Tregs muscle up in the periphery

There is still a lot of confusion regarding role of peripherally generated Foxp3+ Tregs. Data so far indicate that peripherally induced Tregs play limited [if any] role in maintaining check on auto-reactive T cells. However, extra-thymic Treg population could prevent "excesses" of other T helper class differentiation.

For example, transcription factor, Musculin, ordinarily found in muscle cells, appears to stabilize Foxp3+ Tregs phenotype at the mucosal surfaces.  

High level of Musculin expression was detected in in vitro induced Foxp3+ Tregs (iTregs).

Musculin deficient mice harbor fewer Tregs at gut mucosal sites.

Musculin deficiency in T cells shifted their differentiation pathway from Foxp3+ Tregs into Th2 cells.

Indeed, removal of canonical Th2 cytokine IL-4 reversed such Th2 shift in Musculin deficient T cells under iTreg differentiation setting.

Musculin deficient iTregs also failed to inhibit house dust mite allergen induced inflammation in lungs upon adoptive transfer.

For me these data confirm that stability of extra-thymic Foxp3+ T regs in the periphery requires coordinated action of several factors, one of which, appears to be Musculin. Musculin prevents excessive Th2 differentiation in settings that may favor local Tregs generation. Extra-thymic Treg differentiation pathway does not appear to play a significant role in maintaining peripheral tolerance. Rather, their role is mostly, in my view, consist of absorbing and neutralizing any "excesses" in T helper differentiation, in this case Th2 pathway.

David Usharauli

    

A minor subset within CD11c+ dendritic cells is primarily responsible for peripheral Treg expansion

FOXP3+ Tregs play a major role in tolerance maintenance in the periphery. Most of FOXP3+ Tregs are thymus derived. Thymus is a specialized lymphoid tissue that generates novel T cells from their bone marrow precursors. The question whether naive T cells could convert into FOXP3+ Tregs in the periphery has not been settled. 

It is believed that a "steady-state" condition favors FOXP3+ Tregs conversion in the periphery. However, a concept of "steady-state" is an arbitrary one, defined as absence of deliberate immunization or experimentally observed infection. In fact, whether "steady-state" truly exist is an open question as well.

Why this matters? Almost everyone agrees that in absence of so called "steady-state" naive T cells would convert into effector T cells rather than into FOXP3+ Tregs following antigen recognition. For example, if one wants to generate new FOXP3+ Tregs specific for particular antigen to treat autoimmune diseases, this task would be almost impossible to achieve if condition of "steady-state" does not actually exist in the body [from T cells' "point of view"]. 

Also, what cell types are responsible for that supposed FOXP3+ Tregs conversion? A new study in Immunity clarified this question somewhat. It showed that even in "steady-state" condition only minor subset of DCs within CD11c+ population defined by DEC205/CD8 expression were responsible for FOXP3+ Tregs "conversion" in T cell-replete mice [which harbor endogenous FOXP3+ Tregs].

For this study, the authors have used chimeric anti-DEC205 Ab [or anti-CD11c chimeric Ab as a control] that incorporate antigen of interest [MOG or OVA]. When injected into mice chimeric anti-DEC205 Ab, but not control, could "convert" naive MOG or OVA-specific T cells into FOXP3+ Tregs.

It appeared that DEC205+ CD11c+ DCs were also primarily BTLA+ and its expression were required for FOXP3+ Tregs induction.

Interestingly, the authors proposed that BTLA to HVEM signaling in naive T cells up-regulated CD5 and permitted FOXP3+ Tregs conversion even in presence of inflammatory cytokines such as IL-4 and IL-6.  

However, in my view, such mechanism of FOXP3+ Tregs conversion even in presence of inflammation sounds counter-intuitive. Wouldn't it also induce FOXP3+ Tregs conversion from naive T cells specific for nonself antigen derived from pathogens during inflammation? Otherwise, how can system make sure that only self antigens are presented by DEC205+ DCs? The authors could only admit that this tolerance mechanism somehow only affects "self and tolerizing antigens". Also, what about endogenous FOXP3+ Tregs in these mice? Is it possible that endogenous thymus FOXP3+ Tregs are involved in assisting in FOXP3+ Tregs conversion, rather than DEC205+ DCs doing it alone from scratch? If so, implications are very different.  

David Usharauli

Classical DCs are just that classy for oral tolerance and peripheral Tregs

This week I had a hard time to find any good immunology paper that makes me happy to review. Two days ago I read one new paper from immunity with intriguing title about "Organ-Specific Regulatory T Cells" that was so weak (structurally, conceptually and experimentally) that I have no idea how it ended up in that journal. Yesterday I read another new paper from Nature Immunology about dendritic cells (DCs) that had 8 figures in total but only 3 figures were relevant and conclusive, though even that lacked experimental strength. Let me explain my point.

In this paper the authors wanted to find out which type of DCs were involved in oral tolerance (oral tolerance has been used in clinics, for example, to reduce allergy to food products). To do that, the authors compared mice that lacked either monocyte-macrophage–derived DCs [MM^DTR mice] or pre-DC–derived classical DCs [zDC^DTR mice or CD11c^DTR mice as a positive control]. When these mice were fed with OVA protein (to induce oral tolerance) and later challenged with the same antigen in the skin, only mice lacking cDCs showed loss of oral tolerance induction (no reduction in ear swelling or OVA-specific IgG1/2c levels).

Similar results were obtained in upper respiratory airway and lung allergy model (no reduction of eosinophil recruitment in BAL in zDC^DTR mice that lacked classical DCs).

Mechanistically, lack of oral tolerance was "associated" with total lack of OVA-specific peripheral [adoptively transferred OT-II] Treg induction in zDC^DTR mice (please note that though MM^DTR mice showed dramatic reduction of peripheral OVA-Treg generation, it did not affected oral tolerance induction. See Figure 1).

In summary, this study showed that classical DCs were relevant for oral tolerance and antigen-specific peripheral Treg induction, but these two processes were not necessarily connected (at least no experiments were shown to make this connection). So what's the point of showing all these? I don't have an answer to that and probably neither the authors do.

David Usharauli

Some anergic T cells are precursors for Foxp3+ Tregs

T cell anergy is defined as T cell non-responsiveness to [secondary] antigen challenge. This concept has originated  in 1980s to explain T cell behavior following primary encounter with antigen (signal 1) in absence of co-stimulation (signal 2). Later, anergy concept was expanded to include T cell behavior following repetitive antigen encounter, similar to what today we would call T cell exhaustion.    

Generally speaking, anergic T cells are different from canonical thymic Foxp3⁺ Tregs. Anergic T cells do not express Foxp3 marker. While there are several surface and transcription factors that could "identify" anergic T cells, classical test for anergic T cells is to run several functional cytokine secretion assay and if memery/effector T cells do not produce cytokines, especially IL-2, and do not express Foxp3, you can call it anergic T cells. It is primitive but its all we have right now.  

In the past scientists thought that anergic T cells were necessary to maintain peripheral tolerance against self-antigens. It was believed that anergic T cells would compete for antigen access with self-reactive naive T cells (recent thymic emigrants) and would not allow their activation. This model lost its appeal once Foxp3⁺ T cells were re-discovered and re-defined. So, presently, functional significance of anergic T cells is in a kind of limbo.

So, it was interesting to see new paper in Nature Immunology that revisited the role of anergic T cells. In this study the authors tried to make sense of anergic T cells by postulating that some portion of anergic T cells represent precursors for classical Foxp3⁺ T regs.

First, the authors showed that among Foxp3-negative T cells, FR4^hiCD73^hi markers define anergic T cells (by low IL-2 production) that develop following antigen encounter in absence of co-stimulation (for example, pregnant females harbor pregnancy-associated anergic T cells specific for antigen [2W1S] expressed by male fetus). Interestingly, majority of those Foxp3-negative anergic T cells express neuropilin-1 (Nrp-1), a marker for thymus Tregs.

Next, the authors showed that in functional assays, FR4^hiCD73^hi anergic T cells behave very similar to classical Foxp3⁺ T cells.

Furthermore, when polyclonal Foxp3^-FR4^hiCD73^hi anergic T cells were transferred into lymphopenic host, anergic T cells gave rise to classical Foxp3⁺ T cells (compared to naive or effector T cells).

Importantly, if those newly formed Foxp3⁺ T cells [derived from anergic T cells] were selectively deleted it led to systemic autoimmune disease, implying functional role for such daughter Foxp3⁺ T cells.

Finally, the authors showed that new Foxp3⁺ T cells formed from anergic T cells could prevent arthritis development when co-transferred with cartilage antigen-specific KRN T cells (that themselves do not become anergic or develop into Foxp3⁺ T cells).  

In summary, this study showed that some anergic T cells (especially Nrp1+ subpopulation) could give rise to peripheral Foxp3⁺ T cells. However, not all T cells can produce anergic T cell that could serve as a precursors for Foxp3⁺ T cells (for example KRN T cells or 5C.C7 CD4 T cells). It remains seen what role, if any, anergic T cells play in un-manipulated host. So far we lack tools to selectively deplete anergic T cells in un-manipulated host to test their real-world biological significance. Also, since anergic T cells resemble thymic Foxp3⁺ T cells in Npr1 expression, it will be interesting to found out whether anergy induction is a part of thymic Foxp3⁺ T cell development program, in general.

David Usharauli