Thursday, October 27, 2011

A Mechanism for Low Zone Tolerance and Implications for Allergies

Allergic reactions occur when the immune system reacts to an allergen found in the environment, promoting hypersensitivity towards the substance. These reactions are normally quite docile but can become very severe. These allergens can be quite common (such as peanuts, shellfish, gluten, etc.), raising the question: why do some people initiate an immune response to them and others don't? One answer is low zone tolerance (LZT), which involves the repeated exposure to small doses of an antigen. At the other end of the spectrum is high zone tolerance, occurring when individuals are exposed to a high dose of an antigen. LZT is believed to be one of the main routes by which tolerance to an antigen is developed. Failure to install LZT may be due to a high dose exposure during the first initial contact, leading to contact allergies (Luckey et al. 2011). Contact allergies are fairly prevalent in the population, affecting approximately 10% (Cavani et al. 2007). Therefore, allergies are just another example that bolsters the importance of immune tolerance. Autoimmunity is an additional example of when tolerance goes wrong, specifically when the immune system initiates an attack against itself.

Lucky et al. (2011) decided to examine the mechanism by which LZT is initiated, a process which is largely unknown. Previous research by Lucky's group determined that LZT is maintained by CD8+ suppressive T cells, a certain subset of T cells. These cells are induced to develop after stimulation by IL-10, a chemical messenger, which is secreted by helper T cells. The authors connected this information with another chemical messenger hypothesized to regulate the LZT response, Tumor Necrosis Factor (TNF). TNF is a cytokine messenger which is secreted as part of the immune response and has a myriad of functions from promoting inflammation and apoptosis to exerting immunosuppressive effects. TNF binds to two receptors TNFR1(p55) and TNFR2 (p75) (Locksley et al. 2001). These two different receptors are what give TNF such dual-sided functions.

Lucky et al. (2011) used a mouse model of allergic contact dermatitis to study LZT. They used a plethora of transgenic mice (tnf-/-, p55-/-, p75-/-, p55-/- p75-/-, NK cell deficient, Rag1-/-, CD45.1, Thy1.1, and WT). Three different chemicals were used to induce either an allergic reaction or lead to tolerance, picryl chloride (TNCB), picryl sulfonic acid (TNBS), and DNFB. Mice were painted with chemicals every other day for ten days using small doses ( approximately 0.45ug - 4.5ug) or they were sensitized on the 20th day with 450ug of TNCB. Afterwards, they conducted many experiments which required preparation and isolation of immune cells, adoptive transfer, flow cytometry, and cytokine ELISA.

First, mice of the different genetic backgrounds were painted with the allergens to determine which mice developed LZT. The Tnf-/-, p75-/-, and p55-/- p75-/- mice all showed contact hypersensitivity to the allergen, suggesting that these animals did not develop tolerance. The authors concluded that TNFR1 (p55) must not be part of the LZT mechanism. Afterwards, the authors sought to determine which stage of LZT TNF would be important for, either the initiation or the effector phase. The initiation phase was ruled out because when mice were missing either TNFR2 or TNF, there were regular quantities of IL-10 and the helper T cell population necessary for inducing the LZT response. Therefore, the effector phase seemed to be the vital component. To test this, first the researchers isolated CD8+ T cells from mice which were tolerant to the allergen but also lacking TNF. When these T cells were adoptively transferred into WT mice there was normal development of LZT. However, when T cells from WT tolerized mice were transferred to mice lacking TNF there was no LZT, suggesting TNF is important during the effector phase of tolerance and not the initiation. Additional experiments using TNF antibodies during the effector phase of LZT confirmed this finding. They utilized the same adoptive transfer technique except with T cells lacking p75 (TNFR2) and found that p75 was indeed necessary in the effector phase as well.

The authors next asked, which cell is expressing the p75 that seems to be necessary for LZT? They transferred CD8+ effector cells (these are the ones that would be doing the killing) from tolerized mice who lack p75 into WT mice and found that LZT was not established. Other cell types were transferred as controls and did not impede LZT. Thus, the authors concluded that LZT functions by allowing TNF to act on p75 which is expressed by the CD8+ effector cells which are specific to the given antigen. Next, the authors intricately produced a mouse model which contained quantities of both CD8+ effector T cells and CD8+ suppressor T cells (produced by tolerizing Thy1.1 mice and sensitizing Thy1.2 mice, then performing an adoptive transfer) They then used flow cytometry to determine how many of these cells (which are p75 +) would remain in the mice after LZT was established; the effector cells were the only subset which was reduced. The authors concluded that CD8+ effector T cells are specifically targeted for apoptosis via TNF acting on TNFR2.

The next logical question was, where is the TNF coming from? In previous studies, the authors determined B cells and macrophages were not the TNF hoarders. Experiments with a NK cell deficient mouse line proved that NK cells were also not necessary because LZT was successfully induced in these animals. CD8+ T cells and CD8+ Dendritic cells were the authors last choices after they eliminated all other possibilities. Both these cell types were isolated from tolerized mice and stained for TNF; the dendritic cells possessed large quantites. To confirm these results, the authors tolerized mice deficient for TNF and then transferred either CD8+ T cells or CD8+CD11c+ dendritic cells from the skin-draining lymph nodes of naive WT mice; transfer of the dendritic cells led to proper establishment LZT. The authors went into further detail in their supplemental figures to bolster the mechanism. For instance, migration experiments confirmed CD8+CD11c+ DCs migrated to the draining skin lymph nodes during LZT. Also, importantly, they determined that CD8+ suppressor T cells induced TNF expression in the particular subset of DCs.

The ultimate mechanism the authors paint goes like this: LZT causes the accumulation of CD8+ suppressor T cells, these cells promote the upregulation of TNF in CD8+CD11c+ DCs, the TNF binds to TNFR2 on allergen-specific CD8+ effector T cells and induces their apoptosis. Without the CD8+ effector cells inflammation will not occur. The work is important because it further defines TNF's dual-sided role. This cytokine messenger has been implicated in all sorts of immune functions. At the end, the author's discuss the implications of their work. They believe that if tolerogenic dendritic cells can be developed for common allergens then tolerance to these allergens can be brought to people who do have allergies; they do indeed mention that studies utilizing this theory are in progress. However, the authors are also interested in the "cell killing" action of these tolerogenic DCs and wish to study them in another light, beyond LZT.

This research is very promising because it elucidates a mechanism that develops tolerance to allergens. However, there is also promise in autoimmune disorders. It would be interesting to see if such "killer" tolerogenic DCs could be used to combat autoimmune disorders such as Multiple Sclerosis and Rheumatoid Arthritis. Yet, the authors do complete this study in regard to contact allergens, so further research into other types of allergens seems warranted. I would like to know more about the CD8+ suppressor cells they mention. There is not much information about them in the literature and I am curious as to their formation, whether they are anergized or are an entirely different CD8 subset. CD8 T cells seem to function in a manner which is very similar to T-regulatory cells, but these cells are often CD4+ rather than CD8+. Still, these suppressor cells have been previously determined to be activated in response to IL-10 from helper T cells. The exact mechanism by which this activation occurs would be interesting to know; this could be done in a series of in vitro experiments. There are definitely many holes left to fill in the mechanism, but this research is a very promising start to a mechanism which has not been completely identified.

Ulrike Luckey, Marcus Maurer, Talkea Schmidt, Nadine Lorenz, Beate Seebach, Martin Metz, Kerstin Steinbrink
Published in Volume 121, Issue 10
J Clin Invest. 2011; 121(10):3860 doi:10.1172/JCI45963

Other Sources

Cavani A, De Pita O, Girolomoni G. New aspects of the molecular basis of contact allergy. Curr Opin Allergy Clin Immunol. 2007;7(5):404–408.

Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell. 2001;104(4):487–501.

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