Oscar Silva, MD

Abstract

Classic Hodgkin lymphoma (cHL) in young children (ages 0-6) is rare in high income countries (HICs) but is more prevalent in low- and middle-income countries (LMICs) like Guatemala. Given that the majority of cHL studies have evaluated adolescent/adults, and the immune system changes with age, we sought to characterize Epstein-Barr virus (EBV) expression, immune regulatory pathway markers and the tumor microenvironment in 42 children ages 0-6 with cHL from Guatemala. We found a very high frequency of EBV expression (97.5%). Hodgkin cells showed increased expression of PD1 ligands and CD137, indicative of shared immune regulatory mechanisms with adult cHL. Pediatric cHL also showed an increase in CD8+ tumor infiltrating lymphocytes and tumor associated macrophages within the tumor microenvironment. Despite 25 having high risk disease, only 4 patients died from progressive disease, relapse or infection.

View details for DOI 10.1080/10428194.2021.1885666

View details for PubMedID 33627023

View details for PubMedID 30819837

View details for PubMedID 29866887

View details for Web of Science ID 000393724400565

Abstract

Functionally diverse CD8+ T cells develop in response to antigenic stimulation with differing capacities to couple TCR engagement to downstream signals and functions. However, mechanisms of diversifying TCR signaling are largely uncharacterized. Here we identified two alternative splice variants of scaffold protein Dlg1, Dlg1AB and Dlg1B, that diversify signaling to regulate p38-dependent and -independent effector functions in CD8+ T cells. Dlg1AB, but not Dlg1B associated with Lck, coupling TCR stimulation to p38 activation and proinflammatory cytokine production. Conversely, both Dlg1AB and Dlg1B mediated p38-independent degranulation. Degranulation depended on a Dlg1 fragment containing an intact Dlg1SH3-domain and required the SH3-ligand WASp. Further, Dlg1 controlled WASp activation by promoting TCR-triggered conformational opening of WASp. Collectively, our data support a model where Dlg1 regulates p38-dependent proinflammatory cytokine production and p38-independent cytotoxic granule release through the utilization of alternative splice variants, providing a mechanism whereby TCR engagement couples downstream signals to unique effector functions in CD8+ T cells.

View details for DOI 10.1371/journal.pone.0133353

View details for Web of Science ID 000358198700128

View details for PubMedID 26186728

Abstract

CD8(+) T cells respond to TCR stimulation by producing proinflammatory cytokines, and destroying infected or malignant cells through the production and release of cytotoxic granules. Scaffold protein Discs large homolog 1 (Dlg1) specifies TCR-dependent functions by channeling proximal signals toward the activation of p38-dependent proinflammatory cytokine gene expression and/or p38-independent cytotoxic granule release. Two Dlg1 variants are expressed in CD8(+) T cells via alternative splicing, Dlg1AB and Dlg1B, which have differing abilities coordinate TCR-dependent functions. Although both variants facilitate p38-independent cytotoxicity, only Dlg1AB coordinates p38-dependent proinflammatory cytokine expression. In this study, we identify TCR-induced Dlg1 tyrosine phosphorylation as a key regulatory step required for Dlg1AB-mediated p38-dependent functions, including proinflammatory cytokine expression. We find that Dlg1AB but not Dlg1B is tyrosine phosphorylated by proximal tyrosine kinase Lck in response to TCR stimulation. Furthermore, we identify Dlg1 tyrosine 222 (Y222) as a major site of Dlg1 phosphorylation required for TCR-triggered p38 activation and NFAT-dependent expression of proinflammatory cytokines, but not for p38-independent cytotoxicity. Taken together, our data support a model where TCR-induced phosphorylation of Dlg1 Y222 is a key point of control that endows Dlg1AB with the ability to coordinate p38 activation and proinflammatory cytokine production. We propose blocking Dlg1AB phosphorylation as a novel therapeutic target to specifically block proinflammatory cytokine production but not cytotoxicity.

View details for DOI 10.4049/jimmunol.1401196

View details for Web of Science ID 000341859700005

View details for PubMedID 25098293

Abstract

Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.

View details for DOI 10.1126/scitranslmed.3005054

View details for Web of Science ID 000312393900004

View details for PubMedID 23241744

Abstract

The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear.Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1 germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur.These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.

View details for DOI 10.1371/journal.pone.0045276

View details for Web of Science ID 000311313900112

View details for PubMedID 23028902

Abstract

TCR engagement triggers the polarized recruitment of membrane, actin, and transducer assemblies within the T cell-APC contact that amplify and specify signaling cascades and T effector activity. We report that caveolin-1, a scaffold that regulates polarity and signaling in nonlymphoid cells, is required for optimal TCR-induced actin polymerization, synaptic membrane raft polarity, and function in CD8, but not CD4, T cells. In CD8(+) T cells, caveolin-1 ablation selectively impaired TCR-induced NFAT-dependent NFATc1 and cytokine gene expression, whereas caveolin-1 re-expression promoted NFATc1 gene expression. Alternatively, caveolin-1 ablation did not affect TCR-induced NF-B-dependent Ib expression. Cav-1(-/-) mice did not efficiently promote CD8 immunity to lymphocytic choriomeningitis virus, nor did cav-1(-/-) OT-1(+) CD8(+) T cells efficiently respond to Listeria monocytogenes-OVA after transfer into wild-type hosts. Therefore, caveolin-1 is a T cell-intrinsic orchestrator of TCR-mediated membrane polarity and signal specificity selectively employed by CD8 T cells to customize TCR responsiveness.

View details for DOI 10.4049/jimmunol.1101447

View details for Web of Science ID 000295034200018

View details for PubMedID 21849673