Issue #42: In the node - Part 2

Written by Nobel Laureate Professor Peter Doherty

Continuing from last week (#41), Jim Gowans went on to establish that his rat thoracic duct lymphocytes (TDLs) were indeed immune cells by showing that they caused graft-versus-host (GVH) disease – the donor T cells react against the foreign transplant antigens of the recipient – when transferred into mice, or into an MHC-different (#30) rat strain. After Jim moved on to become secretary of the UK Medical Research Council, the Oxford lymphology tradition was continued by his trainees, particularly Bill Ford who was, sadly, killed in a car crash when spending time as a visiting fellow in the ANU, John Curtin School of Medical Research (JCSMR) (#31) group led by Oxford trained lymphologist, Bede Morris. Tragically, the flamboyant and unforgettable Bede also died (1988) in the same way, in France.

Through the 1960s and 70s, Bede, a skilled surgeon, took the lymph node story forward by ‘going big’ and switching from rats to sheep, which were both cheap and in plentiful supply around Canberra. His group did extraordinary experiments cannulating, for example, the tiny lymph node efferent ducts, a study that would have been impossible in rats or mice. Then they drained lymph from sheep foetuses in utero, providing unique insights into the developing immune system. In general, while Bede’s program retained a strong physiology component, their work showed clearly that the lymph nodes are the primary site of immune responsiveness.

Bede was Professor of Immunology at the JCSMR by the time I came there (1972) to learn basic, mouse T cell immunology in Gordon Ada’s Microbiology Department (#32). Before that, working with sheep in Edinburgh, Hugh Reid and I had found evidence of local antibody production in the brains of sheep with encephalitis caused by the tick-borne louping-ill virus. My electron microscopy images of plasmablasts migrating through brain capillary endothelium looked just like those published by the Morris program.

Shortly after arriving in Canberra, I talked to Bede about this, but he wasn’t interested in getting into infectious disease. Still, he raised no objection when I later collaborated with virologist Ian Marshall, his PhD student Andrew Hapel and one of Bede’s postdocs, University of Colorado DVM PhD Len Pearson, to show that sheep injected with Ross River Virus (RRV) in the lower hind limb were, for as long as 12 days, pushing out 10-fold or higher numbers of large, activated lymphocytes in efferent lymph from the draining, popliteal lymph node. Furthermore, at least some of the plasmablasts (#18, #40) were making RRV-specific antibody (#19). I’d almost forgotten that study, which was published (1976) in a solid but minor journal, but it is a very nice piece of work!

At least from the aspect of an experimentalist (I’m not up on human imaging studies), the rest of the lymph node story is all derived from going small, from sheep to mice. The big advances to my mind were made from the latter half of the 1990s when it became possible for investigators to generate accurate numbers for responding lymphocytes of all types as a consequence of having better assay systems available. I’ll limit my account re quantitation to the CD8+ ‘killer’ T cells where we were (with other laboratories) closely involved, but this was also true for B cells and CD4+ T cells where researchers probed using ELISASPOT assays.

That stands for enzyme-linked immunosorbent spot assay: each ‘spot’ that was counted by an automated scanning microscope or a PhD student (depending on the equipment available) represents a single plasmablast secreting Igs (#20, #22) that bind to the relevant antigen – the immunosorbent – or, typically, a CD4+ helper T cell making the cytokine -interferon.

Live CD8+ cytotoxic T lymphocytes (CTLs) were stained with fluorochrome-labelled viral peptide + ‘self’ class I MHC glycoprotein (pMHC1) tetramers (#33) that could be counted, or separated as single cells – each delivered onto one well of a 96 well culture plate – using a fluorescence activated cell sorter (FACS). Over more than a decade of intense effort, experiments using the tetramer technology dominated the work of our SJCRH, then University of Melbourne programs. Major advances beyond just counting the cells and studying their distribution around the body came when Steve Turner introduced the single cell PCR technique for characterising T cell receptor (TCR) beta chain usage (#33), then cytokine gene expression. The TCR sequences ‘marked’ clonally-expanding T cell lineages, with the acuity of that analysis being enhanced when Pradyot Dash and Paul Thomas at SJCRH worked out how to further identify the TCR alpha chains in the same cells.

Focusing mainly on the influenza virus mouse pneumonia model we were able to show that the regional cervical and mediastinal lymph nodes are the primary site of clonal expansion for virus-specific CD8+ CTLs, with after an initial lag phase, these cells dividing every six hours or so. Furthermore, we found that their progressive differentiation to effector function as they start making mRNA for the granzymes, perforin and cytokines that mediate the virus-factory eliminating killer function (#34) is related to cell division. Progressively more of these molecules are expressed as each CTL matures to assassin status before, after about six or seven days, the first responders move out of the nodes and we suddenly find them in the virus-infected lung. Virus titres in the lung then drop rapidly until, by day 10-12 or so after infection, the pathogen is cleared and the process of repair begins.

The next major advance in our understanding of what happens in the nodes came when top researchers like immunologist Ron Germain at the National Institutes of Health (NIH), Bethesda, started to use a revolutionary technique called multi-photon dynamic images to ‘see into’ living tissues, including lymph nodes. If you have 50 minutes to spare, you can watch Ron describe his incisive experiments. The talk is directed at immunologists but, if the technical language is too much, turn off the sound and let the imaging and movies flow by as you sip some calming libation.

There’s still much to learn about what goes on in the lymph node. We know that B cell responses develop in organised germinal centres, but we’re not all that clear where, and how, the different cell populations get together so, for example, the CD4+ T cells can provide ‘help’ (#22) to both B cells and CD8+ CTLs. One colourful speculation is that the CD4+ helpers ‘zip around’ to ‘kiss’, perhaps repeatedly, their ‘partner’ of interest. With that hint of immuno-romance, we will, like the effector and memory T cells and B cells that exit in efferent lymph, leave our lymph node story for now.