Issue #34: Hitting the virus production factories

Setting it Straight - Issue #34

Written by Nobel Laureate Professor Peter Doherty

Now we finally come to the point of discussing how the cellular assassins of immunity, variously known as the ‘killer’ T cells or the CD8+ cytotoxic T lymphocytes (CTLs), do their job in a virus infection like COVID-19. All viruses are ‘obligate intracellular parasites’, meaning that they have to enter our body cells and use their small genomes to take over elements of the big genome that encodes the molecular machinery of us. The only ‘evolutionary aim’ of SARS-CoV-2 is to produce new virus particles (virions) that will ultimately infect other individuals and keep the pathogen in circulation. The more cells that are infected in our respiratory tract, the greater the amount of SARS-CoV-2 that they produce to be coughed and spluttered into the air around us. The CD8+ CTLs interrupt that pathway by killing these virus ‘factories.’ Think of that like an aerial bomb-hit that destroys both a weapons-production facility and any ‘product’ (virus) that hasn’t yet shipped out!

And, as we’ve discussed previously at length, the T cell receptors (TCRs) on our SARS-CoV-2 specific CTLs are specific for one of our very own MHC class I (MHC1) glycoproteins that has an 8-12 amino acid peptide (p) derived from a SARS-CoV-2 protein bound to it. These MHCI molecules are encoded at the HLA-A, B and C loci, so we have a maximum of six options, three from each parent. Each has the capacity to bind a different peptide from the SARS-CoV-2 virus and, as a consequence, present a panel of different pMHCI epitopes for recognition via the available TCR repertoire.

If you still find this confusing, think of the pMHC1 epitopes as a spectrum of coloured single, jellybeans (peptides) stuck in the middle of one of six different, but repeated, icing flowers (MHCI proteins) on the surface of a very elaborate and truly decadent, carbohydrate and cream-cardiac-lethal (virus-infected) kid’s birthday cake. A party game has led to each young (and relatively COVID-resistant) child getting a ‘ticket’ (TCR) that they redeem for a large slice of cake with a particular jellybean/flower complex attached. As, up until that stage, they’ve only been served celery sticks and carrots, they destroy that cake slice immediately and come back for an identical second helping. The kids save us (the adults) by totally eliminating an entity (left-over cream cake) that many should not, under any circumstances, consume!

The fact that the ‘antigen’ (or pMHCI epitope) is a modified cell-surface, transmembrane protein means that the CTLs are not ‘distracted’ by free virus particles floating in blood or mucus as they focus on their job of bringing the infectious process to an end by removing the source of virus production. Unlike the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the ‘target’ for neutralising antibodies (the obsession of vaccinologists), the peptides that form an immunogenic pMHCI epitope by binding to one or other of our MHCI molecules can come from any of the viral proteins. And some of these immunogenic peptides are shared by the common cold coronaviruses that regularly infect us. As a consequence, as many as 80 per cent of people may have some pre-existing, cross reactive T cell-memory to the totally new (to humans) SARS-CoV-2 pathogen. As we’ll discuss in more detail later, that will not prevent us being infected, but it could lead to more rapid recovery and may be part of the explanation for the great diversity in disease severity that we see in COVID-19.

In order for the CTLs to kill, they have to get ‘up close and personal’, then hang on. That intimacy is achieved by the repeated, identical TCRs on the lymphocytes binding to their multiple, ‘cognate’ (complementary) pMHCI epitopes on our virus-infected lung cells. What’s important here is, of course, that anchoring in the ‘deformable fluid-mosaic’ of the CTL (TCRs) and the target cell (pMHCIs) membranes acts to ‘cross-link’, or cluster, multiple TCR/pMHCI binding events and achieve sufficient ‘avidity’ to hold the murderer (the CTL) and the victim (the infected cell) together until the job is done.

Early on, the biochemists who were trying to ‘mark’ virus-specific CD8+CTLs using an isolated (in fluid phase and unattached to cell surface) pMHC1 molecular complex tagged with a fluorescent dye found that this did not attach tightly enough to its ‘cognate’ TCR for the T cells to stay labelled. In effect, the affinity of a single TCR/pMHC-1 binding event was just too low. That problem was solved by John Altman who, working with Mark Davis (one of the co-discoverers of the TCR) at Stanford University, combined the single pMHCI monomers into a tetramer to enable a quadrivalent TCR/p-MHCI interaction. This technical trick revolutionised the whole CTL field.

John’s tetramers bound to an individual T cell with sufficient avidity for the stained CTLs to be counted (or sorted) as, using a complex piece of laboratory equipment called a Flow Cytometer (or Fluorescence Activated Cell Sorter, FACS), they flowed in a fluid stream past a laser beam that caused the fluorescing (positive for p-MHCI tetramer binding) CTLs to be counted and, if desired, separated into ‘pots’ of stained and unstained cells. That allowed us both to measure the kinetics and magnitude of a virus specific CTL response (previous estimates were as much as 10 times too low) and, for example, to isolate a single virus specific CTL in blood taken from the arm of a person with COVID-19 for detailed molecular analysis. The FACS can deposit one such T cell into each well of a plastic, 96-well culture plate.

Apart from massively advancing the science of T cell-mediated immunity in both experimental animals and in people, what this illustrates is the total dependence of modern biomedical research on innovations made by physicists and engineers who design and build sophisticated equipment. Then, the power of such hardware is massively enhanced by advances in computing, both at the level of hardware and software. These themes will resurface from time to time as we further develop our contemporary understanding of infection and immunity.