Behavior of recent thymic emigrant T cells supports 60 year old hypothesis

This week Journal of Experimental Medicine published new study that showed that recent thymic emigrant T cells are highly susceptible to tolerance upon antigen encounter in the periphery.

I selected to review this article because its topic underlies one of the fundamental questions of immunology, namely how T cells are tolerized to peripheral antigens [not expressed in the thymus]?

Let's start from the beginning. T cells generated in the thymus express T cell receptor (TCR) with all kind of specificity. Most of T cells specific for self-antigens are deleted within thymus. Thymus also express some of the peripheral antigens, such as insulin and other proteins, under the guidance of two genes AIRE and Fezl. However, it is conceivable that some of the peripheral antigens are not expressed at all in the thymus or are expressed too little to completely delete auto-reactive T cells. So what those auto-reactive T cells would do once they leave the thymus and encounter self-antigens in the periphery?  

So, this new study suggests that those young T cells leaving thymus for the first time [they are called recent thymic emigrants, RTEs] are highly sensitive to tolerization and display anergic phenotype when stimulated by antigen. Basically there is a "window of opportunity" for peripheral tolerance for RTEs. These results support the hypothesis proposed by Joshua Lederberg 1950s to save Burnet's clonal selection theory (though it was disappointing to see that this study did not refer to it).

To distinguish between RTEs and conventional naive T cells the authors used RAG2p-GFP mouse model that marks RTEs with GFP for ~3 weeks [after leaving thymus]. When OVA-specific RTEs and naive T cells were transferred in OVA-expressing hosts and later re-stimulated, RTEs showed less proliferation and less effector differentiation.

Functional changes in RTEs upon encountering specific antigen in the periphery was confirmed by absence or delay in development of diabetes in OVA-expressing hosts (OVA is expressed in pancreas).

RTEs were also more susceptible to suppression by Tregs.

Finally, the authors showed that if RTEs were exposed to peripheral antigens in context of inflammation (alum or CFA), then they could undergo full effector differentiation equal to that of conventional naive T cells.

In summary, this short paper suggests that RTEs and naive T cells are functionally distinct and that 3 weeks of post-thymus period play physiological role in peripheral tolerance.

I also want to highlight some of the deficiencies of this paper: 

1st, the authors did not test whether RTEs indeed "become" conventional naive T cells in 3 weeks after transfer in antigen-free hosts. For example, I would have transferred RTEs first in antigen-free host and then 3 weeks later transfer again those T cells into antigen-expressed hosts. Prediction would be that 2nd transfer T cells should behave as conventional naive T cells.    

2nd, the authors used alum and CFA immunization to recreate inflammatory context. However, antigen presentation and inflammation in the context of alum or CFA are quite different from inflammation in natural context such as infection. I would have used bacterial or viral infection and then transfer RTEs in infected hosts to see how more natural inflammatory context would have affected their behavior (later I was informed that such experiment was indeed done with the same outcome).

David Usharauli

Imclone's legacy lives on: Cetuximab (Erbitux) induces immunogenic cell death in cancer cells

This week Nature Medicine published new study that suggests that Cetuximab (Erbitux), a chimeric monoclonal antibody directed to the EGFR, induces immunogenic cell death in susceptible cancer cells that carry wild-type form of oncogene KRAS.

Nowadays such research articles are common. Cancer immunotherapy really took off in the past 3 years and many want to jump on the immunotherapy bandwagon. However, I selected this paper for review for entirely different reason. I wanted to highlight how science behind Cetuximab was so advanced and ahead of its time [when it was originally tested] that it deemed ineffective [due to some data inconsistency for reasons that became known only later].  

Let me remind you that Cetuximab was originally developed and tested by the biotech company called Imclone in 1990s.  In clinical trials it was effective in ~20% of patients. However, trials were poorly designed [some did not even show effect] and when FDA refused to accept Imclone's submission for Cetuximab approval in December 2001, company collapsed and its CEO, Samuel Waksal, went to jail for insider trading. 

In retrospect, we can now [based on this new paper] reconstruct to what happened to Cetuximab's original trials. This analysis, in my opinion, clearly vindicates Imclone's and Samuel Waksal's scientific approach, at that time, to explain Cetuximab trials.

The 1st finding from this new study was that mice injected with Cetuximab-treated hEGFR-CT26 cells, a mouse colorectal cancer cell line (mCRC) expressing human EGFR, are protected against secondary challenge with parental CT26 cancer cells. This suggested that Cetuximab treatment of CT26 cancer cell induced immunogenic cell death (F stands for FOLFIRI, a standard chemotherapy regimen for mCRC that does not induce immunogenic cell death. FT stands for freeze-thaw).

The 2nd finding was that Cetuximab + FOLFIRI induced immunogenic cell death in human cancer cells with 

(a) wild-type forms of oncogenes KRAS  and BRAF or
(b) with KRAS^G13D and KRAS^G12D mutations 

(c) but it did not induce immunogenic cell death in cancer cells with with BRAF and KRAS^G12V mutations. In those cell lines immunogenic cell death could be restored by combining Cetuximab + FOLFIRI with inhibitors of mutated BRAF (PLX4032, PLX) and MEK (trametinib, tram).

In summary, these data define molecular markers that make cancer cell susceptible to Cetuximab + chemotherapy combination.

Now lets go back to Imclone's story. At the time of original trials, in late 90s, patients stratification based on tumor genetic makeup was a novel idea and not all mutations were known. In fact, when Imclone's management suggested to conduct trial based on patient stratification, European Medical Agency (FDA equivalent in EU) refused to consider it, such unorthodox and unheard of idea it was at that time.

We can now understand why Cetuximab's original trials gave inconsistent results. Without patient stratification, antibody-based therapy rarely shows statistically significant benefits over placebo in cancer patients. Science behind Cetuximab was so ahead of its time that clinical trials were not ready for it and neither were Imclone's management (note that few years later after Imclone's debacle Cetuximab was approved by FDA and it is marketed by Bristol-Myers Squibb).

David Usharauli

Prevalence of inhibitory LPS within gut microflora could underlie hygiene hypothesis

This week prestigious journal Cell published seminal microbiome study that could explain hygiene hypothesis that suggests that "microbial cleanliness" in developed countries predisposes individuals to immunopathologies such as allergy, multiple sclerosis and other autoimmune diseases. 

This study found that gut microflora in babies from Finland and Estonia, but not from Russian Karelia, are enriched in gut bacteria with inhibitory LPS that failed to properly "educate" newborns innate immune system thus predisposing them to higher incidence of immunopathologies.

The first finding was that microbiota in babies from Finland, but not from Russian Karelia, were enriched in Bacteroides. Now this fact by itself isn't too surprising. 

The second finding was that lipid A component of LPS from these Bacteroides [unlike lipid A component of LPS from E. coli] species were non-stimulatory in TLR4 assay. In fact, molecular analysis showed that Bacteroides harbored tetra- and penta-acylated lipid A structures, as opposed to the hexa-acylated lipid A seen in E. coli. Such structural modification converted Bacteroides lipid A into totally non-stimulatory ligand.

In fact, Bacteroides derived lipid A was inhibitory when combined with stimulatory, E.coli derived lipid A [LPS tolerance assay, where primary exposure to E. coli LPS makes responding cell refractory to secondary exposure].

Finally, third finding was that presence of non-stimulatory lipid A had immunological consequences since it failed to reduce incidence of diabetes in NOD mice [whereas lipid A from E. coli could, as expected].  

In summary, this important study has provided one of the first definite molecular evidence underlying hygiene hypothesis and suggested the path for its prevention.

David Usharauli