Cytotoxic T lymphocyte biology under the microscope

Cytotoxic T lymphocytes (CTLs) are essential agents of the adaptive immune system that seek out and destroy virally infected or cancerous cells. Incapacitate them and you become severely immunocompromised and riddled with tumours (à la prf1-/- mouse model); enhance them and you can beat previously unbeatable cancers (e.g., CAR T cell therapy). More than this though, they are absolutely fascinating little critters. It has been the long-standing goal of Prof Griffiths and her lab to understand the fundamental cell biology underpinning CTL function. Studying a specialized primary cell offers many advantages over working with the cell biology workhorses HeLa or HEK; but at the same time presents challenges. In addition to the unique cell biological insights on offer, studying CTLs also keeps the researcher in touching distance of clinical translatability and relevance. This thesis is an amalgamation of several projects I have undertaken over the past four years in the Griffiths lab, all aimed at understanding different aspects of CTL biology. Throughout these different projects, microscopy has played an important part. Seeing really is believing, and the versatility of microscopic approaches is hopefully on full display throughout this thesis. High resolution live microscopy of individual CTL:target conjugates is perhaps the pinnacle of what the Griffiths lab produces, but many of the more basic analyses of cell function still depend on microscopy, hence its centrality to this thesis. For example, migration assays involve tracking CTLs crawling at 20x magnification, and killing assays require imaging of fluorescent targets being killed over time at 4x magnification are both forms of live cell microscopy. In chapter 1, I introduce CTL biology, both in terms of cell biology and in the context of the adaptive immune response. Microscopy is a crucial technique for understanding much of this biology. I describe the wide array of microscopic imaging modalities as they have been applied to T cell biology. I then focus on the specific problem of imaging the TCR, especially in resting effector T cells (before secondary activation), since this is crucial for understanding the baseline localization of TCR molecules in the T cell membrane. Knowledge of TCR localization is critical for understanding how T cells activate so rapidly, specifically, and sensitively. I compare various imaging techniques that are correlated with particular results, revealing a methodological bias. For example, total internal reflection fluorescence microscopy, which illuminates the cell more brightly the closer it is to the imaging surface, biases detection of TCRs that are at the tips of membrane protrusions, leading researchers to believe that the cell is itself organizing TCRs to be enriched at these tips, when it may be an artifact of the illumination technique. I describe several of these potential biases and synthesize what I believe to be the most accurate interpretation of the available data so far. Chapter 3 introduces biophysics and the idea of forces at the cellular level. These play an important and emerging role in CTL function, highlighted by the fact that high-affinity molecular interactions between pMHC and TCR are not sufficient to drive T cell activation if the surface presenting pMHC is too soft. In this chapter, I present the development and optimization of a new fluorescence microscopy tool to study forces in CTLs. I describe "utrnP2A", which comprises a pair of fluorescence bioprobes based on two truncations of utrophin, an actin-binding protein. These probes bind to actin filaments of the cytoskeleton in a manner proportional to the tension/compression of the filament, since tension affects filament torsion, which allows for ratiometric fluorescence readout of regions of the cytoskeleton with different actin conformations. I implement these probes in CTLs and image CTLs at high spatiotemporal resolution to reveal biased actin conformation during migration on glass. I go on to combine these probes with traction force microscopy, which allows for direct measurements of forces exerted by cells upon the surface on which they are mounted. Via this combination, I confirm that compression-associated probe bias correlates with regions of force exertion by migrating cells. By treating migrating CTLs with various chemical inhibitors, the degree of involvement of key molecular drivers of cytoskeletal turnover and contractility, namely the Arp2/3 complex, formins, and nonmuscle myosin IIa, is determined. Finally, I turn to forces at the cytotoxic immune synapse by creating artificial synapses on traction force gels. This reveals dynamic changes in actin conformation here, which correlate once again with force exertion. I show that these correlates of cytoskeletal force are also present in real cytotoxic immune synapses with cancerous target cells. These data enhance our understanding of intracellular forces, actin conformations, and how they relate to force exertion by CTLs during migration and killing. In Chapter 4 I seek to investigate the role of CDC42. This protein is a master regulator of polarity in many cells, and is thus of key interest in CTLs, which are highly polarised cells. I discover the expression of an unexpected isoform of CDC42 in CTLs, previously only identified in brain tissue. This isoform localises differently to the ubiquitous isoform of CDC42 within the cell, which gives it a completely novel function in these cells that is critical for their ability to kill. Not only do CTLs express the brain isoform of CDC42, but the ubiquitous isoform seems to be unimportant for their function, which I show via use of patient-derived CTLs harboring a loss-of-function mutation in the ubiquitous isoform specifically. I show that the brain isoform of CDC42 is active on secretory lysosomes in CTLs, and controls their polarised secretion at the cytotoxic immune synapse by mediating dynein activation at secretory lysosomes in response to target cell recognition. Whilst attempting to study CDC42 function via CRISPR/Cas9-mediated knockout of Cdc42, I encounter the phenomenon of transcriptional adaptation, which forms a separate and interesting tangent within Chapter 4. Gene deletion of Cdc42 does not result in impaired function, as inhibition of CDC42 does, but rather increased function over time. I show that transcriptional changes are likely the cause of this, with many upregulated genes linked to improved CTL function. Whilst transcriptional adaptation prevents useful insight into the function of CDC42 from Cdc42-deleted CTLs, it still allows for knockout validation of the CDC42 inhibitor CASIN, which I show to be highly specific across a range of functional assays. Chapter 5 takes a step in the direction of clinical immunology, and presents data on the CTLs from two cohorts of patients with mutations in different genes that give rise to primary immunodeficiency, namely RAB27A and ITRP3. Rab27a is a small GTPase involved in the final steps of polarized secretion by CTLs into target cells. The CTLs from these patients are all unable to secrete in this manner, but some of the patients have CTLs that are still able to kill, implying other mechanisms of killing are at play. ITPR3 encodes the inositol trisphosphate receptor, which allows for calcium release from the endoplasmic reticulum in response to TCR activation. I characterise how point mutations in this receptor, present in patients with immunodeficiency, lead to lack of calcium homeostasis and an impaired calcium-derived signalling response. These data contributed to highly collaborative projects that seek to directly impact human patients, whilst also providing key cell biological insights. Finally, I summarize key findings and describe potential future work in chapter 6. As we add, little by little, to our understanding of CTL biology (a tiny fraction of it being within these pages), there is an expanding list of newly uncovered questions that microscopy and the Griffiths lab will surely play a key role in answering.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Rochussen, Adam

Advisor dc:contributor.advisor

• Griffiths, Gillian

• cell biology
• immunology
• T cell
• microscopy
• biophysics

Licence

• https://www.repository.cam.ac.uk/bitstreams/88254026-1fea-40bd-a174-7b10ebb63f35/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

Author Identifier

0000-0001-9439-1206

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/381496