A specific bacteria-infecting virus, bacteriophage, found in gut microflora, augments anti-tumor T cell immunity

Molecular mimicry between microbial and host's antigens could contribute to autoimmunity but also to the protection against tumors through epitope cross-reactivity. A new study in journal Science indicates that those cross-reactive epitopes could come from viruses that infect endogenous microbial species. 

In this study the authors made a surprising observation that only certain Enterococcus hirae microbial strains (E. hirae 13144 or IGR11) augmented anti-cancer effect in experimental cancer model.

 

 

Next, the authors showed that this biological activity was linked to one dominant epitope, TSLARFANI, derived from TMP protein that originated in 39.2-kb prophage only in those specific E. hirae strains. Mice immunized with heat-inactivated E. hirae 13144 strain, or peptide TSLARFANI, or irrelevant E.coli engineered to express TMP, all augmented anti-cancer effect. 

 

 

Mechanistically, the authors showed that epitope, GSLARFRNI, derived from cancer cells used in these experiments, was recognized by the same CD8 T cells which labeled with TSLARFANI epitope tetramers confirming cross-reactivity between these 2 epitopes.

 

 

In summary, this study suggests that microbiota and bacteriophages they carry represent new modality in fight against cancer. In this study overall anti-tumor effect is modest but we need to take into account that this is an effect of just one cross-reactive epitope in one type of MHC inbred mice, and it is likely that many other epitopes will be involved in outbred species such as humans. However, it is still extremely hard to do such analysis in humans in real world scenario due to lack of exact knowledge about human microbiota strains and poor reliability and performances of available bioinformatics approaches. However, once the mechanistic principles underlying anti-cancer effects are uncovered and accepted, then it is much easier to move the field forward.

posted by David Usharauli

Is it possible to engineer Foxp3+ Tregs from primary T cells?

Here is the most recent paper that claims that they can do it. It was published in Science Translational Medicine. This is fairly respectable journal run by Science. This research group is so confident in their data that they even set up a new biotech company to commercialize their approach. I am going to examine how strong are their claims.

Here is a short description what they did: they used a combination of specific nuclease (TALEN) and virus (AAV6) to insert a new promoter into Foxp3 gene in an in vitro activated T cells. They called these Tregs edTregs.

insertion of MND promoter

The edTregs displayed very similar functionality known to occur in thymus-derived Tregs (tTregs) such as no or limited expression of IL-2 and other cytokines in an in vitro stimulation assays. 

The edTregs were suppressive towards effector T cells in a proliferation assay as should be expected from tTregs. Moreover this essential function required endogenous Foxp3 activity as edTregs from IPEX individuals with a defective Foxp3 gene did not show suppression.

proliferation assay

However, edTregs were significantly different from tTregs in Treg-specific demethylated region (TSDR). The point is it is now accepted that Treg identity is not established solely because of Foxp3 expression but requires specific and selective epigenetic modification within and outside of Foxp3 gene. Nonetheless, in in vitro assays, edTregs behaved as bona fide tTregs. 

What about in vivo? There the story gets a little bit murky. The authors used two models to assess edTregs in vivo. First, they co-transferred edTregs with effector T cells into immunodeficient mice to assess if edTregs could prevent graft versus host disease (GvHD). They do see reduction of mice mortality with edTregs.

However, there are some inconsistency between experiments describing GvHD model. In one set of experiments it produced 100% lethality by day 21 (see below, red line) while in other set of experiments it produced only 20% lethality (see above, red line). 

Such inconsistency casts doubts about edTregs ability to inhibit effector T cells in vivo and could explain why the authors did not see much difference in GvHD scores with or without edTregs (see below).

                                                                                                                      

This could also explain why the authors did not see improvement in brain inflammation in mice EAE model when co-transferring antigen-specific edTregs with effector T cells.

In summary, this paper has done a lot of interesting in vitro work trying to convince us that their edTregs work as intended. However, in vivo work lacks consistency. It is not surprising. It has been known for some time now that Tregs behave differently in vitro vs. in vivo. Suppression in vivo appears to be strictly antigen-specific phenomenon unlike in vitro where it could be observed antigen-nonspecific manner (even though Treg activation in itself still require presence of cognate antigen). 

posted by David Usharauli

Do Tregs inhibit or promote allergic responses? How to avoid data misinterpretation

Foxp3+ regulatory T cells (Tregs) are the most essential component of the immune system. No other cell population, taken singularly, have such an indispensable role in the proper functioning of the immune system. Tregs are known to inhibit inappropriate immune response against self-antigens, commensal microbiota or even nonself pathogens.  I strongly believe that data are very clear about it.

So, when I see a well-designed research paper showing something contrary I immediately want to understand its details. Take this new paper from The Journal of Clinical Investigation (JCI) as an example. It claims and data its provided is pretty solid that removal of a subset of Tregs called T follicular regulatory cells (Tfr) from the immune system in Foxp3-cre Bcl6-fl/fl mice paradoxically reduces, rather than increases, peanut-specific IgE responses. 

And if you think maybe their knockout mice are some kind of weirdos, not really. Their model also shows that total IgE is increasing as expected. So, the system the authors are using is within acceptable norms.   

The authors then went on to show that IL-10 derived from Tfr cells are important for promoting peanut allergy-producing IgE production. 

These results are totally against the whole paradigm about the role of Tfr cells and even IL-10 because the authors speculating that we need to block IL-10 to reduce peanut allergy rather than inject IL-10 to inhibit it, as everyone thinks currently (IL-10 is lesser understood cytokine but it is generally accepted as an immunosuppressant.)

So, how to interpret these results? Basically, what's going on? Is there a way to interpret these results within the confines of the established concept?

I think there is at least one possibility the authors did not consider in their discussions. Here the authors are looking for primary adaptive T/B cell response to a novel antigen, peanut antigen in this case. In this scenario, peanut allergy promoting IgE response is clearly reduced without Tfr cells. But at the same time, the level of total IgE (representing unknown antigen-specific IgE responses) has increased. We could say that total IgE is derived from an already established memory T/B system rather than from primary immune response as peanut IgE is. This memory T/B/plasma cells then boost total IgE levels when the brakes applied by Tfrs are removed. But the same principle does not apply for primary T/B response to peanut allergen. Why is that? It is possible that the absence of Tfr specifically messes up with primary T/B cooperation. It is not a direct effect but rather indirect due to the activation of other components of the immune system when brakes are removed. So, primary T/B responses will be undermined by a lack of Tfr cells and it would appear if Tfr cells were promoting primary IgE responses and their absence reduced IgE production. 

The correct interpretation is essential, especially when applied to human clinical data. In humans, peanut allergy IgE is already established as a memory system by the time the allergic individuals are examined by a doctor. In that case, manipulation of the Tfr or IL-10 system the way the authors envisaged could be detrimental rather than beneficial.     

posted by David Usharauli      

Microbiota-derived peptide and autoimmune heart inflammation: a tale of missing data

Papers published in journal Science supposed to undergo thorough high-level vetting process. However, to err is human. Both reviewers and editors are humans and hence they frequently err, for the annoyance of scientists and for the joy of postdocs doing journal clubs.  

Here is an example of a paper that squeezed through the cracks of the Science vetting process. It claims that peptides derived from certain commensal microbiota species cross-react with heart muscle protein, MYH6, causing autoimmune heart inflammation. It has a great Figure 1 showing that MYH6-specific TCR transgenic mice on a germ-free background, lacking microbiota, is protected from heart autoimmunity.

Furthermore, they showed that the re-introduction of microbiota into germ-free makes these mice susceptible to heart inflammation similar to microbiota+ mice.

The authors then tried to identify the microbiota species that contribute to this inflammatory condition. An in silico search identified cross-reactive β-galactosidase (β-gal) mimic peptides in Bacteroides thetaiotaomicron (B. theta) and B. faecis with high similarity to MYH6.

The authors even introduced into germ-free TCR transgenic mice Bacteroides thetaiotaomicron (B. theta) lacking the β-gal. Up to now, it feels that the authors have checked all the boxes necessary for high-quality research. But then for some reason, they do not show survival data comparing Bacteroides thetaiotaomicron (B. theta) with and without the β-gal gene as in figure 1. They just showed how a lack of β-gal Bacteroides thetaiotaomicron modifies MYH6-T cells accumulation in the heart tissue.

So, why the authors don't show survival data of germ-free MYH6 TCR transgenic mice colonized with Bacteroides thetaiotaomicron -/+ β-gal gene? Isn't it the most important result for their hypothesis? Where were reviewers and editors looking?

posted by David Usharauli