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

Tracking deletion of autoreactive clones versus Treg generation for thymically expressed epitopes

So far 3 different outcomes have been identified for developing T cells in the thymus: to develop into naive T cells, get deleted or become Foxp3+ Treg. Both deletion and Treg path require the presence of specific epitopes. However, how a given T cell decides between these pathways is not well understood. 

Here is a new paper in PNAS that tries to tackle this question using the tetramer tracking approach. The authors are using PLP (brain-specific protein) as an endogenous antigen expressed in the thymus. Surprisingly both PLP^WT and PLP^KO mice showed near similar numbers of tetramer-positive T cells in peripheral tissue. However, as expected, only PLP^WT mice that express PLP epitopes in the thymus but not PLP^KO mice that do not express the same epitopes showed Treg development.

   


 Similar results were obtained when thymus tissue was analyzed.

  

To make tetramer tracking for reliable the authors used transgenic mice expressing a fixed TCR beta chain. These mice also showed a similar phenotype.  

As in PLP^WT and PLP^KO mice, fixed:TCR beta mice on PLP^WT but not on PLP^KO background harbored Tregs in the periphery. Notable, the rest of the tetramer-positive Foxp3-negative T cells displayed an anergic phenotype (CD73^HiFR4^Hi).

A similar phenotype was found in the thymus. Note, there was an unexpected and significant reduction of tetramer-positive T cells from the thymus to the periphery in fixed:TCR beta mice on PLP^KO background compared to fixed:TCR beta mice on PLP^WT background. 

So far these data indicated that there is almost no deletion of PLP specific T cells in the thymus on WT mice [compaed KO] but ~2-fold reduction in fixed:TCR beta mice on PLP^WT compared to KO. Almost half of the tetramer-positive T cells ended up in the Treg pool on the WT background. The remaining T cells showed an anergic phenotype. However the dramatic reduction of tetramer-positive T cells from the thymus to the periphery in KO mice raises some serious unanswered questions.

Finally, to find some correlation between TCR specificity and Treg/anergy/deletion phenotype, the authors selected 4 PLP-specific TCRs (denoted here as A, B, C, D). Their analysis showed that some (clone "A") but not other PLP-specific TCRs (clone "C") were able to generate Tregs in the thymus. Notable, TCR "C" displayed the highest affinity to PLP epitope. Also, there is a substantial reduction of clone "C" from the thymus to the periphery in the Foxp3-negative compartment. This possibly reflects the fact that most clones in "C" are anergic and slowly disappear from the periphery.  

In summary, this study re-confirms that tolerance to self-antigens is mostly controlled via Treg generation and that not all antigens/epitopes and their corresponding TCRs are able to participate in this process. There are few unexplained observations in this paper though as discussed above. 

posted by David Usharauli

The auto-reactive CD4+ T cells provide IL-2 to proto-Tregs in the thymus

The T cells expressing the transcription factor Foxp3 called regulatory T cells, abbreviated as Tregs, are the most important cell type in the immune system. Without them, the whole immune system goes haywire. As a result, the body simply dies in a very short time.

The Tregs develop in the thymus and require two things: TCR signaling and IL-2. The thymus expresses a very diverse set of epitopes including that from peripheral tissues such as the pancreas or prostate. The high-affinity interaction between TCR and epitope/MHC II makes proto-Treg sensitive to local IL-2, a necessary step to complete a Treg formation loop.

But what cell provides that crucial IL-2 to proto-Tregs? There hasn't been any consensus with this regard but a new paper in the Journal of Experimental Medicine from Sasha Rudensky's lab indicates that it is mature CD4+ T cells and CD25+Foxp3- CD4+ single-positive (SP) T cells that are the main source of thymic IL-2 required for Treg development.

For this study, they used an IL-2 reporter mouse wherein cells expressing or having a history of the expression of IL-2 are genetically labeled and analyzed. They found that IL-2 expression was restricted to TCRbeta expressing CD4+ population.

Out of CD4+ T cells, the most IL-2 was made by mature CD4 SP and CD25+Foxp3- CD4+ T cell population. Of note, CD25+Foxp3- T cell population contains proto-Tregs.

Interestingly, the authors also detected mature Tregs with the history of IL-2 expression. It implies that bifurcation between Tregs versus IL-2 producer is a stochastic process.

As expected, TCR signaling together with IL-2 was essential for Treg formation. A "bystander" effect on Foxp3 upregulation on antigen-independent proto-Tregs (Vbeta 8- T cells) could be explained by the fact that these T cells were likely TCR activated in vivo before harvesting for ex vivo experimentation.

Based on these data, the authors suggested the following model: among mature SP CD4 T cells, a small pool produces IL-2 that in the context of high-affinity TCR/epitope interaction and CD25 upregulation promotes Foxp3+ Treg formation either autocrine or paracrine manner. Since the thymus is expressing self epitopes we can conclude that those IL-2 producing T cells are auto-reactive T cells.

The following questions remain unanswered:

1. What determines Treg, IL-2-producer or deletion pathways? All three options are open for high-affinity TCR+ CD4 SP cells.

2. Do TCR specificity overlaps between Tregs and IL-2 producers?

3. What cells provide IL-2 to Tregs in the periphery?

4. Is IL-2 delivery TCR/epitope-specific or non-specific event?


We have recently published a new model, called SPIRAL, that provides answers to these questions. The SPIRAL is based on the principle of epitope cross-reactivity.


Shared TCR epitope cross-reactivity could permit dyads of Foxp3+ regulatory and IL-2-producing T cell precursors to escape thymic purge 


posted by David Usharauli


 

Two non-overlapping precursors generate Foxp3+ regulatory T cells in the thymus

New study in @NatImmunol compared #Treg differentiation potential of #CD25+ versus Foxp3(low) precursors in the ##thymus. https://t.co/rIFWqybNnm pic.twitter.com/9ibTGBeHF9

— David Usharauli (@3DiMMUNE) January 22, 2019

2. One interesting #difference between CD25+ vs. Foxp3(low) #precursors was dependency on #IL4 #signaling to generate Tregs only from Foxp3(low) precursors (both responded to IL-2). pic.twitter.com/hNoLxVBfIP

— David Usharauli (@3DiMMUNE) January 22, 2019

in summary, two types of thymic precursors can generate Tregs. Since their #TCR did not overlap with each other but both of them overlapped with mature #Foxp3+ #Tregs it suggests certain #functional bifurcation considering differences in protection against tissue #inflammation.

— David Usharauli (@3DiMMUNE) January 22, 2019

Foxp3+ Tregs suppress other T cells by stripping DCs of specific antigen/MHC complexes

Ag-specific or not Ag-specific, that is [maybe was] question for #Foxp3+ Tregs mechanism of action. New paper in @NatImmunol from #Shevach's lab @NIH says definitely antigen-specific. Lets dive in. BTW, ~20 years ago his lab developed assays that showed it was non-specific 🤫

— David Usharauli (@3DiMMUNE) January 16, 2019

Now his lab made U-turn and concluded that #suppression by #Tregs is actually #antigen-specific and difference between now and then comes down to in #vitro versus in #vivo assays 🤔

— David Usharauli (@3DiMMUNE) January 16, 2019

2. This #antigen-specific #suppression was still active when antigen-specific dendrtic cells were removed from Treg co-culture (1st culture) and exposed to T cells (2nd culture). pic.twitter.com/dj1pzDuNNv

— David Usharauli (@3DiMMUNE) January 16, 2019

So, from now on if someone asks you how Treg suppress other T cells don't hesitate to reply "antigen-specific manner", add Ethan Shevach said so and no one will argue with you. got it 🤨 https://t.co/I4pAYIIGy1

— David Usharauli (@3DiMMUNE) January 16, 2019

CD4 T cells silence innate over-activation to gut microbiota

A simple but very effective study was published in journal Nature from Ron Germain's lab. His group is known for publishing high quality in situ microscopy data combined with cellular analysis. New study continues this trend. 

In this study the authors analyzed pattern of phosphorylation of STAT3 transcription factor in small intestine derived from various immune deficient mouse strains. Compared to WT mice, pSTAT3 staining in RAG1 KO gut tissue (lacking adaptive immune system) was significantly up-regulated. 

Analysis of antibiotic-treated or germ-free mice indicated that pattern of pSTAT3 staining in RAG1-KO was correlated with the presence of gut microbiota.

Interestingly, longitudinal analysis showed that pSTAT3 staining inversely correlated with  maturation of adaptive immune system post weaning (between 4-20 weeks). 

Co-housing experiments showed that T cells, but not B cells, played a role in silencing innate pSTAT3 over-activation.

And out of T cells, it were CD4 T cells and class II antigen-presentation that played the role in pSTAT3 silencing.

Finally, both Tregs and SBF-specific Th17 cells (7B8 transgenic T cells) could mediate silencing of pSTAT3 over-activation. Both T cell type could down-regulate STAT3 phosphorylation in innate and epithelial cells but the mechanisms could be different. 

In summary, this study showed that persistent pSTAT3 over-activation observed in mice deficient for CD4 T cell function could explain some of chronic metabolic shifts observed in clinical settings. 

posted by David Usharauli

Microbiota-generated butyrate works on Aire to amplify Treg numbers

A short but very interesting paper in Journal of Immunology caught my attention this week. In this study the scientists showed that butyrate, a short chain fatty acid derived from fiber fermentation by microbiota acts on GPR41 receptor in the thymus to increase Aire expression and amplify Foxp3+ Treg frequency (of note, compared to WT, GPR41-KO thymus already contains almost 5-fold less Tregs).  

Generally it is believed that microbiota works locally in the gut or other peripheral tissues to either convert naive T cells into Foxp3+ Tregs or expand existing Treg numbers. However no one yet managed to definitely show which pathway is functionally operational in vivo in physiological conditions. This study now could help to further narrow down biological mechanisms responsible for Treg biology. If microbiota-generated butyrate could work on thymus to increase Aire expression that in turn increases level of Thymus-derived Treg generation, then peripheral conversion pathway may play even less relevant role in physiological mechanisms of tolerance.  

The most obvious question after reading this article is why the authors did not test Aire KO mice to verify their conclusions.

posted by David Usharauli

   

How Foxp3+ Tregs and microbiota work together to control immune system

Check out our new paper in PeerJ Preprints that unlocks the mystery of how Foxp3+ regulatory T cells work that enables proper immune functioning.

Usharauli D, Kamala T. (2017) An identical mechanism governs self-nonself discrimination and effector class regulation. PeerJ  Preprints 5:e3081v1  https://doi.org/10.7287/peerj.preprints.3081v1

Prevailing immunological dogma dictates self-nonself discrimination, meaning to respond or not, and effector class regulation, meaning choosing the most effective response, are two separate decisions the immune system makes when faced with a new antigen. Representing a cardinal departure from the past, our model instead predicts both self-nonself discrimination and effector class regulation are in fact one and the same process controlled by Foxp3+ regulatory T cells (Tregs) whose antigen-specific repertoire is entirely maintained by commensal microbiota-derived cross-reactive antigens.

 

 

posted by David Usharauli

antigen-specific Foxp3+ Tregs maintain tolerance in HLA-linked autoimmunity

This week Nature published new paper that has features of textbook studies. In it the authors showed that in human HLA transgenic mice model of Goodpasture disease [HLA+antigen]-specific Foxp3+ Tregs protected against autoimmune disease development.

Goodpasture disease is an "HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cells [reactive to] self-epitope derived from the α3 chain of type IV collagen (α3135–145)". In humans presence of HLA-DR15 allele increases disease risk, while presence of HLA-DR1 allele is shown to be dominantly protective in trans with HLA-DR15.

Interestingly enough the authors reported similar pattern of HLA dependency in mouse model of human Goodpasture disease. Here, DR15+ mice were susceptible to disease development. DR1+ mice were resistant to disease development and DR15+DR1+ double-positive mice were healthy except when Tregs were depleted (all mice were on Fcgr2b−/− background, +/- Treg depletion, + immunization with peptide α 3135–145). 

It is not clear how Treg specific for DR1+peptide protects against autoreactive T cells specific for DR15+peptide. It is possible that there is some similarities between these HLA+peptides (cross-reactivity).

David Usharauli

Minimal threshold for Treg repertoire diversity that prevents autoimmunity

If you have read enough of my blog you most likely noticed the preference for research articles that provide some new insight into Tregs' biology.

Few weeks back I analyzed new paper from Rudensky's lab that showed that 50% of mouse with Tregs expressing single TCR specificity were protected from lethal autoimmunity, though not from non-lethal or less lethal forms of  tissue autoimmunity. 

So what is the minimal Tregs TCRβ repertoire diversity required to suppress autoreactive T cells that escape thymic selection?

New paper from Thomas Malek's lab tried to found it out by serially transferring WT Tregs into Treg-deficient IL-2RβKO mice (1st and 2nd level recipients) and analyzing TCR repertoire pre and post transfer. They observed that after each transfer into IL-2RβKO Tregs TCR repertoire diversity got narrower and when it fell < 7000 unique Treg clonal specificity mice showed autoimmunity.

This threshold also correlated with pathological increase in CD62L^lowCD44^hi population that represents activated T cells typical to autoimmunity.

In summary, this study indicates that there is a minimal threshold for Tregs repertoire diversity that is essential to prevent non-lethal forms of autoimmunity.

David Usharauli
        

Selective Tregs elimination within tumor using CD25 antibody coupled to near-infrared photoimmunotherapy

Regulatory T cells (Tregs) inhibit immune responses. When considering autoimmune diseases or allergies, this function of Tregs has beneficial effect on host. The same function, however, could be "hijacked" by tumors to evade immune destruction. So far, selective depletion of tumor-associated Tregs has not been achieved in clinical settings.

New paper in Science Translational Medicine suggested novel approach to eliminate tumor-associated Tregs: application of anti-CD25 F(ab)2 fragments coupled to photo-active silica-phthalocyanine dye (IR700)  and exposed to near-infrared photo-immunotherapy (NIR-PIT). 

First, in vitro experiments showed that when CD25+ cells are exposed to anti-CD25 F(ab)2-IR700 and subjected to NIR-PIT, they undergo cell death.

Since tumors accumulate high frequency of Tregs, the authors conducted NIR-PIT experiments in vivo. When tumor-challenged mice were exposed to anti-CD25 F(ab)2-IR700 and subjected to NIR-PIT, the authors observed temporal delay in tumor growth and improved survival. 

Of note, tumors located at the distant sites away from direct exposure of NIR-PIT also display growth delay after anti-CD25 F(ab)2-IR700/NIR-PIT application, indicating system-wide after-effect of local elimination of Tregs by NIR-PIT.

Finally, the authors found that anti-tumor effect of anti-CD25 F(ab)2-IR700/NIR-PIT application was mediated by IFN-gamma derived from CD8 T and NK cells.

In summary, the authors claim that this less invasive procedure could selectively eliminate CD25+ Tregs (but not effector T cells) in tumors  and improve survival of tumor-challenged host.

David Usharauli

Foxp3+ Tregs undergo memory-loss following inflammation

Adaptive immune system is characterized by its ability to recall prior antigen encounter and to mount stronger and swifter response for the second time. This is what typically happens to conventional T cells. But what about regulatory T cells (Tregs)? Do Tregs also display enhanced recall response when encountering [antigen] for the second time?

The answer is NO according to new study from Rudensky's lab published this week in journal Cell. It appears that while phenotypically and gene-expression wise Tregs do resemble conventional memory T cells, nonetheless Tregs do not undergo enhanced recall response when going through the second time encounter.  

To test this hypothesis, the authors subjected Tregs to inflammatory environment and tracked their behavior. First, they found that phenotypically Tregs returned to "resting" state within 60 days following activation and resolution of inflammation. 

Gene expression analysis confirmed Tregs tested prior to inflammation (resting Tregs) or following inflammation (memory Tregs) resembled each other, while Tregs going through inflammatory process (activated Tregs) had a distinct gene profile. 

    

Functionally too resting Tregs and memory Tregs showed similar potential to inhibit hyper-proliferation of naive T cells when co-transferred into T cell-deficient host.

However, unlike conventional memory CD4 T cells, memory Tregs did not undergo more robust recall response when subjected to inflammation + undefined antigen for the second time.

Finally, the authors observed that Tregs displayed common gene profile with conventional memory CD4 T cells that differentiate them from naive T cells.

In summary, this study suggests two things: first, Tregs do not acquire secondary enhanced recall response capability [at least when exposed to inflammation and undefined antigen] and second, Tregs and conventional memory CD4 T cells share similar gene expression profile.

David Usharauli 

Division of labor among thymus-derived Foxp3+ regulatory T cells

This week Nature Immunology published study that showed another level of "division of labor" among regulatory Foxp3+ T cells (Tregs). The authors revealed presence of two types of thymic Tregs defined by expression of three receptors, GITR, PD-1 and CD25 (GITR^hiPD-1^hiCD25^hi and GITR^loPD-1^loCD25^lo Tregs cells) that displayed non-overlapping functionality.

Specifically, only GITR^loPD-1^loCD25^lo Tregs cells prevented colitis development in adoptive transfer experiment by converting responding naive T cells into induced Tregs.

On the other hand, only GITR^hiPD-1^hiCD25^hi Tregs cells prevented uncontrolled proliferation of endogenous T cells when transferred into Treg-depleted host.

In summary, this study revealed that even among thymus-derived natural Tregs there is a division of labor. It is possible that difference between these two types of thymic Tregs is also related to their differential migration pattern as it was suggested by one recent study on KLF2.

David Usharauli

Autologous Foxp3+ polyTreg adoptive transfer immunotherapy: phase I study

2nd paper in this week's Science Translational Medicine is more directly related to Foxp3⁺ T_regs. Here, the authors has conducted phase I, dose-escalation study in recent onset T1D patients (16 participants) to determine safety of adoptive transfer of ex-vivo expanded autologous polyclonal Foxp3⁺ T_regs cells.

Unlike CAR-T therapy with its bulk PBMCs as its main ex-vivo target population, working with flow sorted polyT_regs cells is more demanding and few institutions are equipped to conduct such GMP-grade clinical trials. As shown in Table 3, there is variability in ex-vivo expansion potential between polyT_regs cells derived from various donors [since no "healthy" control subjects were included in this trial, it is no clear whether this level of expansion of polyT_regs cells is specific for T1D patients].

The authors conducted several functional/marker analysis on 14-day expanded polyT_regs cells. These results revealed that expanded polyT_regs cells retain the same degree of (a) demethylation at Foxp3 locus, (b) they showed improved phosphorylation of STAT5 in response to IL-2, (c) displayed improved suppressive potential in an in vitro proliferation assay.

After adoptive transfer of expanded polyT_regs cells patients were monitored for any [metabolic] abnormalities. There were few adverse events related to T1D metabolic events that probably were not caused by polyT_regs cell transfer per se. However, due to small sample size, adoptive transfer of polyT_regs cells did not reveal any substantial improvement of T1D in recipients either [though transferred polyT_regs cells labeled with [6,6-²H₂]glucose were detected up to 1 year in cohort 3 and 4].

In summary, this phase I study of polyT_regs cells confirms feasibility of adoptive transfer immunotherapy with Foxp3⁺ polyT_regs cells. In general, it is my opinion that only by understanding Foxp3⁺ T_regs cells can we truly unlock the full benefits of immunotherapy for cancer, allergy and autoimmune diseases.

David Usharauli

Treg-specific expression of amphiregulin prevents immunopathology during viral infection

Foxp3⁺ T cell deficiency leads to lethal immunopathology in mice and severe organ pathology in humans (IPEX syndrome). Over the past 20 years numerous mechanisms of action of Foxp3⁺ T cells have been described. In fact, this number is so numerous that it wouldn't be a "heresy" from my part to claim that no single Foxp3⁺ T cell phenotype could explain it. This leaves us with the hypothesis that multiple versions of Foxp3⁺ T cells exist, each of them using selective path for immune regulation and suppression.    

With this view in mind, it was useful to read a new paper from Rudensky's lab published in journal Cell this week that provided evidence showing the role of Foxp3-positive T cell-specific amphiregulin in preventing excessive tissue pathology during high dose of viral infection.

First, the authors verified that amphiregulin, an epidermal growth factor family member, was expressed by Foxp3⁺ T cells.

Next, the authors showed that amphiregulin deficient Foxp3⁺ T cells displayed normal suppressive functionality when transferred into T cell-deficient host.

Further experiments revealed that amphiregulin deficiency in Foxp3⁺ T cells did not modify the host immune response to intranasal flu infection (mouse PR8 model).

However, the authors noticed that there was excessive lung tissue damage in response to high (but not low) dose of flu infection in the hosts with Foxp3⁺ T cell-specific deletion of amphiregulin.

Finally, the authors showed that amphiregulin up-regulation was primarily restricted to IL-18R⁺ Foxp3⁺ T cells.

In summary, these data tend to reinforce the idea that Foxp3⁺ T cells could function in a tissue selective manner and are most likely controlled by tissue environment as suggested by Polly Matzinger and Tirumalai Kamala. This could explain why there are so mechanisms of suppression by Tregs, at least one mechanism for each tissue.

What are some of the weaknesses of this study? First, injection of amphiregulin to hosts with Foxp3⁺ T cell-specific deletion of amphiregulin would have been useful. Second, data regarding IL-18R deficient Foxp3⁺ T cells could have provided in vivo confirmation for this study (the authors simply mentioned that they have done experiments with bone marrow chimera to test the role of IL-18R in amphiregulin up-regulation and that it supported their conclusions).

Why is this study important? Because without our mastery of Foxp3⁺ T cell biology we will not be able to make any major, predictable advances in treating human immune related conditions (cancer, allergy, autoimmune conditions).

David Usharauli