Identifying regulators of cytotoxic T cell function through molecular and genetic screening

Cytotoxic T lymphocytes (CTL) are crucial components of the adaptive immune system that kill infected and tumourigenic cells. CTL killing requires focused secretion of cytotoxic compounds from lytic granules. This process is known as degranulation. In this study, I aimed to establish the CRISPR-Cas9 gene editing technology in primary T cells and to optimise screening approaches to identify regulators of CTL killing. The first half of the thesis focuses on primary mouse CTL. The CRISPR technology was successfully optimised in CTL using Cas9-ribonucleoprotein complexes resulting in efficient CRISPR-mediated loss of target proteins. Genes encoding known mediators of CTL cytotoxicity, $\it{Rab27a}$, $\it{Munc13-4}$ and $\it{Prf1}$, were targeted using CRISPR. The resulting samples were used to establish a flow cytometry-based assay that simultaneously measures CTL degranulation and target cell death. This assay enabled me to screen for mediators of CTL killing, while providing mechanistic insight by detecting degranulation. The screen was informed by a transcriptomic study that compared naive and effector CD8 T cells. 1803 significantly upregulated differentially expressed genes [log2(fold change)>2] were identified. Functional annotation analysis and literature research were used to select genes for the targeted CRISPR screen, which highlighted the importance of HIF-1$\alpha$ and NFIL3 in CTL killing. The second half of the thesis focuses on primary human CTL. The combined degranulation and killing assay was further validated using patient-derived CTL, indicating its potential as a diagnostic test. I showed that the assay is suitable for mid-sized screens using a library of 64 compounds targeting the NF-$\kappa$B signalling pathway. Further opportunities for increasing the scale of this screening technique are discussed. Finally, I successfully tested CRISPR using Cas9-ribonucleoprotein complexes in the human system. Additionally, stable Cas9 expression through lentiviral transduction was explored in primary CTL and related cell lines. This has the potential to allow selection of cells expressing the CRISPR machinery, providing a cleaner experimental system and the possibility of large-scale screening approaches. In summary, the techniques established in this thesis will be valuable for studying the genetics underlying CTL killing and the combined degranulation and killing assay furthermore shows great potential for diagnostic purposes.This PhD project was funded by the Medical Research Council and the Wellcome Sanger Institute

Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity.

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections

PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes.

How cytotoxic T lymphocytes (CTLs) sense T cell receptor (TCR) signaling in order to specialize an area of plasma membrane for granule secretion is not understood. Here, we demonstrate that immune synapse formation led to rapid localized changes in the phosphoinositide composition of the plasma membrane, both reducing phosphoinositide-4-phosphate (PI(4)P), PI(4,5)P2, and PI(3,4,5)P3 and increasing diacylglycerol (DAG) and PI(3,4)P2 within the first 2 min of synapse formation. These changes reduced negative charge across the synapse, triggering the release of electrostatically bound PIP5 kinases that are required to replenish PI(4,5)P2. As PI(4,5)P2 decreased, actin was depleted from the membrane, allowing secretion. Forced localization of PIP5Kβ across the synapse prevented actin depletion, blocking both centrosome docking and secretion. Thus, PIP5Ks act as molecular sensors of TCR activation, controlling actin recruitment across the synapse, ensuring exquisite co-ordination between TCR signaling and CTL secretion.Wellcome Trus

NFIL3 contributes to cytotoxic T lymphocyte-mediated killing.

Peer reviewed: TruePublication status: PublishedFunder: Bettencourt Schueller FoundationCytotoxic T lymphocytes (CTLs) are key effectors of the adaptive immune system that recognize and eliminate virally infected and cancerous cells. In naive CD8+ T cells, T-cell receptor (TCR) engagement drives a number of transcriptional, translational and proliferation changes over the course of hours and days leading to differentiation into CTLs. To gain a better insight into this mechanism, we compared the transcriptional profiles of naive CD8+ T cells to those of activated CTLs. To find new regulators of CTL function, we performed a selective clustered regularly interspaced short palindromic repeats (CRISPR) screen on upregulated genes and identified nuclear factor IL-3 (NFIL3) as a potential regulator of cytotoxicity. Although NFIL3 has established roles in several immune cells including natural killer, Treg, dendritic and CD4+ T cells, its function in CD8+ CTLs is less well understood. Using CRISPR/Cas9 editing, we found that removing NFIL3 in CTLs resulted in a marked decrease in cytotoxicity. We found that in CTLs lacking NFIL3 TCR-induced extracellular signal-regulated kinase phosphorylation, immune synapse formation and granule release were all intact while cytotoxicity was functionally impaired in vitro. Strikingly, NFIL3 controls the production of cytolytic proteins as well as effector cytokines. Thus, NFIL3 plays a cell intrinsic role in modulating cytolytic mechanisms in CTLs

NFIL3 contributes to cytotoxic T lymphocyte-mediated killing

Cytotoxic T lymphocytes (CTLs) are key effectors of the adaptive immune system that recognize and eliminate virally infected and cancerous cells. In naive CD8+ T cells, T-cell receptor (TCR) engagement drives a number of transcriptional, translational and proliferation changes over the course of hours and days leading to differentiation into CTLs. To gain a better insight into this mechanism, we compared the transcriptional profiles of naive CD8+ T cells to those of activated CTLs. To find new regulators of CTL function, we performed a selective clustered regularly interspaced short palindromic repeats (CRISPR) screen on upregulated genes and identified nuclear factor IL-3 (NFIL3) as a potential regulator of cytotoxicity. Although NFIL3 has established roles in several immune cells including natural killer, Treg, dendritic and CD4+ T cells, its function in CD8+ CTLs is less well understood. Using CRISPR/Cas9 editing, we found that removing NFIL3 in CTLs resulted in a marked decrease in cytotoxicity. We found that in CTLs lacking NFIL3 TCR-induced extracellular signal-regulated kinase phosphorylation, immune synapse formation and granule release were all intact while cytotoxicity was functionally impaired in vitro. Strikingly, NFIL3 controls the production of cytolytic proteins as well as effector cytokines. Thus, NFIL3 plays a cell intrinsic role in modulating cytolytic mechanisms in CTLs

Table S1 from NFIL3 contributes to cytotoxic T lymphocyte-mediated killing

Table showing the significantly differentially expressed genes between day 0 and day 7 CD8+ T cells determined by RNA-se

Table S2 from NFIL3 contributes to cytotoxic T lymphocyte-mediated killing

Table containing the HTSeq counts for all mouse genes (ENSEMBL v84) and ERCC spike in controls across all the samples used in differential expression analysis

Table S3: from NFIL3 contributes to cytotoxic T lymphocyte-mediated killing

Table showing the information of the samples submitted for RNA-sequencing

Supplementary Material from NFIL3 contributes to cytotoxic T lymphocyte-mediated killing

Figures S1, S2 and Table

Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections