For effective adaptive immunity, T lymphocytes must rapidly expand and contract in an antigen-spe... more For effective adaptive immunity, T lymphocytes must rapidly expand and contract in an antigen-specific manner to effectively control invading pathogens and preserve immunological memory, without sustaining excessive collateral damage to host tissues. Starting from initial antigen encounter, carefully calibrated programmed cell death pathways are critical for maintaining homeostasis over distinct phases of the T cell response. Restimulation-induced cell death (RICD), a self-regulatory apoptosis pathway triggered by re-engagement of the T cell receptor (TCR), is particularly important for constraining effector T cell expansion to preclude overt immunopathology; indeed, genetic disorders affecting key molecules involved in RICD execution can manifest in excessive lymphoproliferation, malignancy, and autoimmunity. Herein we review our current knowledge of how RICD sensitivity is ultimately regulated over the course of an immune response, including recent revelations on molecules that tu...
When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immun... more When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immune system relies upon clonal expansion of antigen-specific T cell populations to exercise acquired effector and cytotoxic functions to clear it. However, T cell expansion must be modulated to effectively combat the perceived threat without inducing excessive collateral damage to host tissues. Restimulation-induced cell death (RICD) is an apoptotic program triggered in activated T cells when an abundance of antigen and IL-2 are present, imposing a negative feedback mechanism that constrains the growing T cell population. This autoregulatory process can be detected via increases in caspase activation, Annexin V binding, and loss of mitochondrial membrane potential. However, simple changes in T cell viability through flow cytometric analysis can reliably measure RICD sensitivity in response to T-cell receptor (TCR) restimulation. This protocol describes the in vitro polyclonal activation, expansion, and restimulation of human primary T cells isolated from donor peripheral blood mononuclear cells (PBMC). This simple procedure allows for accurate quantification of RICD via flow cytometry. We also describe strategies for interrogating the role of specific proteins and pathways that may alter RICD sensitivity. This straightforward protocol provides a quick and dependable tool to track RICD sensitivity in culture over time while probing critical factors that control the magnitude and longevity of an adaptive immune response. Graphic abstract: In-vitro simulation of restimulation-induced cell death in activated human T cells.
Genetic testing has increased the number of variants identified in disease genes, but the diagnos... more Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a ''cloning-free'' saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.
In healthy individuals, the balance between immune-mediated pathogen clearance and tissue damage ... more In healthy individuals, the balance between immune-mediated pathogen clearance and tissue damage is tightly regulated through multiple processes. One such mechanism is restimulation-induced cell death (RICD), a propriocidal apoptotic mechanism that constrains T cell responses. Activated T cells must surpass a high threshold of TCR signal strength to induce pro-apoptotic genes responsible for executing RICD. Because co-receptors such as TIM-3 are thought to modulate TCR signal strength, we hypothesized that TIM-3 can shape the T cell response by tuning RICD sensitivity. We utilized siRNA knockdowns and transfection of wild-type and mutant TIM-3 expression constructs to assay for changes in RICD sensitivity in both immortalized and primary human T cells in vitro. Strikingly, we observed that the effect of altering TIM-3 expression varied temporally during the effector T cell response. Whereas TIM-3 promoted RICD resistance in newly activated T cells, TIM-3 enhanced RICD sensitivity in late-stage effector T cells by boosting TCR-induced FASL and BIM expression in a ligand-independent fashion. Preliminary evidence suggests that this dichotomy may relate to substantial temporal changes in the expression, cellular localization, and glycosylation of TIM-3 in early versus late-stage effector T cells. Overall, these results indicate that TIM-3 influences apoptosis sensitivity differently at distinct phases of the human T cell response, with important implications for the use of checkpoint blockade immunotherapies targeting TIM-3. Our findings may also help to clarify discrepancies in the literature regarding TIM-3 signaling and function in T cells.
CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differe... more CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differences in proliferative and protective capacity after infectious challenge. However, their relative sensitivity to apoptosis has been largely overlooked, despite the importance of programmed cell death in regulating effector T cell homeostasis. Here we demonstrate that primary human effector T cells derived from the CD8+ EM subset exhibit significantly higher sensitivity to cytokine withdrawal induced cell death (CWID), a critical intrinsic apoptosis program responsible for culling cells once an infection is cleared and interleukin-2 (IL-2) levels diminish. Interestingly, we found no differences in the expression of IL-2 or IL-2 receptor components in cells originating from either subset. Relative to CM-derived effectors, however, EM-derived T cells display more mitochondrial instability and greater basal caspase activation. Indeed, we found that heightened CWID sensitivity in EM-derived effectors is linked to higher expression of the pro-apoptotic Bcl-2 family protein BIM, both at steady state and with de novo induction following withdrawal of exogenous IL-2. These data point to “imprinted” differences in BIM protein regulation preserved between CD8+ CM and EM progeny that govern their relative sensitivity to CWID. These findings offer new insight into why CM-derived T cells display superior effector cell expansion and more persistent memory responses in vivo relative to EM-derived T cells, based on differential apoptosis sensitivity.
We recently characterized a novel, B-cell specific congenital lymphoproliferative disorder termed... more We recently characterized a novel, B-cell specific congenital lymphoproliferative disorder termed BENTA (B cell Expansion with NF-κB and T-cell Anergy). BENTA disease is caused by heterozygous, germline gain-of-function mutations in the lymphocyte-specific signaling protein CARD11, triggering constitutive NF-κB signaling. BENTA patients present not only with splenomegaly and remarkable polyclonal immature/naive B cell expansion, but also display signs of primary immunodeficiency, including reduced percentages of class-switched/memory B cells, low serum IgM and poor antibody responses to specific vaccines. Using purified naïve B cells from several BENTA patients, we discovered profound intrinsic defects in plasma cell differentiation and antibody secretion in vitro in response to various stimuli. Although BENTA B cells proliferated slightly more than healthy donor B cells in vitro, they failed to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells. Consistent with these defects, BENTA B cells exhibited low IgG secretion, and reduced induction of key proteins involved in class switching (IRF4) and late plasma cell commitment (XBP1). On the other hand, activated BENTA B cells exhibited an overwhelming survival advantage in culture, reflecting apoptosis resistance that may be linked to a CARD11-dependent, NF-κB-independent pathway. Together, these results provide new mechanistic insights into our understanding of both B cell lymphocytosis and humoral immunodeficiency in BENTA disease, which may emerge from impairments in cell death and differentiation programs normally triggered by B cell activation.
Background Approximately 10-20% of patients with critical COVID-19 harbor neutralizing autoantibo... more Background Approximately 10-20% of patients with critical COVID-19 harbor neutralizing autoantibodies (auto-Abs) that target type I interferons (IFN), a family of cytokines that induce critical innate immune defense mechanisms upon viral infection. Studies to date indicate that these auto-Abs are mostly detected in men over age 65. Methods We screened for type I IFN serum auto-Abs in sera collected < 21 days post-symptom onset in a subset of 103 COVID-19 inpatients and 24 outpatients drawn from a large prospective cohort study of SARS-CoV-2 infected patients enrolled across U.S. Military Treatment Facilities. The mean age of this n = 127 subset of study participants was 55.2 years (SD = 15.2 years, range 7.7 – 86.2 years), and 86/127 (67.7%) were male. Results Among those hospitalized 49/103 (47.6%) had severe COVID-19 (required at least high flow oxygen), and nine subjects died. We detected neutralizing auto-Abs against IFN-α, IFN-ω, or both, in four inpatients (3.9%, 8.2% of severe cases), with no auto-Abs detected in outpatients. Three of these patients were white males over the age of 62, all with multiple comorbidities; two of whom died and the third requiring high flow oxygen therapy. The fourth patient was a 36-year-old Hispanic female with a history of obesity who required mechanical ventilation during her admission for COVID-19. Conclusion These findings support the association between type I IFN auto-antibody production and life-threatening COVID-19. With further validation, reliable high-throughput screening for type I IFN auto-Abs may inform diagnosis, pathogenesis and treatment strategies for COVID-19, particularly in older males. Our finding of type I IFN auto-Ab production in a younger female prompts further study of this autoimmune phenotype in a broader population. Disclosures David A. Lindholm, MD, American Board of Internal Medicine (Individual(s) Involved: Self): Member of Auxiliary R&D Infectious Disease Item-Writer Task Force. No financial support received. No exam questions will be disclosed ., Other Financial or Material Support David Tribble, M.D., DrPH, Astra Zeneca (Other Financial or Material Support, HJF, in support of USU IDCRP, funded under a CRADA to augment the conduct of an unrelated Phase III COVID-19 vaccine trial sponsored by AstraZeneca as part of USG response (unrelated work)) Simon Pollett, MBBS, Astra Zeneca (Other Financial or Material Support, HJF, in support of USU IDCRP, funded under a CRADA to augment the conduct of an unrelated Phase III COVID-19 vaccine trial sponsored by AstraZeneca as part of USG response (unrelated work))
After initial stimulation with antigen and exposure to the growth cytokine interleukin-2, activat... more After initial stimulation with antigen and exposure to the growth cytokine interleukin-2, activated T lymphocytes become sensitized to apoptosis upon antigen restimulation through the T cell receptor. This self-regulatory, restimulation-induced cell death (RICD) program constrains the proliferative capacity of activated T cells to help prevent excessive T cell accumulation and associated immunopathology. Here we describe a simple FACS-based approach for measuring RICD sensitivity in activated human T cells following polyclonal restimulation in vitro. This procedure is a straightforward research and clinical diagnostic tool for assessing RICD sensitivity for T cells derived from normal donors and patients suffering from diseases causing dysregulated T cell homeostasis.
European journal of medicinal chemistry, Feb 1, 2019
As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used... more As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used an insilico approach based on chemical homology with the two commercially available DGKα inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKα in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-Iinked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKα activity is deregulated.
Current Opinion in Allergy and Clinical Immunology, Dec 1, 2015
Purpose of review-To present recent advances in the discovery and characterization of new immunod... more Purpose of review-To present recent advances in the discovery and characterization of new immunodeficiency disorders linked to gain-of-function (GOF) mutations in immune signaling molecules. Recent findings-In the past two years, extensive cellular and molecular studies have illuminated the root causes of pathogenesis for several new monogenic primary immunodefiency disorders (PIDs) linked to GOF mutations in signaling molecules. Here we discuss on two disorders (BENTA and APDS/PASLI) featuring shared clinical presentation (e.g. lymphoproliferation, selective antibody deficiencies, recurrent sinopulmonary infections). These findings highlight an emerging theme: both loss and gain-of-function mutations in key molecules can disrupt finely-tuned immunoreceptor signaling modalities, resulting in the dysregulation of lymphocyte differentiation and impaired adaptive immunity. Summary-Continued research on the molecular pathogenesis of PIDs defined by hyperactive signaling molecules will better distinguish these and related disorders, and pinpoint tailored therapeutic interventions for "retuning" the immune response in these patients.
The caspase recruitment domain family member 11 (CARD11 or CARMA1)-B cell CLL/lymphoma 10 (BCL10)... more The caspase recruitment domain family member 11 (CARD11 or CARMA1)-B cell CLL/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) [CBM] signalosome complex serves as a molecular bridge between cell surface antigen receptor signaling and the activation of the NF-κB, JNK, and mTORC1 signaling axes. This positions the CBM complex as a critical regulator of lymphocyte activation, proliferation, survival, and metabolism. Inborn errors in each of the CBM components have now been linked to a diverse group of human primary immunodeficiency diseases termed "CBM-opathies." Clinical manifestations range from severe combined immunodeficiency to selective B cell lymphocytosis, atopic disease, and specific humoral defects. This surprisingly broad spectrum of phenotypes underscores the importance of "tuning" CBM signaling to preserve immune homeostasis. Here, we review the distinct clinical and immunological phenotypes associated with human CBM complex mutations and introduce new avenues for targeted therapeutic intervention.
Antigen receptor (AgR)-induced NF-κB activation is a critical determinant of lymphocyte growth an... more Antigen receptor (AgR)-induced NF-κB activation is a critical determinant of lymphocyte growth and survival. However, constitutive NF-κB activity can drive excessive lymphoproliferation and oncogenesis. We recently described a novel human lymphoproliferative disorder designated as BENTA (B cell Expansion with NF-κB and T cell Anergy), caused by germline gain-of-function mutations in the lymphocyte-specific scaffolding protein CARD11. Similar, somatic CARD11 mutations are found in ~10% of activated B cell-type diffuse large B cell lymphomas (ABC-DLBCL). Normally, AgR ligation converts CARD11 into an active conformation that nucleates a large multi-protein signalosome incorporating BCL10, MALT1, and other components that ultimately facilitate activation of IκB kinase (IKK) and nuclear translocation of NF-κB. We have now identified four different heterozygous CARD11 missense mutations (E134G, G123S, G123D, C49Y) in affected patients. Similar to BENTA patient cells and DLBCL tumors, ectopic expression of each mutant triggers constitutive NF-κB activation in B and T cell lines without AgR stimulation. Using confocal microscopy, we now demonstrate that these CARD11 mutants form large, spontaneous perinuclear aggregates with a novel, shell-like substructure. While BCL10 is largely excluded, other signaling components such as MALT1 and phospho-IκB kinase (IKK) are found within or closely colocalized with CARD11 shells, suggesting these aggregates are sites of active signaling. Our results provide the first evidence that gain-of-function CARD11 mutants assemble into a compartmentalized, active signalosome with a distinct structural organization. Further imaging and biochemical analysis of these aggregates will provide new insights into aberrant CARD11 signaling in BENTA patient lymphocytes and DLBCL tumors harboring active CARD11 mutations. Citation Format: Jeffrey Richard Stinson, Andrew L. Snow. Spontaneous aggregation and novel signalosome organization of BENTA-associated gain-of-function CARD11 mutants in lymphocytes. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A56.
Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr vir... more Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on Bcells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp350 1-470). Tetrameric gp350 1-470 induced ~20-fold higher serum titers of gp350 1-470-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350 1-470. Further, epidermal immunization with plasmid DNA encoding gp350 1-470 tetramer induced 8-fold higher serum titers of gp350 1-470-specific IgG relative to monomer. Tetrameric gp350 1-470 binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350 1-470 had no effect on the gp350 1-470-specific IgG response in naïve mice, and resulted in suppressed gp350 1-470-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350 1-470 is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.
For effective adaptive immunity, T lymphocytes must rapidly expand and contract in an antigen-spe... more For effective adaptive immunity, T lymphocytes must rapidly expand and contract in an antigen-specific manner to effectively control invading pathogens and preserve immunological memory, without sustaining excessive collateral damage to host tissues. Starting from initial antigen encounter, carefully calibrated programmed cell death pathways are critical for maintaining homeostasis over distinct phases of the T cell response. Restimulation-induced cell death (RICD), a self-regulatory apoptosis pathway triggered by re-engagement of the T cell receptor (TCR), is particularly important for constraining effector T cell expansion to preclude overt immunopathology; indeed, genetic disorders affecting key molecules involved in RICD execution can manifest in excessive lymphoproliferation, malignancy, and autoimmunity. Herein we review our current knowledge of how RICD sensitivity is ultimately regulated over the course of an immune response, including recent revelations on molecules that tu...
When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immun... more When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immune system relies upon clonal expansion of antigen-specific T cell populations to exercise acquired effector and cytotoxic functions to clear it. However, T cell expansion must be modulated to effectively combat the perceived threat without inducing excessive collateral damage to host tissues. Restimulation-induced cell death (RICD) is an apoptotic program triggered in activated T cells when an abundance of antigen and IL-2 are present, imposing a negative feedback mechanism that constrains the growing T cell population. This autoregulatory process can be detected via increases in caspase activation, Annexin V binding, and loss of mitochondrial membrane potential. However, simple changes in T cell viability through flow cytometric analysis can reliably measure RICD sensitivity in response to T-cell receptor (TCR) restimulation. This protocol describes the in vitro polyclonal activation, expansion, and restimulation of human primary T cells isolated from donor peripheral blood mononuclear cells (PBMC). This simple procedure allows for accurate quantification of RICD via flow cytometry. We also describe strategies for interrogating the role of specific proteins and pathways that may alter RICD sensitivity. This straightforward protocol provides a quick and dependable tool to track RICD sensitivity in culture over time while probing critical factors that control the magnitude and longevity of an adaptive immune response. Graphic abstract: In-vitro simulation of restimulation-induced cell death in activated human T cells.
Genetic testing has increased the number of variants identified in disease genes, but the diagnos... more Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a ''cloning-free'' saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.
In healthy individuals, the balance between immune-mediated pathogen clearance and tissue damage ... more In healthy individuals, the balance between immune-mediated pathogen clearance and tissue damage is tightly regulated through multiple processes. One such mechanism is restimulation-induced cell death (RICD), a propriocidal apoptotic mechanism that constrains T cell responses. Activated T cells must surpass a high threshold of TCR signal strength to induce pro-apoptotic genes responsible for executing RICD. Because co-receptors such as TIM-3 are thought to modulate TCR signal strength, we hypothesized that TIM-3 can shape the T cell response by tuning RICD sensitivity. We utilized siRNA knockdowns and transfection of wild-type and mutant TIM-3 expression constructs to assay for changes in RICD sensitivity in both immortalized and primary human T cells in vitro. Strikingly, we observed that the effect of altering TIM-3 expression varied temporally during the effector T cell response. Whereas TIM-3 promoted RICD resistance in newly activated T cells, TIM-3 enhanced RICD sensitivity in late-stage effector T cells by boosting TCR-induced FASL and BIM expression in a ligand-independent fashion. Preliminary evidence suggests that this dichotomy may relate to substantial temporal changes in the expression, cellular localization, and glycosylation of TIM-3 in early versus late-stage effector T cells. Overall, these results indicate that TIM-3 influences apoptosis sensitivity differently at distinct phases of the human T cell response, with important implications for the use of checkpoint blockade immunotherapies targeting TIM-3. Our findings may also help to clarify discrepancies in the literature regarding TIM-3 signaling and function in T cells.
CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differe... more CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differences in proliferative and protective capacity after infectious challenge. However, their relative sensitivity to apoptosis has been largely overlooked, despite the importance of programmed cell death in regulating effector T cell homeostasis. Here we demonstrate that primary human effector T cells derived from the CD8+ EM subset exhibit significantly higher sensitivity to cytokine withdrawal induced cell death (CWID), a critical intrinsic apoptosis program responsible for culling cells once an infection is cleared and interleukin-2 (IL-2) levels diminish. Interestingly, we found no differences in the expression of IL-2 or IL-2 receptor components in cells originating from either subset. Relative to CM-derived effectors, however, EM-derived T cells display more mitochondrial instability and greater basal caspase activation. Indeed, we found that heightened CWID sensitivity in EM-derived effectors is linked to higher expression of the pro-apoptotic Bcl-2 family protein BIM, both at steady state and with de novo induction following withdrawal of exogenous IL-2. These data point to “imprinted” differences in BIM protein regulation preserved between CD8+ CM and EM progeny that govern their relative sensitivity to CWID. These findings offer new insight into why CM-derived T cells display superior effector cell expansion and more persistent memory responses in vivo relative to EM-derived T cells, based on differential apoptosis sensitivity.
We recently characterized a novel, B-cell specific congenital lymphoproliferative disorder termed... more We recently characterized a novel, B-cell specific congenital lymphoproliferative disorder termed BENTA (B cell Expansion with NF-κB and T-cell Anergy). BENTA disease is caused by heterozygous, germline gain-of-function mutations in the lymphocyte-specific signaling protein CARD11, triggering constitutive NF-κB signaling. BENTA patients present not only with splenomegaly and remarkable polyclonal immature/naive B cell expansion, but also display signs of primary immunodeficiency, including reduced percentages of class-switched/memory B cells, low serum IgM and poor antibody responses to specific vaccines. Using purified naïve B cells from several BENTA patients, we discovered profound intrinsic defects in plasma cell differentiation and antibody secretion in vitro in response to various stimuli. Although BENTA B cells proliferated slightly more than healthy donor B cells in vitro, they failed to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells. Consistent with these defects, BENTA B cells exhibited low IgG secretion, and reduced induction of key proteins involved in class switching (IRF4) and late plasma cell commitment (XBP1). On the other hand, activated BENTA B cells exhibited an overwhelming survival advantage in culture, reflecting apoptosis resistance that may be linked to a CARD11-dependent, NF-κB-independent pathway. Together, these results provide new mechanistic insights into our understanding of both B cell lymphocytosis and humoral immunodeficiency in BENTA disease, which may emerge from impairments in cell death and differentiation programs normally triggered by B cell activation.
Background Approximately 10-20% of patients with critical COVID-19 harbor neutralizing autoantibo... more Background Approximately 10-20% of patients with critical COVID-19 harbor neutralizing autoantibodies (auto-Abs) that target type I interferons (IFN), a family of cytokines that induce critical innate immune defense mechanisms upon viral infection. Studies to date indicate that these auto-Abs are mostly detected in men over age 65. Methods We screened for type I IFN serum auto-Abs in sera collected < 21 days post-symptom onset in a subset of 103 COVID-19 inpatients and 24 outpatients drawn from a large prospective cohort study of SARS-CoV-2 infected patients enrolled across U.S. Military Treatment Facilities. The mean age of this n = 127 subset of study participants was 55.2 years (SD = 15.2 years, range 7.7 – 86.2 years), and 86/127 (67.7%) were male. Results Among those hospitalized 49/103 (47.6%) had severe COVID-19 (required at least high flow oxygen), and nine subjects died. We detected neutralizing auto-Abs against IFN-α, IFN-ω, or both, in four inpatients (3.9%, 8.2% of severe cases), with no auto-Abs detected in outpatients. Three of these patients were white males over the age of 62, all with multiple comorbidities; two of whom died and the third requiring high flow oxygen therapy. The fourth patient was a 36-year-old Hispanic female with a history of obesity who required mechanical ventilation during her admission for COVID-19. Conclusion These findings support the association between type I IFN auto-antibody production and life-threatening COVID-19. With further validation, reliable high-throughput screening for type I IFN auto-Abs may inform diagnosis, pathogenesis and treatment strategies for COVID-19, particularly in older males. Our finding of type I IFN auto-Ab production in a younger female prompts further study of this autoimmune phenotype in a broader population. Disclosures David A. Lindholm, MD, American Board of Internal Medicine (Individual(s) Involved: Self): Member of Auxiliary R&D Infectious Disease Item-Writer Task Force. No financial support received. No exam questions will be disclosed ., Other Financial or Material Support David Tribble, M.D., DrPH, Astra Zeneca (Other Financial or Material Support, HJF, in support of USU IDCRP, funded under a CRADA to augment the conduct of an unrelated Phase III COVID-19 vaccine trial sponsored by AstraZeneca as part of USG response (unrelated work)) Simon Pollett, MBBS, Astra Zeneca (Other Financial or Material Support, HJF, in support of USU IDCRP, funded under a CRADA to augment the conduct of an unrelated Phase III COVID-19 vaccine trial sponsored by AstraZeneca as part of USG response (unrelated work))
After initial stimulation with antigen and exposure to the growth cytokine interleukin-2, activat... more After initial stimulation with antigen and exposure to the growth cytokine interleukin-2, activated T lymphocytes become sensitized to apoptosis upon antigen restimulation through the T cell receptor. This self-regulatory, restimulation-induced cell death (RICD) program constrains the proliferative capacity of activated T cells to help prevent excessive T cell accumulation and associated immunopathology. Here we describe a simple FACS-based approach for measuring RICD sensitivity in activated human T cells following polyclonal restimulation in vitro. This procedure is a straightforward research and clinical diagnostic tool for assessing RICD sensitivity for T cells derived from normal donors and patients suffering from diseases causing dysregulated T cell homeostasis.
European journal of medicinal chemistry, Feb 1, 2019
As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used... more As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used an insilico approach based on chemical homology with the two commercially available DGKα inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKα in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-Iinked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKα activity is deregulated.
Current Opinion in Allergy and Clinical Immunology, Dec 1, 2015
Purpose of review-To present recent advances in the discovery and characterization of new immunod... more Purpose of review-To present recent advances in the discovery and characterization of new immunodeficiency disorders linked to gain-of-function (GOF) mutations in immune signaling molecules. Recent findings-In the past two years, extensive cellular and molecular studies have illuminated the root causes of pathogenesis for several new monogenic primary immunodefiency disorders (PIDs) linked to GOF mutations in signaling molecules. Here we discuss on two disorders (BENTA and APDS/PASLI) featuring shared clinical presentation (e.g. lymphoproliferation, selective antibody deficiencies, recurrent sinopulmonary infections). These findings highlight an emerging theme: both loss and gain-of-function mutations in key molecules can disrupt finely-tuned immunoreceptor signaling modalities, resulting in the dysregulation of lymphocyte differentiation and impaired adaptive immunity. Summary-Continued research on the molecular pathogenesis of PIDs defined by hyperactive signaling molecules will better distinguish these and related disorders, and pinpoint tailored therapeutic interventions for "retuning" the immune response in these patients.
The caspase recruitment domain family member 11 (CARD11 or CARMA1)-B cell CLL/lymphoma 10 (BCL10)... more The caspase recruitment domain family member 11 (CARD11 or CARMA1)-B cell CLL/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) [CBM] signalosome complex serves as a molecular bridge between cell surface antigen receptor signaling and the activation of the NF-κB, JNK, and mTORC1 signaling axes. This positions the CBM complex as a critical regulator of lymphocyte activation, proliferation, survival, and metabolism. Inborn errors in each of the CBM components have now been linked to a diverse group of human primary immunodeficiency diseases termed "CBM-opathies." Clinical manifestations range from severe combined immunodeficiency to selective B cell lymphocytosis, atopic disease, and specific humoral defects. This surprisingly broad spectrum of phenotypes underscores the importance of "tuning" CBM signaling to preserve immune homeostasis. Here, we review the distinct clinical and immunological phenotypes associated with human CBM complex mutations and introduce new avenues for targeted therapeutic intervention.
Antigen receptor (AgR)-induced NF-κB activation is a critical determinant of lymphocyte growth an... more Antigen receptor (AgR)-induced NF-κB activation is a critical determinant of lymphocyte growth and survival. However, constitutive NF-κB activity can drive excessive lymphoproliferation and oncogenesis. We recently described a novel human lymphoproliferative disorder designated as BENTA (B cell Expansion with NF-κB and T cell Anergy), caused by germline gain-of-function mutations in the lymphocyte-specific scaffolding protein CARD11. Similar, somatic CARD11 mutations are found in ~10% of activated B cell-type diffuse large B cell lymphomas (ABC-DLBCL). Normally, AgR ligation converts CARD11 into an active conformation that nucleates a large multi-protein signalosome incorporating BCL10, MALT1, and other components that ultimately facilitate activation of IκB kinase (IKK) and nuclear translocation of NF-κB. We have now identified four different heterozygous CARD11 missense mutations (E134G, G123S, G123D, C49Y) in affected patients. Similar to BENTA patient cells and DLBCL tumors, ectopic expression of each mutant triggers constitutive NF-κB activation in B and T cell lines without AgR stimulation. Using confocal microscopy, we now demonstrate that these CARD11 mutants form large, spontaneous perinuclear aggregates with a novel, shell-like substructure. While BCL10 is largely excluded, other signaling components such as MALT1 and phospho-IκB kinase (IKK) are found within or closely colocalized with CARD11 shells, suggesting these aggregates are sites of active signaling. Our results provide the first evidence that gain-of-function CARD11 mutants assemble into a compartmentalized, active signalosome with a distinct structural organization. Further imaging and biochemical analysis of these aggregates will provide new insights into aberrant CARD11 signaling in BENTA patient lymphocytes and DLBCL tumors harboring active CARD11 mutations. Citation Format: Jeffrey Richard Stinson, Andrew L. Snow. Spontaneous aggregation and novel signalosome organization of BENTA-associated gain-of-function CARD11 mutants in lymphocytes. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A56.
Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr vir... more Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on Bcells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp350 1-470). Tetrameric gp350 1-470 induced ~20-fold higher serum titers of gp350 1-470-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350 1-470. Further, epidermal immunization with plasmid DNA encoding gp350 1-470 tetramer induced 8-fold higher serum titers of gp350 1-470-specific IgG relative to monomer. Tetrameric gp350 1-470 binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350 1-470 had no effect on the gp350 1-470-specific IgG response in naïve mice, and resulted in suppressed gp350 1-470-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350 1-470 is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.
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Papers by Andrew Snow