Mechanisms of necroptosis in T cells

In caspase 8-deficient mouse T cells, necroptosis occurs via a Ripk3- and Ripk1-dependent pathway independent of autophagy and programmed necrosis

The role of caspase-8 in regulating dendritic cell activation during both homeostasis and chronic viral infection

Chapter I introduces the innate and adaptive immune systems, which serve as the context for my dissertation, as well as the protein of interest in both systems.Chapter II is a reprint of a study on which I was the second author. In this study, we investigated the role of caspase-8 in T cell survival. Caspase-8 deficient T cells were found to accumulate defectively in response to antigenic stimulation in vitro, and mice with caspase-8 deficient T cells (tCasp8-/- mice) were unable to mount a response to an acute viral infection. Inhibition of Ripk1 kinase was shown to rescue this accumulation defect in vitro, and this form of death was termed necroptosis due to its dependence upon Ripk1 and lack of shared morphological characteristics with apoptosis. However, it was unclear whether other proteins were also required for necroptosis. Both programmed necrosis (as defined by a requirement for cyclophilin D) and autophagy (as defined by a requirement for Atg7) had been proposed to play a role in T cell necroptosis. Ripk3 had also been shown to interact with Ripk1 and trigger programmed necrosis. We used genetic studies to show that while neither cyclophilin D nor Atg7 could rescue the accumulation defect of caspase-8 deficient T cells, Ripk3 did. Only mice with caspase-8 deficient T cells and an additional loss of Ripk3 (DKO mice) were able to mount a response to an acute viral infection. We also found that DKO mice developed a lymphoproliferative disease with similarities to lpr mice, which have a lupus-like autoimmune disease.Chapter III examines the role of caspase-8 in another type of immune cell, dendritic cells (DCs). Caspase-8 was shown to negatively regulate the Rig-I innate immune signaling pathway, which produces type I interferons in response to RNA viruses. I found that mice with caspase-8 deficient DCs (dcCasp8-/- mice) develop an age-dependent autoimmunity characterized by hyperactivated DCs and T cells, organ immunopathology, and helper T cells that skew towards a Th1 phenotype. Since DC activation is considered required for a robust T cell response to viral infection, we next infected dcCasp8-/- mice with a chronic virus and found that they had an enhanced virus-specific T cell response characterized by less exhausted T cells and lower viral loads. Caspase-8 deficient DCs appeared to hyperactivate in response to Rig-I stimulation, which was likely dependent on endogenous activation of both IRF3 and NF-kB in the absence of caspase-8

The Effects of Dendritic Cell Hypersensitivity on Persistent Viral Infection.

Caspase-8 (CASP8) is known as an executioner of apoptosis, but more recent studies have shown that it participates in the regulation of necroptosis and innate immunity. In this study, we show that CASP8 negatively regulates retinoic acid-inducible gene I (RIG-I) signaling such that, in its absence, stimulation of the RIG-I pathway in dendritic cells (DCs) produced modestly enhanced activation of IFN regulatory factor 3 with correspondingly greater amounts of proinflammatory cytokines. In addition, mice lacking DC-specific CASP8 (dcCasp8-/- mice) develop age-dependent symptoms of autoimmune disease characterized by hyperactive DCs and T cells, spleen and liver immunopathology, and the appearance of Th1-polarized CD4+ T cells. Such mice infected with chronic lymphocytic choriomeningitis virus, an RNA virus detected by RIG-I, mounted an enhanced lymphocytic choriomeningitis virus-specific immune response as measured by increased proportions of Ag-specific CD4+ T cells and multicytokine-producing CD4+ and CD8+ T cells. These results show that CASP8 subtly modulates DC maturation, which controls the spontaneous appearance of autoimmune T cells while simultaneously attenuating the acquired immune system and its potential to control a persistent viral infection

The Effects of Dendritic Cell Hypersensitivity on Persistent Viral Infection

Caspase-8 (CASP8) is known as an executioner of apoptosis, but more recent studies have shown that it participates in the regulation of necroptosis and innate immunity. In this study, we show that CASP8 negatively regulates retinoic acid-inducible gene I (RIG-I) signaling such that, in its absence, stimulation of the RIG-I pathway in dendritic cells (DCs) produced modestly enhanced activation of IFN regulatory factor 3 with correspondingly greater amounts of proinflammatory cytokines. In addition, mice lacking DC-specific CASP8 (dcCasp8-/- mice) develop age-dependent symptoms of autoimmune disease characterized by hyperactive DCs and T cells, spleen and liver immunopathology, and the appearance of Th1-polarized CD4+ T cells. Such mice infected with chronic lymphocytic choriomeningitis virus, an RNA virus detected by RIG-I, mounted an enhanced lymphocytic choriomeningitis virus-specific immune response as measured by increased proportions of Ag-specific CD4+ T cells and multicytokine-producing CD4+ and CD8+ T cells. These results show that CASP8 subtly modulates DC maturation, which controls the spontaneous appearance of autoimmune T cells while simultaneously attenuating the acquired immune system and its potential to control a persistent viral infection

Neoadjuvant Pembrolizumab and High-Dose IFNα-2b in Resectable Regionally Advanced Melanoma

PurposeNeoadjuvant immunotherapy may improve the clinical outcome of regionally advanced operable melanoma and allows for rapid clinical and pathologic assessment of response. We examined neoadjuvant pembrolizumab and high-dose IFNα-2b (HDI) therapy in patients with resectable advanced melanoma.Patients and methodsPatients with resectable stage III/IV melanoma were treated with concurrent pembrolizumab 200 mg i.v. every 3 weeks and HDI 20 MU/m2/day i.v., 5 days per week for 4 weeks, then 10 MU/m2/day subcutaneously 3 days per week for 2 weeks. Definitive surgery followed, as did adjuvant combination immunotherapy, completing a year of treatment. Primary endpoint was safety of the combination. Secondary endpoints included overall response rate (ORR), pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS). Blood samples for correlative studies were collected throughout. Tumor tissue was assessed by IHC and flow cytometry at baseline and at surgery.ResultsA total of 31 patients were enrolled, and 30 were evaluable. At data cutoff (October 2, 2019), median follow-up for OS was 37.87 months (range, 33.2-43.47). Median OS and RFS were not reached. Radiographic ORR was 73.3% [95% confidence interval (CI): 55.5-85.8], with a 43% (95% CI: 27.3-60.1) pCR rate. None of the patients with a pCR have had a recurrence. HDI and pembrolizumab were discontinued in 73% and 43% of patients, respectively. Correlative analyses suggested that intratumoral PD-1/PD-L1 interaction and HLA-DR expression are associated with pCR (P = 0.002 and P = 0.008, respectively).ConclusionsNeoadjuvant concurrent HDI and pembrolizumab demonstrated promising clinical activity despite high rates of treatment discontinuation. pCR is a prognostic indicator.See related commentary by Menzies et al., p. 4133

Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer

<p>TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOG, we analyzed similar to 480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 x 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 x 10(-8)) and BRCA1 mutation carrier (P = 1.1 x 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 x 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 x 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 x 10(-12)) and BRCA1 mutation carrier (P = 1.6 x 10-14) breast and invasive ovarian (P = 1.3 x 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.</p>