feasibility and usefulness of initiating SCID screening in Türkiye. [ABSTRACT FROM AUTHOR]

A cell’s ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3’ end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease. [ABSTRACT FROM AUTHOR]

Aim. Circular DNA segments TREC (T-cell receptor excision circles) formed during T-lymphocyte maturation in the thymus, are a sensitive marker of thymic lymphocyte production in a broader manner. Quantification using qPCR is proposed as a surrogate marker of T cell malfunction in various primary and secondary conditions in a non-SCID selected risk newborn population. Methods. We collected 207 dry blood spot samples during the years 2015–2018, from newly admitted risk newborns. TREC values calculated per 106 cells were determined and a cut-off values of 5th percentile was set. The positive control group consisted of patients (n=13) with genetically confirmed SCID. Results. The median TREC value was 34,591.56 (18,074.08−60,228.58) for girls resp. 28,391.20 (13,835.01−51,835.93) per 106 cells for boys, P=0.046. Neonates born by C-section have been found to have higher TREC levels compared to neonates born by spontaneous delivery (P=0.018). In the group of preterm newborns (n=104), 3.8% had TREC value < 5th percentile, half of them died due to sepsis as opposed to no fatalities in preterm newborns with sepsis and TREC value > 5th percentile. In the group of term newborns (n=103) 9 children (8.7%) had TREC < 5th percentile, half of them were treated for asphyxia, with no fatal complications. Conclusion. TREC levels calculated for the 5th percentile of a risk neonatal group is suggested as a surrogate marker for increased risk of fatal septic complication. Early recognition of these newborns within a risk scoring system using TREC levels could lead to potentially lifesaving interventions. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To conduct clinical and genetic analysis in a patient with severe combined immunodeficiency disease (SCID). METHODS: A 3.5-year-old female patient with chronic diarrhea, fever, and failure to thrive was examined at the Pakistan Institute of Medical Sciences, Islamabad. Ethical approval was obtained, and informed consent was secured for genetic studies. Flow cytometry was performed on blood samples to evaluate T, B, and NK cell concentrations. Genomic DNA was extracted from peripheral blood samples of the patient, her mother, and healthy siblings. Sanger sequencing of the RAG1 gene was conducted, followed by mutational analysis using BioEdit and bioinformatics tools for pathogenicity assessment. RESULTS: The proband, the youngest girl born to first-cousin parents presented with intractable diarrhea with failure to thrive and skin and nappy rashes on clinical evaluation. The immunological assessment revealed total absence of B and T lymphocytes, normal NK cells (47% with 85% CD16 and CD56 respectively: No CD4, CD8 and CD19), and significant hypogammaglobulinemia (IgG;165 mg/dl, IgM; 9 mg/dl, IgA;8 mg/dl), while normal level of IgE (2 mg/dl). On the other hand, targeted Sanger sequencing of RAG1 exon 2 region revealed a new homozygous deleterious mutation (NM_000448.2: c.2876 G>A; p.Trp959*) in the RAG1 gene resulting in complete loss of function due to the production of truncated protein. CONCLUSION: We identified a potentially pathogenic nonsense mutation in the RAG1 gene (c.2876 G>A; p.Trp959*) in a child with SCID. This finding emphasizes the significance of early detection for timely treatment and genetic counseling.

Aim. Circular DNA segments TREC (T-cell receptor excision circles) formed during T-lymphocyte maturation in the thymus, are a sensitive marker of thymic lymphocyte production in a broader manner. Quantification using qPCR is proposed as a surrogate marker of T cell malfunction in various primary and secondary conditions in a non-SCID selected risk newborn population. Methods. We collected 207 dry blood spot samples during the years 2015-2018, from newly admitted risk newborns. TREC values calculated per 106 cells were determined and a cut-off values of 5th percentile was set. The positive control group consisted of patients (n=13) with genetically confirmed SCID. Results. The median TREC value was 34,591.56 (18,074.08-60,228.58) for girls resp. 28,391.20 (13,835.01-51,835.93) per 106 cells for boys, P=0.046. Neonates born by C-section have been found to have higher TREC levels compared to neonates born by spontaneous delivery (P=0.018). In the group of preterm newborns (n=104), 3.8% had TREC value < 5th percentile, half of them died due to sepsis as opposed to no fatalities in preterm newborns with sepsis and TREC value > 5th percentile. In the group of term newborns (n=103) 9 children (8.7%) had TREC < 5th percentile, half of them were treated for asphyxia, with no fatal complications. Conclusion. TREC levels calculated for the 5th percentile of a risk neonatal group is suggested as a surrogate marker for increased risk of fatal septic complication. Early recognition of these newborns within a risk scoring system using TREC levels could lead to potentially lifesaving interventions.

Early diagnosis and effective management of Primary immunodeficiency diseases (PIDs), particularly severe combined immunodeficiency (SCID), play a crucial role in minimizing associated morbidities and mortality. Newborn screening (NBS) serves as a valuable tool in facilitating these efforts. Timely detection and diagnosis are essential for swiftly implementing isolation measures and ensuring prompt referral for definitive treatment, such as allogeneic hematopoietic stem cell transplantation. The utilization of comprehensive protocols and screening assays, including T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC), is essential in facilitating early diagnosis of SCID and other PIDs, but their successful application requires clinical expertise and proper implementation strategy. Unfortunately, a notable challenge arises from insufficient funding for the treatment of PIDs. To address these issues, a collaborative approach is imperative, involving advancements in technology, a well-functioning healthcare system, and active engagement from stakeholders. The integration of these elements is essential for overcoming the existing challenges in NBS for PIDs. By fostering synergy between technology providers, healthcare professionals, and governmental stakeholders, we can enhance the efficiency and effectiveness of early diagnosis and intervention, ultimately improving outcomes for individuals with PIDs. [ABSTRACT FROM AUTHOR]

Background: Severe combined immunodeficiency (SCID) is a life-threatening genetic disorder caused by critical defects of the immune system. Almost all cases are lethal if not treated within the first two years of life. Early diagnosis and intervention are thus essential for improving patient outcomes. In 2013, Ontario became the first Canadian province to perform newborn screening (NBS) for SCID by T cell receptor excision circles (TRECs) analysis, a surrogate marker of thymic function and lymphocyte maturation. Methods: This retrospective study reports on nearly 10 years of NBS for SCID at a quaternary referral centre. Results: From August 2013 to April 2023, our centre's densely populated catchment area flagged 162 newborns with low TRECs levels, including 10 cases with SCID. Follow-up revealed other causes of low TRECs, including non-SCID T cell lymphopenia (secondary/reversible or idiopathic causes, and syndromic conditions) and prematurity. A small number of cases with normal repeat TRECs levels and/or T cell subsets were also flagged. Province-wide data from around this period revealed at least 24 diagnosed cases of SCID or Leaky SCID. Conclusions: This is the first report of NBS outcomes in a Canadian province describing the causative genetic defects, and the non-SCID causes of a positive NBS for SCID. [ABSTRACT FROM AUTHOR]

PurposeThe measurement of T-cell receptor excision circle (TREC) is used for newborn screening (NBS) in dried blood spot (DBS) samples from Guthrie card for severe combined immunodeficiency (SCID). Here, we report the results of first newborn screening pilot program for SCID conducted in Türkiye.MethodsThe study was carried out together with Ankara University School of Medicine and The Ministry of Health, Public Health General Directorate, Pediatric and Adolescent Health Department. TREC measurements were performed in randomly selected Guthrie card samples obtained from 20253 babies born between October 2018 and October 2020. The TREC analyses were performed together with beta Actin (β-Actin) via RT-PCR (Real Time Polymerase Chain Reaction).ResultsTRECs found to be normal (≥15 copies/µl) in 98,6% of the newborns (n: 19975) but low (

The development of new treatment agents in recent decades has significantly improved the survival of patients with multiple myeloma (MM). Nonetheless, MM remains an incurable disease; therefore, novel combination therapies are required. Natural killer (NK) cells are one of the safest immunotherapeutic options. In this study, we found that the anti-myeloma activity of expanded NK cells (eNKs) was improved by daratumumab, lenalidomide, and dexamethasone (DRd) in an MM xenograft mouse model. NK cells expanded from peripheral blood mononuclear cells collected from MM patients were highly cytotoxic against DRd pretreated tumor cells in vitro. To mimic the clinical protocol, a human MM xenograft model was developed using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. MM bearing mice were randomly divided into six groups: no treatment, eNK, Rd, Rd + eNKs, DRd, and DRd + eNKs. DRd significantly enhanced the cytotoxicity of eNKs by upregulating NK cell activation ligands and effector function. DRd in combination with eNKs significantly reduced the serum M-protein level and prolonged mouse survival. In addition, DRd significantly increased the persistence of eNK and homing to MM sites. These results show that the anti-myeloma activity of ex vivo-expanded and activated NK cells is augmented by the immunomodulatory effect of DRd in MM-bearing mice, suggesting the therapeutic potential of this combination for MM patients.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.

Alamin Mustafa,1 Rogaia Hasap Alrasoul Ahmed,2 Hala Hamza Eltayeb,2 Malaz Elsadeg,2 Omaima Abdel Majeed Mohamed Salih,3 Nahla HH Erwa4 1Al-Neelain University, Faculty of Medicine, Khartoum, Sudan; 2Faculty of Medicine, Omdurman Islamic University, Omdurman, Sudan; 3Departments of Pediatrics and Child Health, Tropical and Infectious Diseases Consultant, Clinical Immunologist, Faculty of Medicine and Health Sciences, Omdurman Islamic University, Omdurman, Sudan; 4Clinical Immunology Consultant, Faculty of Medicine, University of Khartoum, Khartoum, SudanCorrespondence: Alamin Mustafa, Email alamin900005@gmail.comIntroduction: A class of disorders known as inborn errors of immunity (IEI) is defined by a compromised or missing immune response, which increases the vulnerability to infections, immunological dysregulation, and cancer. Severe combined immunodeficiencies (SCIDs), affecting both T and B-cell function are rare but often severe diseases. In this report, we describe a 10-month-old SCID patient from Sudan with disseminated BCG infection.Case Presentation: A 10-month-old boy whose parents were first degree relatives, presented with a six-month history of repeated chest infections and fever. Physical examination revealed a very ill-looking boy with respiratory distress dependent on oxygen, had slight abdominal distention and hepatomegaly. Investigations revealed positive polymerase chain reaction (PCR) for M. tuberculosis complex infection and low CD4+ and CD8+ cells. Genetic testing showed compound heterozygosity in trans for two variants in the Zeta-chain Associated Protein Kinase 70 (ZAP70) gene associated with autosomal recessive SCID. The patient was started on BCG-related infection treatment, intravenous immunoglobulin (IVIG) replacement and trimethoprim/sulfamethoxazole prophylaxis with an excellent response and the patient responded well to the treatment.Conclusion: SCIDs are rare, and early management is crucial. In this case, a diagnosis of ZAP70 deficiency was based on next-generation sequencing and inhouse bioinformatic computational analysis of the ZAP70 gene, highlighting the importance of genetic testing in the workup of immunodeficiencies in low resource settings.Keywords: Inborn errors of immunity, SCID, disseminated BCG infection, ZAP70 deficiency, pediatric immunology

Abstract Background Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing. Methods The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1’s role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation. Results Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling. Conclusions Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

BACKGROUND: Shearer et al in 2014 articulated well-defined criteria for the diagnosis and classification of severe combined immunodeficiency (SCID) as part of the Primary Immune Deficiency Treatment Consortiums (PIDTCs) prospective and retrospective studies of SCID. OBJECTIVE: Because of the advent of newborn screening for SCID and expanded availability of genetic sequencing, revision of the PIDTC 2014 Criteria was needed. METHODS: We developed and tested updated PIDTC 2022 SCID Definitions by analyzing 379 patients proposed for prospective enrollment into Protocol 6901, focusing on the ability to distinguish patients with various SCID subtypes. RESULTS: According to PIDTC 2022 Definitions, 18 of 353 patients eligible per 2014 Criteria were considered not to have SCID, whereas 11 of 26 patients ineligible per 2014 Criteria were determined to have SCID. Of note, very low numbers of autologous T cells (

Severe combined immunodeficiency (SCID) results from defects in the differentiation of hematopoietic stem cells into mature T lymphocytes, with additional lymphoid lineages affected in particular genotypes. In 2014, the Primary Immune Deficiency Treatment Consortium published criteria for diagnosing SCID, which are now revised to incorporate contemporary approaches. Patients with typical SCID must have less than 0.05 × 109 autologous T cells/L on repetitive testing, with either pathogenic variant(s) in a SCID-associated gene, very low/undetectable T-cell receptor excision circles or less than 20% of CD4 T cells expressing naive markers, and/or transplacental maternally engrafted T cells. Patients with less profoundly impaired autologous T-cell differentiation are designated as having leaky/atypical SCID, with 2 or more of these: low T-cell numbers, oligoclonal T cells, low T-cell receptor excision circles, and less than 20% of CD4 T cells expressing naive markers. These patients must also have either pathogenic variant(s) in a SCID-associated gene or reduced T-cell proliferation to certain mitogens. Omenn syndrome requires a generalized erythematous rash, absent transplacentally acquired maternal engraftment, and 2 or more of these: eosinophilia, elevated IgE, lymphadenopathy, hepatosplenomegaly. Thymic stromal defects and other causes of secondary T-cell deficiency are excluded from the definition of SCID. Application of these revised Primary Immune Deficiency Treatment Consortium 2022 Definitions permits precise categorization of patients with T-cell defects but does not imply a preferred treatment strategy.

Mice reconstituted with human immune systems are instrumental in the investigation of HIV-1 pathogenesis and therapeutics. Natural killer (NK) cells have long been recognized as a key mediator of innate anti-HIV responses. However, established humanized mouse models do not support robust human NK cell development from engrafted human hematopoietic stem cells (HSCs). A major obstacle to human NK cell reconstitution is the lack of human interleukin-15 (IL-15) signaling, as murine IL-15 is a poor stimulator of the human IL-15 receptor. Here, we demonstrate that immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice expressing a transgene encoding human IL-15 (NSG-Tg(IL-15)) have physiological levels of human IL-15 and support long-term engraftment of human NK cells when transplanted with human umbilical-cord-blood-derived HSCs. These Hu-NSG-Tg(IL-15) mice demonstrate robust and long-term reconstitution with human immune cells, but do not develop graft-versus-host disease (GVHD), allowing for long-term studies of human NK cells. Finally, we show that these HSC engrafted mice can sustain HIV-1 infection, resulting in human NK cell responses in HIV-infected mice. We conclude that Hu-NSG-Tg(IL-15) mice are a robust novel model to study NK cell responses to HIV-1.

Maternal obesity adversely impacts the in utero metabolic environment, but its effect on fetal hematopoiesis remains incompletely understood. During late development, the fetal bone marrow (FBM) becomes the major site where macrophages and B lymphocytes are produced via differentiation of hematopoietic stem and progenitor cells (HSPCs). Here, we analyzed the transcriptional landscape of FBM HSPCs at single-cell resolution in fetal macaques exposed to a maternal high-fat Western-style diet (WSD) or a low-fat control diet. We demonstrate that maternal WSD induces a proinflammatory response in FBM HSPCs and fetal macrophages. In addition, maternal WSD consumption suppresses the expression of B cell development genes and decreases the frequency of FBM B cells. Finally, maternal WSD leads to poor engraftment of fetal HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2rγ-/- mice. Collectively, these data demonstrate for the first time that maternal WSD impairs fetal HSPC differentiation and function in a translationally relevant nonhuman primate model.

This article presents case reports of two siblings diagnosed with severe neonatal acute respiratory distress syndrome (ARDS) and immune deficiencies. The cases highlight the challenges of managing neonatal ARDS and the severity of viral infections in patients with immune system disorders. The article emphasizes the importance of innate immunity in antiviral responses and provides valuable insights for researchers and healthcare professionals studying neonatal ARDS and immune deficiencies. The text also discusses a case study of a patient with neonatal ARDS and a potential underlying immune deficiency called adenosine deaminase (ADA) deficiency. The text emphasizes the importance of detailed evaluation and early diagnosis and treatment of ADA-SCID. The text concludes by discussing the challenges in managing neonatal ARDS and the lack of definitive guidelines. [Extracted from the article]

Summary: Severe combined immunodeficiency (SCID) is a rare and life‐threatening genetic disorder that severely impairs the immune system's ability to defend the body against infections. Often referred to as the "bubble boy" disease, SCID gained widespread recognition due to the case of David Vetter, a young boy who lived in a sterile plastic bubble to protect him from germs. SCID is typically present at birth, and it results from genetic mutations that affect the development and function of immune cells, particularly T cells and B cells. These immune cells are essential for identifying and fighting off infections caused by viruses, bacteria, and fungi. In SCID patients, the immune system is virtually non‐existent, leaving them highly susceptible to recurrent, severe infections. There are several forms of SCID, with varying degrees of severity, but all share common features. Newborns with SCID often exhibit symptoms such as chronic diarrhea, thrush, skin rashes, and persistent infections that do not respond to standard treatments. Without prompt diagnosis and intervention, SCID can lead to life‐threatening complications and a high risk of mortality. There are over 20 possible affected genes. Treatment options for SCID primarily involve immune reconstitution, with the most well‐known approach being hematopoietic stem cell transplantation (HSCT). Alternatively, gene therapy is also available for some forms of SCID. Once treated successfully, SCID patients can lead relatively normal lives, but they may still require vigilant infection control measures and lifelong medical follow‐up to manage potential complications. In conclusion, severe combined immunodeficiency is a rare but life‐threatening genetic disorder that severely compromises the immune system's function, rendering affected individuals highly vulnerable to infections. Early diagnosis and appropriate treatment are fundamental. With this respect, newborn screening is progressively and dramatically improving the prognosis of SCID. [ABSTRACT FROM AUTHOR]

Early diagnosis and effective management of Primary immunodeficiency diseases (PIDs), particularly severe combined immunodeficiency (SCID), play a crucial role in minimizing associated morbidities and mortality. Newborn screening (NBS) serves as a valuable tool in facilitating these efforts. Timely detection and diagnosis are essential for swiftly implementing isolation measures and ensuring prompt referral for definitive treatment, such as allogeneic hematopoietic stem cell transplantation. The utilization of comprehensive protocols and screening assays, including T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC), is essential in facilitating early diagnosis of SCID and other PIDs, but their successful application requires clinical expertise and proper implementation strategy. Unfortunately, a notable challenge arises from insufficient funding for the treatment of PIDs. To address these issues, a collaborative approach is imperative, involving advancements in technology, a well-functioning healthcare system, and active engagement from stakeholders. The integration of these elements is essential for overcoming the existing challenges in NBS for PIDs. By fostering synergy between technology providers, healthcare professionals, and governmental stakeholders, we can enhance the efficiency and effectiveness of early diagnosis and intervention, ultimately improving outcomes for individuals with PIDs.

We examined whether women with prenatal mood and anxiety disorders would exhibit differential pro- and anti-inflammatory marker trajectories during the prenatal and postpartum periods compared to women without these disorders. Approximately 179 pregnant women participated in a longitudinal study conducted in two urban areas. Blood samples for inflammatory markers were collected at six study visits. The Structured Clinical Interview for the DSM-IV (SCID) was administered to participants scoring above cutoffs on anxiety and depression. Pregnant women with SCID Axis I diagnoses of mood and/or anxiety disorders were compared to other participants on inflammatory markers. Multilevel modeling tested associations between SCID diagnoses and within-person interleukin (IL)6 and IL10 trajectories. Prenatal SCID diagnoses were associated with linear, quadratic and cubic change in IL6 from prenatal to postpartum timepoints. Women with a prenatal SCID diagnosis had steeper decreases and increases in IL6 during prenatal and postpartum periods. SCID diagnoses were associated with lower IL10 in mid-pregnancy to postpartum (b = −0.078, SE = 0.019; p =.015). Future studies would benefit from a larger sample size and a larger number of participants with SCID diagnoses. Future research should also examine whether different prenatal Axis 1 diagnoses are associated with different patterns of immune response in pregnancy. Pregnant women with prenatal mood and anxiety disorders had greater fluctuations in IL6 across prenatal and postpartum periods and lower IL10 through pregnancy and postpartum. They may have different proinflammatory states that remain after birth without a reciprocal anti-inflammatory response. • Prenatal mood and anxiety disorders are linked to postpartum inflammation. • Women with prenatal SCID diagnoses had greater fluctuations of inflammatory markers. • Lower anti-inflammatory markers in pregnancy associated with prenatal SCID diagnoses [ABSTRACT FROM AUTHOR]

Patients with severe primary immunodeficiency are at risk for complications from live-attenuated vaccines. Here, we report a case of a vaccine-associated paralytic polio and Bacille Calmette-Guérin disease in a 6-month-old girl with severe combined immunodeficiency resulting from homozygous recombinant activating gene 1 deficiency. The patient was successfully treated with intravenous immunoglobulins and oral pocapavir for poliovirus, and antimycobacterial therapy for regional Bacille Calmette-Guérin disease, allowing stem cell transplant. Following transplantation, poliovirus type 3 with 13 mutations was detected from cerebrospinal fluid but not from stool, indicating ongoing viral evolution in the central nervous system despite pocapavir treatment. Clinical improvement and immune reconstitution allowed the patient to be successfully discharged with no further detection of poliovirus. [ABSTRACT FROM AUTHOR]

The Primary Immune Deficiency Treatment Consortium (PIDTC) enrolled children in the United States and Canada onto a retrospective multicenter natural history study of hematopoietic cell transplantation (HCT). We investigated outcomes of HCT for severe combined immunodeficiency (SCID). We evaluated the chronic and late effects (CLE) after HCT for SCID in 399 patients transplanted from 1982 to 2012 at 32 PIDTC centers. Eligibility criteria included survival to at least 2 years after HCT without need for subsequent cellular therapy. CLE were defined as either conditions present at any time before 2 years from HCT that remained unresolved (chronic), or new conditions that developed beyond 2 years after HCT (late). The cumulative incidence of CLE was 25% in those alive at 2 years, increasing to 41% at 15 years after HCT. CLE were most prevalent in the neurologic (9%), neurodevelopmental (8%), and dental (8%) categories. Chemotherapy-based conditioning was associated with decreased-height z score at 2 to 5 years after HCT (P <.001), and with endocrine (P <.001) and dental (P =.05) CLE. CD4 count of ≤500 cells/μL and/or continued need for immunoglobulin replacement therapy >2 years after transplantation were associated with lower-height z scores. Continued survival from 2 to 15 years after HCT was 90%. The presence of any CLE was associated with increased risk of late death (hazard ratio, 7.21; 95% confidence interval, 2.71-19.18; P <.001). Late morbidity after HCT for SCID was substantial, with an adverse impact on overall survival. This study provides evidence for development of survivorship guidelines based on disease characteristics and treatment exposure for patients after HCT for SCID. [ABSTRACT FROM AUTHOR]

The Structured Clinical Interview for Gambling Disorder (SCI-GD) has the potential to bridge a diagnostic clinical gap, but psychometric evaluations have been scarce, in particular in relation to self-reported diagnostic criteria. This study analyzed existing data, including Swedish gamblers (N = 204) from treatment- and help-seeking contexts, self-help groups, and the general population, who were interviewed with the SCI-GD and completed self-report measures. The results indicated that fewer individuals fulfilled the diagnostic criteria for gambling disorder (GD) with the SCI-GD (n = 110, 54%), compared to a self-report Diagnostic and Statistical Manual of Mental Disorders:5th Edition (DSM-5) questionnaire on GD (n = 145, 71%; p <.001). Agreement between interviews and self-reported criteria was generally low (Fleiss kappa range: 0.31–0.52; r range: 0.35–0.55). A Rasch analysis showed that specific diagnostic criteria varied in difficulty, indicating a general pattern of higher item difficulty for the SCI-GD compared to self-reported DSM-5 criteria. Both the SCI-GD and the self-reported DSM-5 criteria performed well in terms of internal consistency, convergent, and discriminant validity. We conclude that the SCI-GD is a reliable and valid diagnostic tool to assess GD among individuals with various gambling behavior patterns. Further research-related and clinical implications are discussed. [ABSTRACT FROM AUTHOR]

CC-90002 is an anti-CD47 antibody that inhibits CD47-SIRPα interaction and enables macrophage-mediated killing of tumor cells in hematological cancer cell lines. In this first clinical, phase 1, dose-escalation and -expansion study (CC-90002-AML-001; NCT02641002), we evaluated CC-90002 in patients with relapsed/refractory acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS). CC-90002 was administered in escalating doses of 0.1-4.0 mg/kg, using a modified 3 + 3 design. Primary endpoints included dose-limiting toxicities (DLTs), non-tolerated dose (NTD), maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary endpoints included preliminary efficacy, pharmacokinetics, and presence/frequency of anti-drug antibodies (ADAs). Between March 2016 and July 2018, 28 patients were enrolled (24 with AML and 4 with MDS) at 6 sites across the USA. As of July 18, 2018, all patients had discontinued, mainly due to death or progressive disease. The most common treatment-emergent adverse events were diarrhea (46.4%), thrombocytopenia (39.3%), febrile neutropenia (35.7%), and aspartate aminotransferase increase (35.7%). Four patients experienced DLTs (1 patient had grade 4 disseminated intravascular coagulation and grade 5 cerebral hemorrhage, 1 had grade 3 purpura, 1 had grade 4 congestive cardiac failure and grade 5 acute respiratory failure, and another had grade 5 sepsis). The NTD and MTD were not reached. No objective responses occurred. CC-90002 serum exposure was dose-dependent. ADAs were present across all doses, and the proportion of ADA-positive patients in cycle 1 increased over time. Despite no unexpected safety findings, the CC-90002-AML-001 study was discontinued in dose escalation for lack of monotherapy activity and evidence of ADAs. However, as other anti-CD47 agents in clinical trials are showing promising early results for AML and MDS, understanding preclinical and clinical differences between individual agents in this class will be of high importance.

Various combination treatments have been considered to attain the effective therapy threshold by combining independent antitumor mechanisms against the heterogeneous characteristics of tumor cells in malignant brain tumors. In this study, the natural killer (NK) cells associated with bevacizumab (Bev) plus irinotecan (Iri) against glioblastoma multiforme (GBM) were investigated. For the experimental design, NK cells were expanded and activated by K562 cells expressing the OX40 ligand and membrane-bound IL-18 and IL-21. The effects of Bev and Iri on the proliferation and NK ligand expression of GBM cells were evaluated through MTT assay and flow cytometry. The cytotoxic effects of NK cells against Bev plus Iri-treated GBM cells were also predicted via the LDH assay in vitro. The therapeutic effect of different injected NK cell routes and numbers combined with the different doses of Bev and Iri was confirmed according to tumor size and survival in the subcutaneous (s.c) and intracranial (i.c) U87 xenograft NOD/SCID IL-12Rγnull mouse model. The presence of injected-NK cells in tumors was detected using flow cytometry and immunohistochemistry ex vivo. As a result, Iri was found to affect the proliferation and NK ligand expression of GBM cells, while Bev did not cause differences in these cellular processes. However, the administration of Bev modulated Iri efficacy in the i.c U87 mouse model. NK cells significantly enhanced the cytotoxic effects against Bev plus Iri-treated GBM cells in vitro. Although the intravenous (IV) injection of NK cells in combination with Bev plus Iri significantly reduced the tumor volume in the s.c U87 mouse model, only the direct intratumorally (IT) injection of NK cells in combination with Bev plus Iri elicited delayed tumor growth in the i.c U87 mouse model. Tumor-infiltrating NK cells were detected after IV injection of NK cells in both s.c and i.c U87 mouse models. In conclusion, the potential therapeutic effect of NK cells combined with Bev plus Iri against GBM cells was limited in this study. Accordingly, further research is required to improve the accessibility and strength of NK cell function in this combination treatment.

Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy.

Background/aimThe therapeutic potential of bromodomain and extra-terminal motif (BET) inhibitors in hematological cancers has been well established in preclinical and early-stage clinical trials, although as of yet, no BETtargeting agent has achieved approval. To add insight into potential response to mivebresib (ABBV-075), a broadspectrum BET inhibitor, co-clinical modeling of individual patient biopsies was conducted in the context of a Phase I trial in acute myeloid leukemia (AML).Materials and methodsCo-clinical modeling involves taking the patient's biopsy and implanting it in mice with limited passage so that it closely retains the original characteristics of the malignancy and allows comparisons of response between animal model and clinical data. Procedures were developed, initially with neonate NOD/Shi-scid-IL2rγnull (NOG) mice and then optimized with juvenile NOG-EXL as host mice, eventually resulting in a robust rate of engraftment (16 out of 26, 62%).ResultsResults from the co-clinical AML patient-derived xenograft (PDX) modeling (6 with >60% inhibition of bone marrow blasts) were consistent with the equivalent clinical data from patients receiving mivebresib in monotherapy, and in combination with venetoclax. The modeling system also demonstrated the activity of a novel BD2-selective BET inhibitor (ABBV-744) in the preclinical AML setting. Both agents were also highly effective in inhibiting blast counts in the spleen (10/10 and 5/6 models, respectively).ConclusionThese findings confirm the validity of the model system in the co-clinical setting, establish highly relevant in vivo models for the discovery of cancer therapy, and indicate the therapeutic value of BET inhibitors for AML and, potentially, myelofibrosis treatment.

Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality from PC. Thus, there is growing recognition that preventing or delaying PC metastasis holds great potential for substantially improving patient outcomes. Here we show receptor-interacting protein kinase 2 (RIPK2) is a clinically actionable target for inhibiting PC metastasis. RIPK2 is amplified/gained in ~65% of lethal metastatic castration-resistant PC. Its overexpression is associated with disease progression and poor prognosis, and its genetic knockout substantially reduces PC metastasis. Multi-level proteomics analyses reveal that RIPK2 strongly regulates the stability and activity of c-Myc (a driver of metastasis), largely via binding to and activating mitogen-activated protein kinase kinase 7 (MKK7), which we identify as a direct c-Myc-S62 kinase. RIPK2 inhibition by preclinical and clinical drugs inactivates the noncanonical RIPK2/MKK7/c-Myc pathway and effectively impairs PC metastatic outgrowth. These results support targeting RIPK2 signaling to extend metastasis-free and overall survival.

Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-β1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1hi/TGFB1hi fibroblasts. Our study indicates that hAEC2 loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through an hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.

About 15-20% of breast cancer (BCa) is triple-negative BCa (TNBC), a devastating disease with limited therapeutic options. Aberrations in the PI3K/PTEN signaling pathway are common in TNBC. However, the therapeutic impact of PI3K inhibitors in TNBC has been limited and the mechanism(s) underlying this lack of efficacy remain elusive. Here, we demonstrate that a large subset of TNBC expresses significant levels of MAPK4, and this expression is critical for driving AKT activation independent of PI3K and promoting TNBC cell and xenograft growth. The ability of MAPK4 to bypass PI3K for AKT activation potentially provides a direct mechanism regulating tumor sensitivity to PI3K inhibition. Accordingly, repressing MAPK4 greatly sensitizes TNBC cells and xenografts to PI3K blockade. Altogether, we conclude that high MAPK4 expression defines a large subset or subtype of TNBC responsive to MAPK4 blockage. Targeting MAPK4 in this subset/subtype of TNBC both represses growth and sensitizes tumors to PI3K blockade.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in human PDA cell lines, which completely blocked proliferation in vitro and led to cell death. In contrast, GFAT1 knockout did not preclude the growth of human tumor xenografts in mice, suggesting that cancer cells can maintain fidelity of glycosylation precursor pools by scavenging nutrients from the tumor microenvironment. We found that hyaluronic acid (HA), an abundant carbohydrate polymer in pancreatic tumors composed of repeating N-acetyl-glucosamine (GlcNAc) and glucuronic acid sugars, can bypass GFAT1 to refuel the HBP via the GlcNAc salvage pathway. Together, these data show HA can serve as a nutrient fueling PDA metabolism beyond its previously appreciated structural and signaling roles.

BackgroundCurrent understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment.MethodsHumanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias.ResultsA total of 897 HSPC clones were longitudinally tracked in hu-BLT mice-providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions.ConclusionsHuman repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

Receptor clustering is the first and critical step to activate apoptosis by death receptor-5 (DR5). The recent discovery of the autoinhibitory DR5 ectodomain has challenged the long-standing view of its mechanistic activation by the natural ligand Apo2L. Because the autoinhibitory residues have remained unknown, here we characterize a crucial patch of positively charged residues (PPCR) in the highly variable domain of DR5. The PPCR electrostatically separates DR5 receptors to autoinhibit their clustering in the absence of ligand and antibody binding. Mutational substitution and antibody-mediated PPCR interference resulted in increased apoptotic cytotoxic function. A dually specific antibody that enables sustained tampering with PPCR function exceptionally enhanced DR5 clustering and apoptotic activation and distinctively improved the survival of animals bearing aggressive metastatic and recurrent tumors, whereas clinically tested DR5 antibodies without PPCR blockade function were largely ineffective. Our study provides mechanistic insights into DR5 activation and a therapeutic analytical design for potential clinical success.

Cell-based immunotherapy has become the new-generation cancer medicine, and "off-the-shelf" cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

The Hippo and mTOR signaling cascades are major regulators of cell growth and division. Aberrant regulation of these pathways has been demonstrated to contribute to gliomagenesis and result in enhanced glioblastoma proliferation and invasive characteristics. Several crosstalk mechanisms have been described between these two pathways, although a complete picture of these signaling interactions is lacking and is required for effective therapeutic targeting. Here we report the ability of mTORC2 to directly phosphorylate YAP at serine 436 (Ser436) positively regulating YAP activity. We show that mTORC2 activity enhances YAP transcriptional activity and the induction of YAP-dependent target gene expression while its ablation via genetic or pharmacological means has the opposite affects on YAP function. mTORC2 interacts with YAP via Sin1 and mutational analysis of serine 436 demonstrates that this phosphorylation event affects several properties of YAP leading to enhanced transactivation potential. Moreover, YAP serine 436 mutants display altered glioblastoma growth, migratory capacity and invasiveness both in vitro and in xenograft experiments. We further demonstrate that mTORC2 is able to regulate a Hippo pathway resistant allele of YAP suggesting that mTORC2 can regulate YAP independent of Hippo signaling. Correlative associations between the expression of these components in GBM patient samples also supported the presence of this signaling relationship. These results advance a direct mTORC2/YAP signaling axis driving GBM growth, motility and invasiveness.

Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2-CAR T cells activate patient-derived endogenous T cells and monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. SIGNIFICANCE: Our findings highlight the critical role of IFNγ signaling for a productive CAR T-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors.This article is highlighted in the In This Issue feature, p. 2113.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with the development of hematopoietic cancers of B-lymphocyte origin, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Primary infection of B-cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. TfR1 is highly expressed in proliferating cells, including activated lymphocytes and malignant cells. We developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has previously shown significant antitumor activity in immunosuppressed mouse models bearing human malignant B-cells, including multiple myeloma and AIDS-NHL cells. In this article, we examined the effect of targeting TfR1 to inhibit EBV-driven activation and growth of human B-cells in vivo using an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ [NOD/SCID gamma (NSG)] mouse model. Mice were implanted with T-cell-depleted, human peripheral blood mononuclear cells (PBMCs), either without EBV (EBV-), or exposed to EBV in vitro (EBV+), intravenously via the tail vein. Mice implanted with EBV+ cells and treated with an IgG1 control antibody (400 μg/mouse) developed lymphoma-like growths of human B-cell origin that were EBV+, whereas mice implanted with EBV+ cells and treated with ch128.1/IgG1 (400 μg/mouse) showed increased survival and significantly reduced inflammation and B-cell activation. These results indicate that ch128.1/IgG1 is effective at preventing the growth of EBV+ human B-cell tumors in vivo, thus, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent the development of EBV-associated B-cell malignancies. SIGNIFICANCE: An anti-TfR1 antibody, ch128.1/IgG1, effectively inhibits the activation, growth, and immortalization of EBV+ human B-cells in vivo, as well as the development of these cells into lymphoma-like tumors in immunodeficient mice.

From 2011, 37 children were referred to a hospital due to low levels of T cell receptor excision circles (TRECs) from newborn screening. Among them, three children were immunologically characterized and followed up to show that postnatal corticosteroid usage may be among the causes of false positivity in TRECs screening.

Abstract Background X-linked severe combined immunodeficiency is caused by IL2RG gene mutation. Several variations have been identified in the IL2RG gene, which potentially can prevent the production of nonfunctional proteins. Herein, a novel X-linked variant in the IL2RG gene is reported in twin brothers, associated with inflammatory bowel symptoms. Case presentation The patients were 26-month-old monozygotic twin middle-eastern males with failure to thrive and several inpatient admissions due to severe chronic nonbloody diarrhea that started at the age of 12 months. Pancolitis was revealed after performing upper and lower gastrointestinal endoscopies on the twin with more severe gastrointestinal symptoms. Flow cytometric evaluation of the peripheral blood cells showed low levels of CD4+ cells in both patients. Next generation sequencing-based gene panel test results of the two patients proved a novel heterozygous missense X-linked IL2RG mutation (70330011 A > G, p.Trp197Arg) in one of the patients, which was predicted to be deleterious (CADD score of 28), which soon after was confirmed by Sanger segregation in his twin brother. Both parents were wild types and had never experienced similar symptoms. The patients received an human leukocyte antigen (HLA)-matched cord blood transplant. The twin with more severe gastrointestinal symptoms died 1 month after transplantation. In his brother, watery diarrhea eventually subsided after transplantation. Conclusion Intestinal involvement in X-linked severe combined immunodeficiency is a rare presentation that might be neglected. The increasing availability of genetic screening tests worldwide could be helpful for early detection of such lethal primary immunodeficiency diseases and in implementing effective interventions to handle the severe outcomes.

A significant complication of HSCT is graft failure, although few studies focus on this problem in patients with inborn errors of immunity (IE). We explored outcome of second HSCT for IEI by a retrospective, single-centre study between 2002 and 2022. Four hundred ninety-three patients underwent allogeneic HSCT for severe combined immunodeficiency (SCID; n = 113, 22.9%) or non-SCID IEI (n = 380, 77.1%). Thirty patients (6.0%) required second HSCT. Unconditioned infusion or no serotherapy at first HSCT was more common in patients who required second transplant. Median interval between first and second HSCT was 0.97 years (range: 0.19–8.60 years); a different donor was selected for second HSCT in 24/30 (80.0%) patients. Conditioning regimens for second HSCT were predominately treosulfan-based (with thiotepa: n = 18, 60.0%; without, n = 6, 20.0%). Patients received grafts from peripheral blood stem cell (n = 25, 83.3%) or bone marrow (n = 5, 16.7%) with median stem cell dose 9.5 × 106 CD34 + cells/kilogram (range: 1.4–32.3). Median follow-up was 1.92 years (0.22–16.0). Overall survival was 80.8% and event-free survival was 64.7%. Four patients died, two of early-transplant related complications, and two of late sepsis post-second HSCT. Three patients required third HSCT; all are alive with 100% donor chimerism. Cumulative incidence of acute graft-versus-host disease was 28.4%, (all grade I–II). Viral reactivation was seen in 13/30 (43.3%) patients, including HHV6 (n = 6), CMV (n = 4), and adenovirus (n = 2). At latest follow-up, 25/26 surviving patients have donor chimerism ≥ 90% and 16/25 (64.0%) have discontinued immunoglobulin replacement. Second HSCT offers IEI patients with graft failure curative treatment with good overall survival and immunological recovery. [ABSTRACT FROM AUTHOR]

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In the patient, however, this trend reversed dramatically, TCR- / CD8+ lymphocytes formed the majority subset, accounting for 63% while / CD8+ lymphocytes reduced to 36.7% (Figure 2C). While B cells typically express the Syk (ZAP70 homolog) instead of ZAP70, mean ZAP70 fluorescent staining in B cells yielded a background MFI of 220. {gamma}{delta} T cell homeostasis is established in competition with {alpha}{beta} T cells and NK cells. Keywords: NBS; newborn screening; pathogenic variants; PJP; pneumocystis jirovecii; SCID; ZAP70; T cells EN NBS newborn screening pathogenic variants PJP pneumocystis jirovecii SCID ZAP70 T cells 1 5 5 10/27/23 20231001 NES 231001 PEER REVIEW The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/pai.14035. [Extracted from the article]