Combined Immunodeficiency Research Articles

MLH1 Inhibits Metastatic Potential of Pancreatic Ductal Adenocarcinoma via Downregulation of GPRC5C

DNA mismatch repair gene MutL homolog-1 (MLH1) has divergent effects in many cancers; however, its impact on the metastasis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, MLH1 stably overexpressed (OE) and knockdowned (KD) sublines were established. Wound healing and transwell assays were used to evaluate cell migration/invasion. In vivo metastasis was investigated in orthotopic implantation models (severe combined immunodeficiency mice). RT-qPCR and western blotting were adopted to show gene/protein expression. MLH1 downstream genes were screened by transcriptome sequencing. Tissue microarray–based immunohistochemistry was applied to determine protein expression in human specimens. In successfully generated sublines, OE cells presented weaker migration/invasion abilities, compared with controls, whereas in KD cells, these abilities were significantly stronger. The metastasis-inhibitory effect of MLH1 was also observed in mice. Mechanistically, G protein–coupled receptor, family C, group 5, member C (GPRC5C) was a key downstream gene of MLH1 in PDAC cells. Subsequently, transient GPRC5C silencing effectively inhibited cell migration/invasion and remarkably reversed the proinvasive effect of MLH1 knockdown in KD cells. In animal models and human PDAC tissues, tumoral GPRC5C expression, negatively associated with MLH1 expressions, was positively correlated with histologic grade, vessel invasion, and poor cancer-specific survival. In conclusion, MLH1 inhibits the metastatic potential of PDAC via downregulation of GPRC5C.

MHC Class II Deficiency: Clinical, Immunological, and Genetic Insights in a Large Multicenter Cohort

BackgroundMajor Histocompatibility Complex Class II (MHC-II) deficiency, a combined immunodeficiency, results from loss of Human Leukocyte Antigen class II expression on antigen-presenting cells. Currently, hematopoietic stem cell transplantation (HSCT) stands as the sole curative approach, though factors influencing patient outcomes remain insufficiently explored. ObjectiveOur aim was to elucidate the clinical, immunological, and genetic profiles associated with MHC-II deficiency and identify prognostic indicators that affect survival rates. MethodsIn this multicenter retrospective analysis, we gathered data from 35 patients diagnosed with MHC-II deficiency across 12 centers in Turkey. We recorded infection histories, gene mutations, immune cell subsets, and surface MHC-II expression on blood cells. We conducted survival analyses to evaluate the impact of various factors on patient outcomes. ResultsPredominant symptoms observed were pneumonia (n=29, 82.9%), persistent diarrhea (n=26, 74.3%), and severe infections (n=26, 74.3%). The RFXANK gene mutation (n=9) was the most frequent, followed by mutations in RFX5 (n=8), CIITA (n=4), and RFXAP (n=2) genes. Patients with RFXANK mutations presented with later onset and diagnosis compared to those with RFX5 mutations (p=0.0008 and p=0.0006, respectively), alongside a more significant diagnostic delay (p=0.020). A notable founder effect was observed in 5 patients with a specific RFX5 mutation (c.616G>C). The overall survival rate for patients was 28.6% (n=10), showing a significantly higher proportion in individuals with HSCT (n=8, 80%). Early demise (p=0.006) and higher CD8+ T-cell counts were observed in patients with the RFX5 mutations compared to RFXANK-mutant patients (p=0.006 and p=0.009, respectively). ConclusionThe study delineates the genetic and clinical panorama of MHC-II deficiency, emphasizing the prevalence of specific gene mutations such as RFXANK and RFX5. These insights facilitate early diagnosis and prognosis refinement, significantly contributing to the management of MHC-II deficiency.

Immunological and molecular study in children with combined immunodeficiency.

Background. Severe combined immunodeficiency (SCID) is a form of immunodeficiencies (PID), caused by molecular defects. These defects can restrict the development and function of lymphocytes. Early diagnosis and treatment of SCID can lead to disease-free survival. This study aims to investigate some of the possible underlying genetic defects in a group of Egyptian infants and children with clinical and immunological profiles suggestive of SCID. Methods. This study included eighty patients who showed clinical warning signs of immunodeficiency. Subjects were thoroughly examined clinically. Laboratory evaluation included immunoglobulins serum levels and flow cytometric assessment of immune cells. This testing showed an altered immune profile in thirty patients. They had decreased T and/or B lymphocytes or natural killer cells. DNA extraction was done for those cases. The coding regions of the RAG1 gene and RAG2 gene was investigated for hot spot mutations by sequencing technique guided by the patient clinical evaluation, inheritance pattern, immunophenotyping by flow cytometric analysis of lymphocyte subsets, and serum immunoglobulins level detection. Results. Results showed novel and previously reported variants (mutation, polymorphism), they were found in 18 cases which include variants in the RAG1 gene (E880K, A960A, H249R, S913R, K820R, V782G), and variants in the RAG2 gene (P501T, L514M, rs10836573, cDNA.2129A>T). Conclusions. To evaluate SCID patients completely, mutation gene analysis is highly required and recommended.

Human DNA-dependent protein kinase catalytic subunit deficiency: A comprehensive review and update

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has an essential role in the non-homologous end-joining pathway that repairs DNA double-strand breaks in V(D)J recombination involved in the expression of T- and B-cell receptors. Whereas homozygous mutations in PRKDC define the scid mouse, a model that has been widely used in biology, human mutations in PRKDC are extremely rare and the disease spectrum has not been described so far. To provide an update on the genetics, clinical spectrum, immunological profile, and therapy of DNA-PKcs deficiency in human. The clinical, biological, and treatment data from the 6 cases published to date and from 1 new patient were obtained and analyzed. Rubella PCR was performed on available granuloma material. We report on 7 patients; Six patients displayed the autosomal recessive p.L3062R mutation in PRKDC gene encoding DNA-PKcs. Atypical severe combined immunodeficiency with inflammatory lesions, granulomas, and autoimmunity was the predominant clinical manifestation (n=5/7). Rubella viral strain was detected in the granuloma of 1 patient over the 2 tested. T-cell counts, including naïve CD4+CD45RA+ T cells and T-cell function were low at diagnosis for 6 patients. For most patients with available values naïve CD4+CD45RA+ T cells decreased over time (n=5/6). Hematopoietic stem cell transplantation (HSCT) was performed in 5 patients, of whom 4 are still alive without transplant-related morbidity. Sustained T- and B-cell reconstitution was respectively observed for 4 and 3 patients, after a median follow-up of 8 years (range 3-16 y). DNA-PKcs deficiency mainly manifests as an inflammatory disease with granuloma and autoimmune features, along with severe infections.

Photo-metallo-immunotherapy: Fabricating Chromium-Based Nanocomposites to Enhance CAR-T Cell Infiltration and Cytotoxicity against Solid Tumors.

The infiltration and cytotoxicity of chimeric antigen receptor (CAR)-T cells are crucial for effective elimination of solid tumors. While metallo-immunotherapy is a promising strategy that can activate the antitumor immunity, its role in promoting CAR-T cell therapy remains elusive. The first single-element nanomaterial based on chromium nanoparticles (Cr NPs) for cancer photo-metallo-immunotherapy has been reported previously. Herein, an extended study using biodegradable polydopamine as a versatile carrier for these nanoparticles, enabling synergistic CAR-T cell therapy, is reported. The results show that these nanocomposites with or without further encapsulation of the anticancer drug alpelisib can promote the CAR-T cell migration and antitumor effect. Upon irradiation with near-infrared light, they caused mild hyperthermia that can "warm" the "cold" tumor microenvironment (TME). The administration of B7-H3 CAR-T cells to NOD severe combined immunodeficiency gamma mice bearing a human hepatoma or PIK3CA-mutated breast tumor can significantly inhibit the tumor growth after the induction of tumor hyperthermia by the nanocomposites and promote the secretion of serum cytokines, including IL-2, IFN-γ, and TNF-α. The trivalent Cr3+ ions, which are the major degradation product of these nanocomposites, can increase the CXCL13 and CCL3 chemokine expressions to generate tertiary lymphoid structures (TLSs) in the tumor tissues, facilitating the CAR-T cell infiltration.

Promotion of perineural invasion of cholangiocarcinoma by Schwann cells via nerve growth factor.

Cholangiocarcinoma (CCA), a highly lethal tumor of the hepatobiliary system originating from bile duct epithelium, can be divided into the intrahepatic, hilar, and extrahepatic types. Due to its insidious onset and atypical early clinical symptoms, the overall prognosis is poor. One of the important factors contributing to the poor prognosis of CCA is the occurrence of perineural invasion (PNI), but the specific mechanisms regarding how it contributes to the occurrence of PNI are still unclear. The main purpose of this study is to explore the molecular mechanism leading to the occurrence of PNI and provide new ideas for clinical treatment. CCA cell lines and Schwann cells (SCs) were stimulated to observe the changes in cell behavior. SCs cocultured with tumor supernatant and SCs cultured in normal medium were subjected to transcriptome sequencing to screen the significantly upregulated genes. Following this, the two types of tumor cells were cultured with SC supernatant, and the changes in behavior of the tumor cells were observed. Nonobese diabetic-severe combined immunodeficiency disease (NOD-SCID) mice were injected with cell suspension supplemented with nerve growth factor (NGF) via the sciatic nerve. Four weeks later, the mice were euthanized and the tumor sections were removed and stained. Nerve invasion by tumor cells was common in CCA tissues. SCs were observed in tumor tissues, and the number of SCs in tumor tissues and the degree of PNI were much higher than were those in normal tissues or tissues without PNI. The overall survival time was shorter in patients with CCA with PNI than in patients without PNI. SCs were enriched in CCA tissues, indicating the presence of PNI and associated with poor prognosis in CCA patients. CCA was found to promote NGF secretion from SCs in vitro. After the addition of exogenous NGF in CCA cell culture medium, the proliferation activity and migration ability of CCA cells were significantly increased, suggesting that SCs can promote the proliferation and migration of CCA through the secretion of NGF. NGF, in turn, was observed to promote epithelial-mesenchymal transition in CCA through tropomyosin receptor kinase A (TrkA), thus promoting its progression. Tumor growth in mice shows that NGF can promote PNI in CCA. In CCA, tumor cells can promote the secretion of NGF by SCs, which promotes the progression of CCA and PNI by binding to its high-affinity receptor TrkA, leading to poor prognosis.

Quality considerations and major pitfalls for high throughput DNA-based newborn screening for severe combined immunodeficiency and spinal muscular atrophy

Quality considerations and major pitfalls for high throughput DNA-based newborn screening for severe combined immunodeficiency and spinal muscular atrophy

Novel Mutation in the Moesin (MSN) Gene Leads to Immunodeficiency with Epstein-Barr Virus (EBV) Infection and Dermatomyositis-Like Symptoms.

Moesin (MSN) deficiency is a recently reported combined immunodeficiency, and few cases have been reported to date. We describe a Chinese patient with a novel mutation causing MSN deficiency and a novel phenotype. Clinical and immunological data were collected. Whole-exome sequencing was performed to identify gene mutations. MSN protein expression and T cell proliferation and activation were determined by flow cytometry. Cell migration was confirmed with a Transwell assay. Autoantibody levels were analyzed using antigen microarrays. The patient was a 10-year-old boy who presented with recurrent fever, oral ulcers and dermatomyositis-like symptoms, such as periorbital edema, facial swelling, elevated creatine kinase levels, and abnormal electromyography and muscle biopsy results. Epstein-Barr virus (EBV) DNA was detected in the serum, cells and tissues of this patient. He further developed nasal-type NK/T-cell lymphoma. A novel hemizygous mutation (c.68A > G, p.N23S) in the MSN gene was found. The immunological phenotype of this patient included persistent decreases in T and B lymphocyte counts but normal immunoglobulin IgG levels. The patient had attenuated MSN protein expression and impaired T-cell proliferation and migration. The proportions of Tfh cells and CD21low B cells in the patient were higher than those in the controls. Moreover, 82 IgG and 102 IgM autoantibodies were more abundant in the patient than in the healthy controls. The novel mutation N23S is pathogenic and leads to a severe clinical phenotype. EBV infection, tumor, and dermatomyositis-like autoimmune symptoms may be associated with MSN deficiency, further expanding the understanding of the disease.

Paving the way in implementation of SCID newborn screening in developing nations: feasibility study and strategies to move forward in Malaysia.

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.

Opening SCID newborn screening for novel exon genetic variants through whole-exome sequencing in China

BackgroundSevere combined immunodeficiency (SCID) is the most fatal form of inherited primary immunodeficiency disease. Known molecular defect mutations occur in most children with SCID. MethodsHerein, we report Adenosine Deaminase-SCID (ADA-SCID) using whole-exome sequencing (WES), explore exome mutational landscape and significance for 17 SCID samples, and verify the mutated exon genes using the Gene Expression Omnibus (GEO) datasets. A total of 250 patients, who were hospitalized at the Neonatal Intensive Care Unit (NICU) of The Seventh Medical Center of the PLA General Hospital for 3 years (from 2017 to 2020), were screened for SCID. We collected mutated genes from the WES data of 17 SCID children. GSE609 and GSE99176 cohorts were used to identify the expressions of mutated exon genes and molecular features in SCID. Gene set variation analyses (GSVA) and correlation analyses were performed. ResultsThe detection rate with approximately 6.8 % (17/250) of SCID is high in the NICU. A total of 16 genes were identified among 17 SCID samples, of which the Top 2 genes (MUC6 and RP11-683L23.1) might be crucial in the progression of SCID with 94 % mutation frequency. Furthermore, CNN2 and SCGB1C1 had significant co-mutations and may cooperate to affect SCID development. Importantly, the phylogenetic tree classification results of 17 SCID samples are more correlated to MUC6 with the most significant mutations. Expression profiles of seven mutated genes and five mutated genes were documented in GSE609 and GSE99176 cohorts based on microarray, respectively. Several immune-related pathways were significantly enriched, and Foxd4, differing from the other four mutated genes, was inversely correlated with the GSVA-enriched pathway. ConclusionDue to its high detection rate (6.8%) and fatality rate (100%), the inclusion of SCID in newborn screening (NBS) is urgent for children in China. The WES successfully identified several common exonic variants (e.g., MUC6) and depicted the feature of mutations and evolution, which will help develop new diagnostic methods for SCID.

18q Deletion Syndrome Presenting with Late-Onset Combined Immunodeficiency

Patients with chromosome 18q deletion syndrome generally experience hypogammaglobulinemia. Herein, we describe two patients with chromosome 18q deletion syndrome who presented with late-onset combined immune deficiency (LOCID), which has not been previously reported. Patient 1 was a 29-year-old male with 18q deletion syndrome, who was being managed for severe motor and intellectual disabilities at the Yamabiko Medical Welfare Center for 26 years. Although the patient had few infections, he developed Pneumocystis pneumonia at the age of 28. Patient 2, a 48-year-old female with intellectual disability and congenital malformations, was referred to Tokyo Medical and Dental University Hospital with abnormal bilateral lung shadows detected on her chest radiography. Computed tomography showed multiple lymphadenopathies and pneumonia. A lymph node biopsy of the inguinal region revealed granulomatous lymphadenitis, and a chromosomal examination revealed 18q deletion. Array-based genomic hybridization analysis revealed deletion at 18q21.32-q22.3 for patient 1 and at 18q21.33-qter for patient 2. Immune status work-up of the two patients revealed panhypogammaglobulinemia, decreased number of memory B cells and naïve CD4+ and/or CD8+ cells, reduced response on the carboxyfluorescein diacetate succinimidyl ester T-cell division test, and low levels of T-cell receptor recombination excision circles and Ig κ-deleting recombination excision circles. Consequently, both patients were diagnosed with LOCID. Although patients with 18q deletion syndrome generally experience humoral immunodeficiency, the disease can be further complicated by cell-mediated immunodeficiency, causing combined immunodeficiency. Therefore, patients with 18q deletion syndrome should be regularly tested for cellular/humoral immunocompetence.

Bone Marrow Mesenchymal Stem Cells Promote Ovarian Cancer Cell Proliferation via Cytokine Interactions.

Bone marrow mesenchymal stem cells (BMSCs) are key players in promoting ovarian cancer cell proliferation, orchestrated by the dynamic interplay between cytokines and their interactions with immune cells; however, the intricate crosstalk among BMSCs and cytokines has not yet been elucidated. Here, we aimed to investigate interactions between BMSCs and ovarian cancer cells. We established BMSCs with a characterized morphology, surface marker expression, and tri-lineage differentiation potential. Ovarian cancer cells (SKOV3) cultured with conditioned medium from BMSCs showed increased migration, invasion, and colony formation, indicating the role of the tumor microenvironment in influencing cancer cell behavior. BMSCs promoted SKOV3 tumorigenesis in nonobese diabetic/severe combined immunodeficiency mice, increasing tumor growth. The co-injection of BMSCs increased the phosphorylation of p38 MAPK and GSK-3β in SKOV3 tumors. Co-culturing SKOV3 cells with BMSCs led to an increase in the expression of cytokines, especially MCP-1 and IL-6. These findings highlight the influence of BMSCs on ovarian cancer cell behavior and the potential involvement of specific cytokines in mediating these effects. Understanding these mechanisms will highlight potential therapeutic avenues that may halt ovarian cancer progression.

Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.

Brown Adipose Tissue as a Unique Niche for Islet Organoid Transplantation: Insights From In Vivo Imaging.

Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery. A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase. Luciferase-expressing hiPSCs were differentiated into islet organoids. The islet organoids were transplanted into the scapular brown adipose tissue (BAT) of nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice as the BAT group and under the left kidney capsule (KC) of NOD/SCID mice as a control group, respectively. Bioluminescence imaging (BLI) of the organoid grafts was performed on days 1, 7, 14, 28, 35, 42, 49, 56, and 63 posttransplantation. BLI signals were detected in all recipients, including both the BAT and control groups. The BLI signal gradually decreased in both BAT and KC groups. However, the graft BLI signal intensity under the left KC decreased substantially faster than that of the BAT. Furthermore, our data show that islet organoids transplanted into streptozotocin-induced diabetic mice restored normoglycemia. Positron emission tomography/MRI verified that the islet organoids were transplanted at the intended location in these diabetic mice. Immunofluorescence staining revealed the presence of functional organoid grafts, as confirmed by insulin and glucagon staining. Our results demonstrate that BAT is a potentially desirable site for islet organoid transplantation for T1D therapy.

Clinical features and lymphocyte immunophenotyping analysis in primary immunodeficiency patients with non-transplant lymphoproliferative disorders

Lymphoproliferative disorders (LPD) comprise a heterogeneous group and are originally classified into the “Disease of immune dysregulation” category. Of 96 Taiwanese patients during 2003–2022, 31 (median 66, range 0.03–675 months) developed LPD, mainly including palpable lymphadenopathy (in 10 patients), intestinal lymphadenopathy associated with refractory inflammatory bowel disease (IBD in 8) and hepatosplenomegaly (in 7) during long-term follow-up (median 144, range 3–252 months). They distributed in the categories of antibody deficiency (2 CVID, 2 TTC37, PIK3CD, PIK3R1 and AICDA each), phagocyte (4 CYBB, 1 STAT1 and 1 IFNRG1), immune dysregulation (2 FOXP3, 2 XIAP and 2 HLH), combined immunodeficiencies (2 IL2RG; CD40L, ZAP70 and unknown each), syndromic features (2 STAT3-LOF, 1 WAS and 1 ATM) and three with anti-IFN-γ autoantibodies. An increased senescent (CD8 + CD57+) and CD21-low, disturbed transitional B (CD38 + IgM++), plasmablast B (CD38++IgM-), memory B (CD19 + CD27+) and TEMRA (CD27-IgD-) components were often observed in cross-sectional immunophenotyping and trended to develop LPD.

Characterization of thymic architecture and lymphocyte populations in X-MAID due to an underlying pathogenic moesin mutation

Background: X-linked Moesin Associated Immunodeficiency (X-MAID) is a combined immunodeficiency caused by deficiency in the moesin protein. Moesin, which is encoded by the MSN gene, is part of the ezrin-radixin-moesin (ERM) family of transmembrane proteins that interact with the actin cytoskeleton and regulate the shape and migration of cells. Deficiency of moesin is associated with aberrant T cell migration and inadequate immune synapse formation, leading to significant immunodeficiency and recurrent infections. While the clinical presentation of X-MAID is diverse, to date, no thymus histopathology findings have been reported. Aim: Describe the thymus histopathology of a patient with X-MAID. Results: Our patient is a 10-year-old male who presented early in life with recurrent infections, dysmorphic features, and severe pulmonary venous stenosis which required a double lung transplant at the age of 4 years. Prior to transplant, he was referred to Immunology for assessment and was subsequently found to harbour a hemizygous variant in the MSN gene (c.278dupT; p.L93FfsX21). Thymus histopathology findings showed significant cortical atrophy and dysplasia and was accompanied by reduction in CD3+ cells in the cortex. Abnormally low numbers of suppressor T cells and T helper cells in the thymic cortex and medulla were noted. Conclusion: Thymic findings in X-MAID can include cortical atrophy, dysplasia, and decreased cellularity. This provides further evidence for the importance of moesin on T cell development and migration in the thymus. Statement of novelty: Description of thymus histopathology in a patient with X-MAID.

Artemis deficiency: A large cohort including a novel variant with increased radiosensitivity.

Artemis deficiency is an autosomal recessive disorder characterized by a combined immunodeficiency with increased cellular radiosensitivity. In this review, the clinical and genetic characteristics of 15 patients with DCLRE1C variants are presented. The demographic, clinical, immunologic, and genetic characteristics of patients with confirmed DCLRE1C variants diagnosed between 2013 and 2023 were collected retrospectively. Three patients were evaluated for radiosensitivity by the Comet assay, compared with age- and sex-matched healthy control. Seven patients who had severe infections in the first 6 months of life were diagnosed with T-B-NK+ SCID (severe combined immunodeficiency). Among them, four individuals underwent transplantation, and one of those died due to post-transplant complications in early life. Eight patients had hypomorphic variants. Half of them were awaiting a suitable donor, while the other half had already undergone transplantation. The majority of patients were born into a consanguineous family (93.3%). Most patients had recurrent sinopulmonary infections (73.3%), and one patient had no other infection than an acute respiratory infection before diagnosis. Two patients (13.3%) had autoimmunity in the form of autoimmune hemolytic anemia. Growth retardation was observed in only one patient (6.6%), and no malignancy was detected in the surviving 11 patients during the median (IQR) of 21.5 (12-45) months of follow-up. Three patients who had novel variants exhibited increased radiosensitivity and compromised DNA repair, providing a potential vulnerability to malignant transformation. Early diagnosis, radiation avoidance, and careful preparation for transplantation contribute to minimizing complications, enhancing life expectancy, and improving the patient's quality of life.

Understanding clonal heterogeneity and mechanisms of methotrexate resistance in two osteosarcoma PDX models.

e23512 Background: Osteosarcoma is a primary malignant bone tumor that is known to have significant intratumoral heterogeneity with branched cancer evolution. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % children and young adults. It has been revealed that the metastatic ability and resistance of cancers including osteosarcoma might be due to the presence of a subset of cells within the intra-tumorally heterogeneous population. We investigated the emergence of resistant clonal subpopulations following targeted methotrexate treatment in two patient derived xenograft (PDX) models in vitro and in vivo. Our aim was to not only track the subclonal population of cells that establish in the lungs but also to identify clonal drivers and the mutational signature of the genes and pathways involved in MTX resistance. Methods: We previously established 20 barcoded osteosarcoma PDX models and demonstrated that the application of methotrexate (MTX-IC95) treatment as a selective pressure in seven models produces clonal subpopulations that were more resistant to MTX. In the current work we used barcoded next generation and RNA sequencing techniques to map the clonal and transcriptomic architecture of MTX resistant single clones in M36 and OS252 PDX models. We next injected the barcoded PDX cells intratibially in twenty severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize to the lungs. Following primary tumor growth, limb amputation was carried out and mice were treated with methotrexate or vehicle for two weeks. Primary tumor, blood and lung samples were collected after death from metastasis or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -800C. DNA and RNA was extracted and analyzed by PCR, NGS or WES to map clonality, mutational and evolutionary profiles. Results: MTX treated single clones showed diversity and barcode abundance when compared to vehicle; barcode abundance reduced in the resistant clonal subpopulations when compared to the vehicles. Ten unique subclones were shared in both vehicle and MTX resistant clones in M36 while OS252 had only one shared subclone respectively. More than 50% of the injected mice developed primary tumors and matched lung micrometastases. We are currently analyzing both targeted and RNA sequencing results to further understand clonality, the mutational signature of the genes (DHFR, MTR, FPGS, GGH) and pathways involved in MTX resistance. Conclusions: We investigated mechanism of methotrexate resistance in two barcoded PDX models in vitro and in vivo using cytotoxicity, next generation and RNA sequencing techniques. Targeted amplicon sequencing of key genes involved in antifolate pathway will yield new information on clonal evolution and mechanisms of resistance.

Packaging of supplemented urokinase into alpha granules of in vitro–grown megakaryocytes for targeted nascent clot lysis

AbstractFibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce “antithrombotic” platelets from CD34+ hematopoietic stem cell–derived in vitro–grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor–related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro–generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

NUDCD3 deficiency disrupts V(D)J recombination to cause SCID and Omenn syndrome.

Inborn errors of T cell development present a pediatric emergency in which timely curative therapy is informed by molecular diagnosis. In 11 affected patients across four consanguineous kindreds, we detected homozygosity for a single deleterious missense variant in the gene NudC domain-containing 3 (NUDCD3). Two infants had severe combined immunodeficiency with the complete absence of T and B cells (T -B- SCID), whereas nine showed classical features of Omenn syndrome (OS). Restricted antigen receptor gene usage by residual T lymphocytes suggested impaired V(D)J recombination. Patient cells showed reduced expression of NUDCD3 protein and diminished ability to support RAG-mediated recombination in vitro, which was associated with pathologic sequestration of RAG1 in the nucleoli. Although impaired V(D)J recombination in a mouse model bearing the homologous variant led to milder immunologic abnormalities, NUDCD3 is absolutely required for healthy T and B cell development in humans.