Thursday, August 6, 2020

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