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

Selective PD-L1/2 expression in lymphoid tissues allows tumor escape during allo-HSCT

The goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to achieve graft-versus-leukemia (GVL) effect to eliminate residual tumor cells in host [left after irradiation]. Since most allo-HSCs are derived from HLA-compatible donors, GVL reaction is mediated by donor T cells reacting against host minor histocompatibility antigens (miHAg; self or tumor-specific Ags). However, even reaction to self-miHAg could still induce graft-versus-host disease (GVHD) because self-miHAgs are broadly expressed in different tissues. Interestingly, GVHD preferentially affects peripheral tissues such as liver or gut. But why?

A new [and very good] paper in journal Immunity may have an answer for this question. In this study, the authors found that selective expression of PD-1 ligands, PD-L1 and PD-L2, in allo-HSCT recipients' lymphoid tissues [but not in peripheral tissues] provide a "cover" against cytotoxic activity exerted by donor CD8 T cells.

To mimic allo-HSCTs, the authors have used female-to-male BM stem cell transplantation model. In this model, self-miHAg is represented by male antigen, HY. To induce GVHD, female BM cells were transplanted alongside with HY-specific transgenic MataHari CD8 T cells. As expected, only male recipients of female HSCs + MataHari CD8 T cells developed GVHD (in liver, gut and skin tissue).

To address the question why only those peripheral tissues were affected by GVHD, the authors examined the hypothesis that HY specific CD8 T cell cytotoxic activity were differentially affected by different tissues. Indeed, co-transfer of labeled male and female targets revealed that recipients of allo-HSCs + MataHari CD8 T cells showed selective reduction of cytotoxic activity against male targets in lymphoid tissues (but not in liver).

This observation was supported by the fact that in contrast to peripheral tissues, MataHari CD8 T cells obtained from lymphoid tissues expressed low level of granzyme B (molecule involved in cytotoxic activity).

To understand why it is the case, the authors examined expression of inhibitory molecules on CD8 T cells. This revealed that while PD-1 on CD8 T cells were similarly expressed irrespective of tissue origin, its ligands, PD-L1 and PD-L2 were selectively up-regulated in lymphoid tissues.

The role of PD-L1 and PD-L2 in inhibition of CD8 T cell cytotoxicity in lymphoid tissues were confirmed in  experiment with anti-PD1 antibody.

Finally, using anti-PD-1 antibody injection, the authors showed that B cell leukemia cells that were hiding in lymphoid tissues of allo-HSCs male recipients could be now eliminated by MataHari CD8 T cells.

In summary, this study showed that (a) during GVHD donor CD8 T cell activity is differentially regulated by different tissues based on availability of inhibitory PD-1 signaling and that (b) this is exploited by leukemia cells to hide in lymphoid tissues but it could be overcome by anti-PD1 antibody injection.

David Usharauli