Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

A familial case of Kikuchi-Fujimoto disease in dizygotic twins.

BACKGROUND: Kikuchi-Fujimoto disease (KFD) or necrotizing histiocytic lymphadenitis, was described separately by both Kikuchi and Fujimoto in Japan in the early 1970's. Despite its rarity in the pediatric population, it is an important differential in persistent lymphadenopathy. Familial cases of KFD in the literature are rare. Here we describe the first reported case of KFD in non-identical twin sisters. CASE PRESENTATION: Twin 1 presented with a 3-week history of worsening right-sided cervical lymphadenopathy, daily fevers, significant lethargy, weight loss and arthralgia of her knees and ankles at the age of 12 years in 2015. She had had an unremarkable medical history. A biopsy of her lymph nodes showed histiocytic necrosis consistent with KFD. Twin 2 presented with a three-week history of lethargy, fatigue, weight loss and left-sided posterior cervical chain lymphadenopathy at 16 years of age in 2018. She had a history of frequently relapsing nephrotic syndrome and celiac disease. A biopsy of her lymph nodes was undertaken and showed histiocytic necrosis consistent with KFD. CONCLUSIONS: KFD is a rare but self-limiting pathological process of necrotizing histiocytic lymphadenitis. Although further research is needed, there is an increasing amount of evidence which suggests a multifactorial pathological basis of disease. The two cases we document here are the first reported cases of familial KFD in dizygotic HLA-identical twins which reinforces the likely HLA-linkage in the etiology of KFD.

Expanding TREC and KREC Utility in Primary Immunodeficiency Diseases Diagnosis.

Primary immunodeficiency diseases (PID) area heterogeneous group of disorders caused by genetic defects of the immune system, which manifest clinically as recurrent infections, autoimmune diseases or malignancies. Early detection of PID remains a challenge, particularly in older children with milder and less specific symptoms. This study aimed to assess TREC and KREC diagnostic ability in PID. Data from children assessed by clinical immunologists at Speransky Children's Hospital, Moscow, Russia with suspected immunodeficiencies were analyzed between May 2013 and August 2016. Peripheral blood samples were sent for TREC/KREC, flow cytometry (CD3, CD4, CD8 and CD19), IgA and IgG analysis. A total of 434 children [189 healthy, 97 with group I and II PID (combined T and B cell immunodeficiencies & well-defined syndromes with immunodeficiency) and 148 group III PID (predominantly antibody deficiencies)] were included. Area under the curve (AUC) for TREC in PID groups I and II diagnosis reached 0.82 (CI = 0.75-0.90), with best model providing sensitivity of 65% and specificity of 92%. Neither TREC, nor KREC had added value in PID group III diagnosis. In this study, the predictive value of TREC and KREC in PID diagnosis was examined. We found that the TREC had some diagnostic utility for groups I and II PID. Possibly, addition of TREC measurements to existing clinical diagnostic algorithms may improve their predictive value. Further investigations on a larger cohort are needed to evaluate TREC/KREC abilities to be used as diagnostic tools on a wider scale.

Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands.

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α1D-adrenergic receptors (α1D-ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.

Hematopoietic stem cell transplant effectively rescues lymphocyte differentiation and function in DOCK8-deficient patients.

Bi-allelic inactivating mutations in DOCK8 cause a combined immunodeficiency characterised by severe pathogen infections, eczema, allergies, malignancy and impaired humoral responses. These clinical features result from functional defects in most lymphocyte lineages. Thus, DOCK8 plays a key role in immune cell function. Hematopoietic stem cell transplantation (HSCT) is curative for DOCK8 deficiency. While previous reports have described clinical outcomes for DOCK8 deficiency following HSCT, the effect on lymphocyte reconstitution and function has not been investigated. Our study determined whether defects in lymphocyte differentiation and function in DOCK8-deficient patients were restored following HSCT. DOCK8-deficient T and B lymphocytes exhibited aberrant activation and effector function in vivo and in vitro. Frequencies of αß T and MAIT cells were reduced while γδT cells were increased in DOCK8-deficient patients. HSCT improved, abnormal lymphocyte function in DOCK8-deficient patients. Elevated total and allergen-specific IgE in DOCK8-deficient patients decreased over time following HSCT. Our results document the extensive catalogue of cellular defects in DOCK8-deficient patients, and the efficacy of HSCT to correct these defects, concurrent with improvements in clinical phenotypes. Overall, our findings provide mechanisms at a functional cellular level for improvements in clinical features of DOCK8 deficiency post-HSCT, identify biomarkers that correlate with improved clinical outcomes, and inform the general dynamics of immune reconstitution in patients with monogenic immune disorders following HSCT.

Activating PIK3CD mutations impair human cytotoxic lymphocyte differentiation and function and EBV immunity.

BACKGROUND: Germline gain-of function (GOF) mutations in PIK3CD, encoding the catalytic p110δ subunit of phosphoinositide 3-kinase (PI3K), result in hyperactivation of the PI3K-AKT-mechanistic target of rapamycin pathway and underlie a novel inborn error of immunity. Affected subjects exhibit perturbed humoral and cellular immunity, manifesting as recurrent infections, autoimmunity, hepatosplenomegaly, uncontrolled EBV and/or cytomegalovirus infection, and increased incidence of B-cell lymphoproliferation, lymphoma, or both. Mechanisms underlying disease pathogenesis remain unknown. OBJECTIVE: Understanding the cellular and molecular mechanisms underpinning inefficient surveillance of EBV-infected B cells is required to understand disease in patients with PIK3CD GOF mutations, identify key molecules required for cell-mediated immunity against EBV, and develop immunotherapeutic interventions for the treatment of this and other EBV-opathies. METHODS: We studied the consequences of PIK3CD GOF mutations on the generation, differentiation, and function of CD8+ T cells and natural killer (NK) cells, which are implicated in host defense against infection with herpesviruses, including EBV. RESULTS: PIK3CD GOF total and EBV-specific CD8+ T cells were skewed toward an effector phenotype, with exaggerated expression of markers associated with premature immunosenescence/exhaustion and increased susceptibility to reactivation-induced cell death. These findings were recapitulated in a novel mouse model of PI3K GOF mutations. NK cells in patients with PIK3CD GOF mutations also exhibited perturbed expression of differentiation-associated molecules. Both CD8+ T and NK cells had reduced capacity to kill EBV-infected B cells. PIK3CD GOF B cells had increased expression of CD48, programmed death ligand 1/2, and CD70. CONCLUSIONS: PIK3CD GOF mutations aberrantly induce exhaustion, senescence, or both and impair cytotoxicity of CD8+ T and NK cells. These defects might contribute to clinical features of affected subjects, such as impaired immunity to herpesviruses and tumor surveillance.

Germline-activating mutations in PIK3CD compromise B cell development and function.

Gain-of-function (GOF) mutations in PIK3CD, encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with PIK3CD GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in Pik3cd In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery. Furthermore, PI3K GOF B cells exhibited intrinsic defects in class-switch recombination (CSR) due to impaired induction of activation-induced cytidine deaminase (AID) and failure to acquire a plasmablast gene signature and phenotype. Importantly, defects in CSR, AID expression, and Ig secretion were restored by leniolisib, a specific p110δ inhibitor. Our findings reveal key roles for balanced PI3K signaling in B cell development and long-lived humoral immunity and memory and establish the validity of treating affected individuals with p110δ inhibitors.

TREC and KREC Levels as a Predictors of Lymphocyte Subpopulations Measured by Flow Cytometry.

Primary immunodeficiency diseases (PID) is a heterogeneous group of disorders caused by genetic defects of the immune system, which manifests clinically as recurrent infections, autoimmune diseases, or malignancies. Early detection of other PID remains a challenge, particularly in older children due to milder and less specific symptoms, a low level of clinician PID awareness and poor provision of hospital laboratories with appropriate devices. T-cell recombination excision circles (TREC) and kappa-deleting element recombination circle (KREC) in a dried blood spot and in peripheral blood using real-time polymerase chain reaction (PCR) are used as a tool for severe combined immune deficiency but not in PID. They represent an attractive and cheap target for a more extensive use in clinical practice. This study aimed to assess TREC/KREC correspondence with lymphocyte subpopulations, measured by flow cytometry and evaluate correlations between TREC/KREC, lymphocyte subpopulations and immunoglobulins. We carried out analysis of data from children assessed by clinical immunologists at Speransky Children's Hospital, Moscow, Russia with suspected immunodeficiencies between May 2013 and August 2016. Peripheral blood samples were sent for TREC/KREC, flow cytometry (CD3, CD4, CD8, and CD19), IgA, IgM, and IgG analysis. A total of 839 samples were analyzed for using TREC assay and flow cytometry and 931 KREC/flow cytometry. TREC demonstrated an AUC of 0.73 (95% CI 0.70-0.76) for CD3, 0.74 (95% CI 0.71-0.77) for CD4 and 0.67 (95% CI 0.63-0.70) for CD8, respectively, while KREC demonstrated an AUC of 0.72 (95% CI 0.69-0.76) for CD19. Moderate correlation was found between the levels of TREC and CD4 (r = 0.55, p < 0.01) and KREC with CD19 (r = 0.56, p < 0.01). In this study, promising prediction models were tested. We found that TREC and KREC are able to moderately detect abnormal levels of individual lymphocyte subpopulations. Future research should assess associations between TREC/KREC and other lymphocyte subpopulations and approach TREC/KREC use in PID diagnosis.

Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets.

Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rß1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rß1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation.

Spontaneous Subcapsular Hematoma of the Fetal Liver: A Case Report and Review of Literature.

We report a case of a cystic abdominal mass on ultrasound which presented a diagnostic dilemma at 32 weeks gestation. A presumptive antenatal diagnosis of a subcapsular liver hematoma (SCH) was made based on the location and ultrasound appearance similar to SCH seen in pediatric and adult patients. Sequential evaluation of the mass showed an evolving ultrasound appearance which reinforced our initial impression of a subcapsular hematoma of the fetal liver. Postnatal ultrasound confirmed the resolving SCH as well as a previously undetected echo bright lesion characteristic of an infantile focal hemangioma directly adjacent to the resolving SCH. A review of the literature is provided.

IL-10 Potentiates Differentiation of Human Induced Regulatory T Cells via STAT3 and Foxo1.

Foxp3(+) regulatory T cells (Tregs) play essential roles in maintaining the immune balance. Although the majority of Tregs are formed in the thymus, increasing evidence suggests that induced Tregs (iTregs) may be generated in the periphery from naive cells. However, unlike in the murine system, significant controversy exists regarding the suppressive capacity of these iTregs in humans, especially those generated in vitro in the presence of TGF-ß. Although it is well known that IL-10 is an important mediator of Treg suppression, the action of IL-10 on Tregs themselves is less well characterized. In this article, we show that the presence of IL-10, in addition to TGF-ß, leads to increased expansion of Foxp3(+) iTregs with enhanced CTLA-4 expression and suppressive capability, comparable to that of natural Tregs. This process is dependent on IL-10R-mediated STAT3 signaling, as supported by the lack of an IL-10 effect in patients with IL-10R deficiency and dominant-negative STAT3 mutation. Additionally, IL-10-induced inhibition of Akt phosphorylation and subsequent preservation of Foxo1 function are critical. These results highlight a previously unrecognized function of IL-10 in human iTreg generation, with potential therapeutic implications for the treatment of immune diseases, such as autoimmunity and allergy.

Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies.

BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.

Idiopathic systemic capillary leak syndrome in children.

Adult subjects with systemic capillary leak syndrome (SCLS) present with acute and recurrent episodes of vascular leak manifesting as severe hypotension, hypoalbuminemia, hemoconcentration, and generalized edema. We studied clinical disease characteristics, serum cytokine profiles, and treatment modalities in a cohort of children with documented SCLS. Six children with SCLS were recruited from the United States, Australia, Canada, and Italy. Serum cytokines from SCLS subjects and a group of 10 healthy children were analyzed. Children with SCLS (aged 5-11 years old) presented with at least 1 acute, severe episode of hypotension, hypoalbuminemia, and hemoconcentration in the absence of underlying causes for these abnormalities. In contrast to what is observed in adult SCLS, identifiable infectious triggers precipitated most episodes in these children, and none of them had a monoclonal gammopathy. We found elevated levels of chemokine (C-C motif) ligand 2 (CCL2), interleukin-8, and tumor necrosis factor α in baseline SCLS sera compared with the control group. All patients are alive and well on prophylactic therapy, with 4 patients receiving intravenous or subcutaneous immunoglobulins at regular intervals. The clinical manifestations of pediatric and adult SCLS are similar, with the notable exceptions of frequent association with infections and the lack of monoclonal gammopathy. Prophylactic medication, including high dose immunoglobulins or theophylline plus verapamil, appears to be safe and efficacious therapy for SCLS in children.

Expansion of CD4(+) HLA-G(+) T Cell in human pregnancy is impaired in pre-eclampsia.

PROBLEM: The role of CD4(+) HLA-G(+) T cells in healthy pregnancy and pre-eclampsia is unclear. METHOD OF STUDY: CD4(+) HLA-G(+) T cells were analysed from peripheral blood and decidual samples from healthy pregnant and pre-eclamptic women. In vitro T-cell induction, trogocytosis and suppression assays were performed. RESULTS: In peripheral blood, CD4(+) HLA-G(+) T cells were significantly higher in pregnant women (mean ± S.E.M.: 7.98 ± 1.10%), compared with non-pregnant controls (mean ± S.E.M.: 1.78 ± 0.30%) and pre-eclamptic women (mean ± S.E.M.: 3.69 ± 0.51%). The presence of CD4(+) HLA-G(+) T cells is even more prominent in the decidua, suggestive of local induction and accumulation. Decidual CD14(+) DC-SIGN(+) antigen-presenting cells (APCs) enhance the HLA-G expression of cocultured CD4(+) naïve T cells in vitro. IL-10 augments expression of HLA-G, ILT4 and DC-SIGN in monocyte-derived DCs (MoDCs), endowing them with a phenotype analogous to decidual CD14(+) DC-SIGN(+) APCs of healthy pregnancy. Furthermore, naïve T cells acquire HLA-G from these IL-10-treated MoDCs via the process of trogocytosis. CONCLUSIONS: Our data indicate that in addition to Foxp3(+) Treg cells, CD4(+) T cells acquire HLA-G from decidual DCs and may play an important role in immune tolerance induction in pregnancy, a process which is impaired in pre-eclampsia.

Induction of a tumor-associated activating mutation in protein tyrosine phosphatase Ptpn11 (Shp2) enhances mitochondrial metabolism, leading to oxidative stress and senescence.

Activating mutations in Ptpn11 (Shp2), a protein tyrosine phosphatase involved in diverse cell signaling pathways, are associated with pediatric leukemias and solid tumors. However, the pathogenic effects of these mutations have not been fully characterized. Here, we report that induction of the Ptpn11(E76K/+) mutation, the most common and active Ptpn11 mutation found in leukemias and solid tumors, in primary mouse embryonic fibroblasts resulted in proliferative arrest and premature senescence. As a result, apoptosis was markedly increased. These cellular responses were accompanied and mediated by up-regulation of p53 and p21. Moreover, intracellular levels of reactive oxygen species (ROS), byproducts of mitochondrial oxidative phosphorylation, were elevated in Ptpn11(E76K/+) cells. Since Shp2 is also distributed to the mitochondria (in addition to the cytosol), the impact of the Ptpn11(E76K/+) mutation on mitochondrial function was analyzed. These analyses revealed that oxygen consumption of Ptpn11(E76K/+) cells and the respiratory function of Ptpn11(E76K/+) mitochondria were significantly increased. Furthermore, we found that phosphorylation of mitochondrial Stat3, one of the substrates of Shp2 phosphatase, was greatly decreased in the mutant cells with the activating mutation Ptpn11(E76K/+). This study provides novel insights into the initial effects of tumor-associated Ptpn11 mutations.