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

Gut IgA is mostly T-independent and mostly microbiota-centric

The most IgA is produced at mucosal surfaces in response to antigenic exposure. While IgA is considered to be part of adaptive immune system, controversy still persists regarding signals needed for its production.

New article in journal Immunity may put an end to this discussion, finally. The authors, led by Albert Bendelac (he was one of the discoverer of NKT cells), have used IgA-seq methodology and showed that gut IgA production is mostly T-independent and mostly specific for gut microbiota. 

Initially, the authors showed that proportion of gut microbiota both in small or large intestine are covered with IgA (there are IgA^- and IgA⁺).  

Next, the authors have used IgA-seq approach to phenotype microflora. This method consists of capturing of IgA from the feces followed by sequencing of antigens attached to IgA (in this case microbial 16S rRNA).

Interestingly, unlike IgA^- microflora, there was a clear overlap between IgA⁺ microflora both from large and small intestine, implying common origin.

Next, the authors elucidated the origin of gut IgA. First, both wild-type and T cell-deficient mice found to produce comparable gut microbiota-centric IgA.

Second, no difference was found between wild-type and T_FH-deficient mice (Bcl-6^ΔT) either.

However, the authors showed that few selected microflora members, such as SFB and Mucispirillium could elicit T-dependent IgA response.

Finally, the authors' experiments revealed that most of gut IgA was produced by subset of non-canonical B1 cells called B1b cells in a T-independent manner.

In summary, this study showed that (1) most of small intestine microflora is coated with IgA (2) these IgA are produced in absence of T cell help (3) these IgA is derived from B1b cells whose repertoire was broad and of comparable diversity to that of canonical, spleen B cells and (4) some members of microflora, SFB and Mucispirillium  did appear to induce IgA production via canonical T-dependent manner.

Why is this study relevant? First, it suggests that gut immune health is established in absence of T helper cells via B1b cells. Not much is known about B1b population. Is it possible that many gut immune pathologies (ulcerative colitis, celiac disease, crohn's disease) originate because of alteration in B1b cells?      

David Usharauli

Pair socks from a pile: reconstructing TCRβ and TCRα pairs through sequencing

T cell receptor (TCR) β and α chains are generated in the thymus (some in the gut) via randomly rearranged V, J, D, C segments (unlike TCRβ chains, TCRα chains lack D segments). After rearrangement, some TCRβ and TCRα chains form productive pairs, we call TCRs. Note, not every TCRβ and TCRα chains can form productive pair. Afterwards, T cells expressing surface TCR undergo positive and negative selection and enter blood circulation to start patrolling the body.

Advances in personalized medicine created a niche to study TCR specificity in clinical setting. This is especially true for cancer therapy where adoptive transfer of ex vivo expanded tumor antigen specific T cells show protection against various lymphoid malignancies.

Ordinarily TCR specificity are elucidated by tetramer or hybridoma technologies, followed, if necessary, by TCR sequencing. These two, separate steps ensure selective expansion of T cell clones of interests. However, these methods are (1) technically difficult to accomplish (few scientists have "good" hands for in vitro experiments), (2) time consuming and (3) limited in scale (10-100 specificity). 

Another method is a direct TCRβ and TCRα massive-parallel sequencing. In this regard, sequencing technologies developed by Adaptive biotechnologies from Seattle has been cited by many high profile academic papers. 

However, TCR sequencing (typically only TCRβ sequencing) per se cannot provide an answer about TCR specificity, i.e. which TCRβ chain pairs with what TCRα chain. The knowledge of genetic structure of both chains in TCR, however, would allow reconstruction of 3D structure of TCR and it's specificity determination with algorithms that work similar to MHC+peptide algorithms.

In a recent article from Science Translational Medicine, Adaptive's team tried, in collaboration with the scientists from the local Cancer Center, to develop a method that would allow such TCRβ and TCRα chain pairing determination.

The methods itself is based on simple idea, if I understood it correctly. It requires some statistical analysis but principle works the following way: 

1. T cells are collected and distributed among 96 wells. Number of T cells per well can very based on statistical analysis. 

2. In 96 well plate, each well contains an unique small oligo DNA barcode that will be amplified along side with T cells derived cDNA. 

3. T cell derived cDNA is amplified with primers specific for TCR V and C regions and then sequenced.

Afterwards, if every time a particular Vβ and Vα genes are detected together in the same randomly barcoded wells, it is assumed that those two are pairs. This is in principle. Since there are potentially hundreds of of thousands unique TCR pairs, validation of experimental design is critical. In my view, strict validation of this type of readout would require actual determination of pairing using tetramer catch or hybridoma based expansion of T cells and then specific sequencing of recovered clones. 

Validation step the authors suggested is not entirely clear from paper description and may not be sufficiently robust to be accept as a gold standard for pairing. Another weakness is amplification step since it is not clear whether every V and C primer could amplify the target region with the same optimal rate.

David Usharauli

Two distinct, mutually exclusive immune signatures, TH2 and TH17, determine therapy responsiveness in asthma

Asthma is a chronic condition manifested in episodes of airway hypersensitivity (inflammation, smooth muscle constriction) to innocuous signals that ordinarily would cause no tissue response in average individual. Eventually this chronic state leads to tissue remodeling and reduction of oxygen-rich air available for lung. 

Classical, allergic asthma episodes are driven by type 2 (TH2) immune response dominated by IL-4 and IL-13. However, more recently another category of asthma characterized by TH17 signature has been described. It appears that TH17 signature asthma may be more resistant to current asthma therapy.

The new study in Science Translational Medicine provided additional support for asthma stratification based on immune signature. This Genentech study showed that asthma immune profile can be stratified into mutually exclusive, non-overlapping TH2^high and TH17^high signatures.

This is a short paper. Basically, the authors analyzed airway gene signature from 51 asthma patients. They found that asthma patients' immune signature fall in 2 categories: either TH2^high or TH17^high. There is additional double negative category but its immune signature is not known.

Interestingly, both TH2^high and TH17^high signatures were associated with eosinophil infiltration.

Since current asthma therapies mainly target TH2 signature, the authors tested the outcome of α-IL-4/IL-13 blockade on animal asthma model (house dust mite antigen sensitization). As expected, dual blockade of TH2 cytokines significantly reduced airway inflammation.

However, TH2 cytokine blockade also induced TH17 signature, as would have been expected from in vitro studies (though it is not entirely clear from the data if this shift to TH17 pathway induced any clinically-relevant airway inflammation here).

Finally, the authors showed that blockade of both TH2 and TH17 pathways may be necessary to avoid inverse increase in TH2-driven airway hypersensitivity during anti-IL-17 therapy.

In summary, this study suggests that clinical trial design for α-IL-17 target therapy in asthma patients may need modification based on this findings. For example, α-IL-17 therapy alone may be not sufficient or that eosinophil signature could not be used as a exclusion factor for α-IL-17 therapy.

David Usharauli

IL-9 puts the brakes on tumor growth

TNF family receptors control the magnitude and functionality of immune response. Several of them have been targeted for therapeutic purpose (4-1BB, CD40L, BAFF-R). 

GITR (Glucocorticoid-Induced TNFR-related protein), a member of TNF superfamily, was initially implicated in inhibiting regulatory T cell (Tregs) function. However, work on this molecule receded into oblivion for these past few years due to overall failure of immunological research to develop Tregs based therapies.

Now, new study in Nature Medicine put forward novel mechanism of action for GITR. This study showed that GITR stimulation amplified IL-9 production from T helper cells and contributed to tumor protection.

First, the authors showed that GITR stimulating antibody inhibited tumor growth in a CD4-dependent manner.

Next, they showed that GITR stimulation improved tumor protection in an IL-4Rα-dependent manner.

This observation led to discovery that GITR stimulating antibody improved tumor protection via IL-9 production [that requires IL-4Rα signaling].

In vitro experiments showed that GITR stimulating antibody indeed promoted Th9 differentiation (Th2 cells showed minimal cancer protection). 

Finally, the authors showed that GITR stimulating antibody shifted the balance in favor of CD8 T cells in tumor environment.

These results suggest that GITR targeting could provide additional layer of protection against solid tumors. 

David Usharauli 

Endogenous gut flora drives colonic RORγ+ Treg development

Foxp3 transcription factor drives development of specialized CD4 T cell subset called regulatory T cells (Tregs) which can protect against immune pathology. 20 years has passed since their re-discovery in 1995 and 10 years has passed since identification of Foxp3 transcription factor as a master regulator of Tregs induction. Still, there is not a single drug or cell therapy protocol approved that involves Tregs. Why? In my opinion it has to do with the fact that we don't how Tregs work.    

Here is another example published yesterday in journal Science. In this article, the authors led by Mathis-Benoist team have reported that colonic gut microbiota induced RORγ, a transcription factor commonly associated with Th17 cell development, in colonic Foxp3+ Tregs and its expression played a beneficial role in maintaining colonic tissue health.  

First, the authors showed that colonic but not spleen Foxp3+ T regs expressed RORγ transcription factor.

However, unlike RORγ+ inflammatory Th17 cells, RORγ+ Foxp3+ Tregs do not express IL-17.

Next, the authors showed that induction of colonic RORγ+ Foxp3+ Tregs was linked to multiple gut microbiota species (of note, the authors did not find any correlation between SCFA expression and RORγ+ Foxp3+ Tregs).

Finally, the authors showed that specific deletion of RORγ in colonic Foxp3+ T regs tipped the balance in favor of IL-17 and IFN-γ and worsened gut inflammation.

In summary, these results indicate that antagonistic transcription factors RORγ and Foxp3 co-expressed and are indeed necessary for proper functioning of tissue specific Tregs. This even more complicates already messy field.

David Usharauli

Stimulating CTLA4-Ig mimetic abatacept relieves autoimmune inflammation

It is quite counter-intuitive to believe that human common immune deficiencies are characterized by autoimmune inflammations in various organs. Such observations suggest that what we call immune deficiencies are actually immune disregulations. Immune disregulation would imply that such patient is not able to produce for example sufficient amount of IgA to mucosal antigens but will have excessive response dominated with TNF-alpha that would appear as autoimmune inflammation.

New paper in journal Science provided an example for such immune deficiency, LRBA, and its rescue by abatacept, a drug that mimics natural CTLA4 action.       

The authors described several patients with deficiency in LRBA (lipopolysaccharide responsive beige-like anchor protein) expression and showing signs of autoimmune inflammation.

The authors found that these LRBA deficient patients had reduced CTLA4 protein level (but not other molecules such as CD40L, CD107a), a phenotype that could be reproduced in healthy cells with LRBA siRNA. 

Interestingly, chloroquine, a drug that reduces lysosomal degradation could improve CTLA4 expression in cells from LRBA patients implying that excessive degradation of CTLA4 protein in LRBA deficient patients.

Finally, the authors showed that CTLA4 and LRBA colocalize in endosomal vesicles, providing a mechanistic explanation for low levels of CTLA4 protein in LRBA patients.

In summary, these results provide a rationale for use of stimulating CTLA4-Ig mimetic in treatment of LRBA deficiency.

David Usharauli