Differential Requirement for the Cell Wall Integrity Sensor Wsc1p in Diploids Versus Haploids.

The primary role of the Cell Wall Integrity Pathway (CWI) in Saccharomyces cerevisiae is monitoring the state of the cell wall in response to general life cycle stresses (growth and mating) and imposed stresses (temperature changes and chemicals). Of the five mechanosensor proteins monitoring cell wall stress, Wsc1p and Mid2p are the most important. We find that WSC1 has a stringent requirement in zygotes and diploids, unlike haploids, and differing from MID2's role in shmoos. Diploids lacking WSC1 die frequently, independent of mating type. Death is due to loss of cell wall and plasma membrane integrity, which is suppressed by osmotic support. Overexpression of several CWI pathway components suppress wsc1∆ zygotic death, including WSC2, WSC3, and BEM2, as well as the Rho-GAPS, BEM3 and RGD2. Microscopic observations and suppression by BEM2 and BEM3 suggest that wsc1∆ zygotes die during bud emergence. Downstream in the CWI pathway, overexpression of a hyperactive protein kinase C (Pkc1p-R398P) causes growth arrest, and blocks the pheromone response. With moderate levels of Pkc1p-R398P, cells form zygotes and the wsc1∆ defect is suppressed. This work highlights functional differences in the requirement for Wsc1p in diploids Versus haploids and between Mid2p and Wsc1p during mating

Mitochondrial translation is required for sustained killing by cytotoxic T cells.

T cell receptor activation of naïve CD8+ T lymphocytes initiates their maturation into effector cytotoxic T lymphocytes (CTLs), which can kill cancer and virally infected cells. Although CTLs show an increased reliance on glycolysis upon acquisition of effector function, we found an essential requirement for mitochondria in target cell–killing. Acute mitochondrial depletion in USP30 (ubiquitin carboxyl-terminal hydrolase 30)–deficient CTLs markedly diminished killing capacity, although motility, signaling, and secretion were all intact. Unexpectedly, the mitochondrial requirement was linked to mitochondrial translation, inhibition of which impaired CTL killing. Impaired mitochondrial translation triggered attenuated cytosolic translation, precluded replenishment of secreted killing effectors, and reduced the capacity of CTLs to carry out sustained killing. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing

Mitochondrial regulation of CD8+ T cell cytotoxicity

Recent discoveries in the field of immunometabolism have emphasised the importance of mitochondria in the context of T cell development, differentiation, signalling and exhaustion. However, the question of whether mitochondria can actively participate to the killing activity of differentiated, cytotoxic CD8+ T cells (CTLs) remains unanswered. In this work I analyse CTLs derived from USP30-deficient mice, which have been previously characterised as part of the Infection, Immunity and Immunophenotype Consortium and shown to display altered CTL killing. USP30 is a deubiquitinase localised on the outer mitochondrial membrane and on the peroxisomal membrane, where it counteracts mitophagy and pexophagy by cleaving ubiquitin chains from target proteins. Here I characterise T cell motility, signalling, energy requirements and cytotoxicity in CD8+ T cells derived from splenocytes of Usp30-/- mice. Furthermore, I test different mitochondrial functions using both genetic and pharmacological manipulation of oxidative phosphorylation, mitochondrial calcium flux and mitochondrial translation. I highlight mitochondrial translation as a previously uncharacterised mitochondrial function that allows for optimal CTL cytotoxicity. Specifically, I show that both Usp30-/- CTLs and CTLs in which mitochondrial translation is inhibited display impaired cytosolic protein synthesis, which results in lower abundance of cytolytic molecules essential for killing. This requirement was especially evident when CTLs were limiting in numbers or when they needed to sustain killing for prolonged time, underscoring the importance of efficient mitochondrial translation during an immune challenge. Finally, I investigate mechanisms that could link mitochondrial translation to a loss of cytosolic protein synthesis, including mTOR signalling, mitochondrial reactive oxygen species generation and the integrated stress response. Overall, this work uncovers a novel role for mitochondrial translation in CTL cytotoxicity and adds to the growing body of evidence revealing the multifaceted and crucial roles of mitochondria in T cell function.CIMR PhD programme (Miriam Lisci). Wellcome Trust grants: 10390, 10040, 217100 (Gillian M. Griffiths); 220543 (Claire Y. Ma); 100156 (www.immunophenotype.org); 205023 (Doreen A. Cantrell). Medical Research Council: MC_UU_0015/7 (Julien Prudent). Australian National Health and Medical Research Council CJ Martin early Career Fellowship (Julia M. Marchingo)

Arming a killer: mitochondrial regulation of CD8+ T cell cytotoxicity

While once regarded as ATP factories, mitochondria have taken the spotlight as important regulators of cellular homeostasis. The last two decades have witnessed an intensifying interest in the study of mitochondria in cells of the immune system, with many new and unexpected roles for mitochondria emerging. Immune cells offer intriguing insights as mitochondria appear to play different roles at different stages of T cell development, matching the changing functions of the cells. Here we briefly review the multifaceted roles of mitochondria during T cell differentiation, focusing on CD8+ cytotoxic T lymphocytes (CTLs) and we consider how mitochondrial dysfunction can contribute to CTL exhaustion. In addition, we highlight a newly appreciated role for mitochondria as homeostatic regulators of CTL-mediated killing and explore the emerging literature describing mechanisms linking cytosolic and mitochondrial protein synthesis

Differential Requirement for the Cell Wall Integrity Sensor Wsc1p in Diploids Versus Haploids

The primary role of the Cell Wall Integrity Pathway (CWI) in Saccharomyces cerevisiae is monitoring the state of the cell wall in response to general life cycle stresses (growth and mating) and imposed stresses (temperature changes and chemicals). Of the five mechanosensor proteins monitoring cell wall stress, Wsc1p and Mid2p are the most important. We find that WSC1 has a stringent requirement in zygotes and diploids, unlike haploids, and differing from MID2’s role in shmoos. Diploids lacking WSC1 die frequently, independent of mating type. Death is due to loss of cell wall and plasma membrane integrity, which is suppressed by osmotic support. Overexpression of several CWI pathway components suppress wsc1∆ zygotic death, including WSC2, WSC3, and BEM2, as well as the Rho-GAPS, BEM3 and RGD2. Microscopic observations and suppression by BEM2 and BEM3 suggest that wsc1∆ zygotes die during bud emergence. Downstream in the CWI pathway, overexpression of a hyperactive protein kinase C (Pkc1p-R398P) causes growth arrest, and blocks the pheromone response. With moderate levels of Pkc1p-R398P, cells form zygotes and the wsc1∆ defect is suppressed. This work highlights functional differences in the requirement for Wsc1p in diploids Versus haploids and between Mid2p and Wsc1p during mating

Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A).

Peer reviewed: TrueINTRODUCTION: Inborn errors of immunity (IEI) are characterized by a dysfunction of the immune system leading to increased susceptibility to infections, impaired immune regulation and cancer. We present a unique consanguineous family with a history of Hodgkin lymphoma, impaired EBV control and a late onset hemophagocytic lymphohistiocytosis (HLH). METHODS AND RESULTS: Overall, family members presented with variable impairment of NK cell and cytotoxic T cell degranulation and cytotoxicity. Exome sequencing identified homozygous variants in RAB27A, FBP1 (Fructose-1,6-bisphosphatase 1) and ACAD9 (Acyl-CoA dehydrogenase family member 9). Variants in RAB27A lead to Griscelli syndrome type 2, hypopigmentation and HLH predisposition. DISCUSSION: Lymphoma is frequently seen in patients with hypomorphic mutations of genes predisposing to HLH. We hypothesize that the variants in FBP1 and ACAD9 might aggravate the clinical and immune phenotype, influence serial killing and lytic granule polarization by CD8 T cells. Understanding of the interplay between the multiple variants identified by whole exome sequencing (WES) is essential for correct interpretation of the immune phenotype and important for critical treatment decisions

Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A)

Inborn errors of immunity (IEI) are characterized by a dysfunction of the immune system leading to increased susceptibility to infections, impaired immune regulation and cancer. We present a unique consanguineous family with a history of Hodgkin lymphoma, impaired EBV control and a late onset hemophagocytic lymphohistiocytosis (HLH). Overall, family members presented with variable impairment of NK cell and cytotoxic T cell degranulation and cytotoxicity. Exome sequencing identified homozygous variants in RAB27A, FBP1 (Fructose-1,6-bisphosphatase 1) and ACAD9 (Acyl-CoA dehydrogenase family member 9). Variants in RAB27A lead to Griscelli syndrome type 2, hypopigmentation and HLH predisposition. Lymphoma is frequently seen in patients with hypomorphic mutations of genes predisposing to HLH. We hypothesize that the variants in FBP1 and ACAD9 might aggravate the clinical and immune phenotype, influence serial killing and lytic granule polarization by CD8 T cells. Understanding of the interplay between the multiple variants identified by whole exome sequencing (WES) is essential for correct interpretation of the immune phenotype and important for critical treatment decisions