By regfbec, on 19 January 2014
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Welcome to this new blog where I propose to write occasional pieces related to immunology in general, and quantitative immunology in particular. I hope you will feel free to post REPLIES, COMMENTS and ESPECIALLY CRITICISMS. The blog is meant to be an informal place to share ideas and perhaps results which are still not sufficiently well formed, complete or formal enough to merit publication through the usual peer-reviewed channels. I therefore rely on my readership, if they exist, to correct errors which will undoubtedly creep in !
I would like to start with a few thoughts on cell numbers. In conversation with Yaron Antebi, then a postdoc in Nir Freidman’s lab at the Weizmann Institute (http://www.weizmann.ac.il/immunology/NirFriedman/) where I was spending a Sabbatical a few years ago (and where I am again now, writing this), we agreed that the immunology literature, though vast, is remarkably number free. We needed a site like those collecting the physical constants of the universe (see for example http://physics.nist.gov/cuu/Constants/index.html) , but for immunologists. Perhaps there are no immunological constants and the quest for a quantitative immunology is a mirage. But for the moment, let us consider the question of cell numbers.
One of the basic experimental protocols of a cellular immunologist is to take cells from blood, spleen, lymph node or some other organ and culture these cells in vitro, in the presence of an appropriate stimulus such as an antigen, an antibody or a cytokine and measure some response of the cells: proliferation, cytokine production, change in surface phenotype etc. But how many cells should one culture, at what density and how might these relate to the situation one might observe in lymphoid tissue for example? In the section following, I focus on T cells.
Of course, lymph nodes vary widely in size. But as an approximation, lymph nodes from an immunised mouse typically have a diameter in the order of 1 mm, equivalent to a volume of about 0.5 µl. The number of T cells in one such lymph node is in the order of 5×105. So the density, or concentration of T cells in a lymph node is very high: in the order of 109 per ml! Such concentrations are never even remotely achieved by conventional in vitro culture. However, consider now the concentration of antigen specific cells that might be found in vivo. Precursor numbers for a naïve antigen (as measured so elegantly by Marc Jenkins for example (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334329/) are probably in the order of 10-6 (1 cell in a million). Following immunisation, these frequencies rise dramatically, reaching typically 10-4 for CD4 T cells, and as high as 10-3, or even 10-2 for CD8 cells.
Now the numbers make a lot more sense ! The concentration of naïve T cells in a lymph node specific for a particular antigen approximates to around 103 /ml (=109/106). Considered in relation to in vitro experiments, these are very low concentrations, and cytokine production in the supernatant at these cell numbers is negligible. But if an antigen-specific response comprises 0.1% of the T cell population (a high, but not unrealistic number for the peak of a response), the concentration of such antigen specific cells in the lymph node rises to 106/ml (=109/103). In the context of in vitro experiments, this is usually towards the top end of the experimental range, and stimulation of cells at this concentration can results in high levels of cytokines in the culture supernatant.
So it turns out that the range of typical in vitro cell concentrations (104-106/ml) correspond rather nicely to the range of cell concentrations which might occur in vivo in a lymph node. These calculations are based on mouse immunology. But since cell size does not scale between organisms significantly, the number of cells in a lymph node will probably scale with the volume of the lymph node. So similar considerations probably hold in a human lymph node as well.
The relationship between these cell numbers and cytokine concentrations in a lymph node is a bit more difficult to establish. Diffusion over a range of 1 mm is going to be very fast, and probably not limiting. Cytokines are remarkably stable molecules, and spontaneous breakdown is unlikely to be significant either. Lymphatic flow will of course dramatically increase the effective volume in which the cytokine is diluted. A recent attempt to quantify lymph flow in mice using flourescent imaging (http://ajpheart.physiology.org/content/302/2/H391) suggested a rate of approximately 3 µl/min, suggesting the whole lymph node fluid volume is turned over 3 times per minute ! So cytokines produced under these conditions will rarely be able to accumulate to significant concentrations. However, after an immune response lymph flow is rapidly decreased, potentially allowing cytokine concentrations within a lymph node to rise to active levels. An additional complexity is the possibility of high local concentrations at the site of cytokine secretion within the immunological synapse, although the extent to which the synapse structure prevents cytokine diffusion remains debatable. It seems not unreasonable that, at the peak of a response, some cytokine levels may rise above their activity threshold globally throughout a lymph node. This could have important implications in terms of bystander activation, antigen linkage and cellular cooperation.
To conclude, a rough and ready estimate of cell numbers and tissue volumes suggests that total cell concentrations in lymphoid tissue is extremely high. It is not surprising that polyclonal activation of cells under these conditions results in pathological cytokine storms. And, reassuringly, culturing cells at concentrations similar to those of naïve precursors results in little or no measurable cytokine release. On the other hand, stimulating cells in culture at concentrations which mirror those which exist in vivo after immunisation result in biologically active levels of cytokines.
Finally, and somewhat paradoxically, in vitro experiments in which cells respond to polyclonal activation (e.g. via anti-TcR antibodies) or experiments using monoclonal TcR transgenic T cells all responding to the same epitope, capture rather well the range of cell concentrations likely to exist in lymphoid tissue in vivo. But more “realistic” in vitro models looking at antigen specific responses within polyclonal T cell populations (classical recall antigen responses measured in human PBMC for example) will enormously underestimate the real T cell concentrations in vivo. It seems that studying responses of antigen specific responses in vitro will require more sophisticated models such as artificial lymph nodes or organ cultures. Or, perhaps, computational models in which raising concentrations to 10^9 is no problem at all.