Forden's Immunology Home page

The novel assertion of the danger-difference-sensing hypothesis is that reference cells affect lymphocyte cytotoxicity in a reverse manner, i.e. that low-levels of danger-signals on non-cognate targets increase T cell (Tc) cytotoxicity, and conversely, that high-levels of danger signals on non-cognate targets decrease cytotoxicity.

Sensitivity of these tests for disproving theories
The three-party cytotoxicity tests described herein will discover whether the level of danger-signals on non-cognate targets affects cytotoxicity against cognate targets. The known reverse-signalling effect of FasL suggests that adding Fas^hi or MICA^hi bystanders would increase cytotoxicity. The conventional danger-model would predict that adding NKG2D ligands to a cytotoxicity assay would always increase cytotoxicity, due to the general, proinflammatory effect of danger signals. In contrast, the reference-cell theory predicts that such additional cells would decrease cytotoxicity.

The experiments will resemble bystander toxicity assays where non-cognate targets are added to standard two-party (effector and target) toxicity assays. Heretofore the additional cells have been called "bystanders", and cytotoxicity against them has been what was measured. I propose measuring how the non-cognate targets affect cytotoxicity against cognate targets. If the presence of non-cognate targets bearing high levels of danger signals, reduces cytotoxicity against cognate targets, then we have confirmation that we can call the non-cognate targets "reference" cells instead of just "bystanders". In order to reduce the complexity of interpretation I would recommend that all three cell parties (cognate targets, effectors, and reference targets) be syngeneic with each other. As referenced at length in my paper on Ag correlation, many cytotoxicity assays have shown that syngeny is mandatory for FasL probing of (non-cognate) "bystanders" after T cells (Tc) engage cognate targets, so reference cells at least must be syngeneic with effectors.

CD8+ effectors are more likely than CD4+ Tc, to produce comprehensible results in these experiments. There are both specific and general reasons for the preference for killer rather than helper T cells. Suzuki and Fink's data show that CD8+ cells proliferate in response to ligating their FasL with cognate targets' Fas, whereas CD4+ cells do not. One simple explanation for that difference is that antibody-mediated responses are directed against necrotically lysing viruses, whereas cytotoxicity is directed against viruses that do not burst open their host cells in order to escape. A simple (perhaps old-fashioned and overly simplistic) model of T cell compartments would consider CD8+ Tc to be killers, and CD4+ Tc to be helpers of B cells.

Target cells
The cognate (primary) and non-cognate (reference) targets should be as similar as possible to each other, except for cognate-antigen and danger-signal presentation. Tissue similarity between primary targets and reference targets is necessary because in-vivo Tc face many extraneous local cell-types that should not be used to measure the reference levels of danger signals of the local tissue of interest. For example, if Tc must evaluate the danger-signals presented by muscle cells, nearby nerve fibers and blood vessel cells would be likely to display different levels of danger signals than healthy muscle cells. Therefore it is quite likely that Tc have evolved to be unresponsive to background levels of danger-signals as presented by nearby non-cognate cells of dramatically different tissue types.

Ideally none of the assay cells would be transformed cell lines, but if culture of transfected, non-transformed cells is too difficult or slow, then parental and transfected immortalized cell lines could be used. A parental line could be transfected to express Fas receptor, and a ligand of NKG2D. H-Y would serve as the cognate antigen; Fas and/or a ligand of NKG2D would be danger signals. Eight cell lines could result, ranging from cells expressing no more than background levels of Fas and MICA, and no H-Y, to cells expressing large quantities of all three molecules. More cell lines could be created if transfected cells express varying amounts of the transfected genes. The estimate of eight cell lines assumes the transfection success would be binary for each of the three transfected genes; i.e. that transfected cells either express uniform amount of the targeted protein, or they express or increase their expression not at all. Intermediate expression of transfected genes would be very useful for advanced experiments, but the simple assays listed below require cells with just high or low (or zero in the case of H-Y) expression levels. (I deem it unlikely, but there is a possibility that Tc might regard tissue cells absolutely devoid of danger signals, as being unreliable due to possible compromise by a pathogen. In that case, cells displaying low, but not completely negligible levels of danger signals would be necessary for enhancing reference targets.)

Preparation of effector cells
Ensure T cells are primed, but not by dendritic cells that matured when they were embedded in tissue that began secreting danger signals before necrosis. Priming by dendritic cells that absorbed H-Y with necrotic debris would be acceptable. It might be helpful to allow two months (rather than two weeks) of rest after priming, to allow Tc stimulated by DC to recover from that acute stimulation; When highly stimulated by dendritic cells, Tc need to temporarily ignore the lack of danger-correlation to early viral proteins because some viruses have evolved to temporally separate danger-signals from their epitopes. By allowing the responder Tc to shift into a memory-state instead of testing them during high-activation after priming, they are more likely to respond to relatively lower levels of danger-signals on cognate targets, by reducing cytotoxicity. Appended 2007 June 25: The vaccination methods are obviously good things to vary to ascertain how autonomous differential danger sensing by Tc, interacts with response to activation by APCs.

Assay guidelines
As per standard practice, cytotoxicity should be measured with varying ratios of effectors to targets. My hope is that when low-danger reference cells are added, high cytotoxicity can be achieved even when targets greatly outnumber effectors. The fact that effectors usually need to outnumber targets in order to kill a substantial fraction of them in four hours, seems an indication that something has been missing from our assays. My hope is that the missing ingredient is reference targets. It is desirable to test high ratios of targets to effectors for another reason, too; then effectors will be less likely to use other effectors as references. Duke shows that effectors seem to prefer non-lymphocytes as reference targets (bystanders affected by FasL on effectors that kill cognate targets). However, restricting reference effects to cells that we specifically add as references, will make our data easier to interpret. I recommend that the initial tests use equal numbers of reference and cognate targets.

In addition to these described assays, it would be helpful to at least at first, simultaneously run assays of cytotoxicity against bystanders, just to verify that the experiment successfully duplicates conditions previously published in which syngeny was found necessary for bystander lysis. Descriptions of such assays can be found in:

• Smyth MJ, Krasovskis E, Johnstone R; "Fas Ligand-Mediated Lysis of Self Bystander Targets by Human Papillomavirus-Specific CD81 Cytotoxic T Lymphocytes" Virology, July 1998, p. 5948–5954)
• Smyth MJ, "Fas Ligand-Mediated Bystander Lysis of Syngeneic Cells in Response to an Allogeneic Stimulus" The Journal of Immunology, 1997,158: 5765-5772.

Assay cell sets
The following discussion uses the notation (low) for cells that display low levels of a given danger signal. Ideally such levels would be those typical of normal, mature cells in a given tissue type. However it may be difficult to culture healthy, mature cells, so blast cells might be a reasonable alternative. If necessary, transformed cell lines might be used, in which case (low) might mean absolutely zero expression of a given danger signal. I do not know whether complete extinction of danger signals will be interpreted by immune cells as a danger signal in itself. 

All primary targets will express the cognate Ag, e.g. H-Y. No reference target will express the cognate Ag. 

Confounding effects of competition
A positive result from the cytotoxicity inhibition assays, could be interpreted as due to competition for the attentions of effectors by high-danger bystander targets, rather than as due to effectors comparing the danger signals on cells serving as references.