If T cell clones are so diverse, what prevents anti-tumor immune response?

Identifying cancer and pathogen-specific epitopes or TCRs may sound intuitive, but it is a futile approach. Diversity of T cell or B cell clones guarantees that the adaptive immune system will always have relevant clones to detect cancer or pathogen.  

Epitopes have no meaning attached to them with one exception.  It is when the said epitope is self. Each body will have different sets of self-epitopes. Every self-epitopes relevant for host's survival are encoded in the thymus, and thymic Tregs are trained to prevent any T cell activity against those epitopes in the periphery. This is called tolerance, and it is antigen[epitope]-specific. 

Then what prevents effective responses to cancers or pathogens? It is commonly but mistakenly believed that Tregs prevent effective T or B cell responses to cancers or pathogens. But Tregs only prevent anti-self response, and it is epitope-specific action.  So, by definition, if Tregs do their job as required, we cannot blame them. But it has nothing to do with cancer or pathogens, which obviously have other epitopes different from self, we call nonself. So, if cancer cells or pathogens express nonself epitopes that are always detected by adaptive immune system, why not everyone can fight it off effectively?

This is because T cells themselves prevent it. Yes, T cells, not Tregs, prevent effective response to cancers or pathogens in certain conditions. What are those conditions? These are condition when polarized T helper cells prevent other T cells functions. Polarization is a pathological state. A Polarized T cell's effect on other T cells is epitope non-specific, meaning, a polarized T helper cell specific to cancer or pathogen nonself epitope A will prevent T cells specific to epitope B, C, D, E, F, etc., to function properly. It is exactly Treg's job to shut down those polarized T helper cells to allow other T cells to manifest their functions and get rid of either cancer or pathogen. And Treg do it, as we already said, epitope-specific manner.

For this reason, it is not so important to identify any cancer or pathogen-specific nonself epitope, but rather to identify an epitope that could activate polarized T helper cells.

To make things even more complicated, one may ask if Tregs are self-specific and act epitope-specific manner, how can Tregs shut down polarized T helper cells that are nonself-specific? It is possible because Tregs are cross-reactive and can inhibit only those polarized T helper cells which share TCR specificity with Tregs.

In other words, control of self-tolerance and control of effective anti-nonself response are one and the same.  

These are 3 papers that together provide a full discussion related to the SPIRAL model we have developed to explain how Tregs work within an adaptive immune system:

 

 

Concurrent cross-reactivity of microbiota-derived epitopes to both self and pathogens may underlie the "Hygiene hypothesis"  

 

https://onlinelibrary.wiley.com/doi/10.1111/sji.12708 

 

Could cross-reactivity rescue Foxp3+ regulatory T cell precursors from thymic deletion? 

 

https://onlinelibrary.wiley.com/doi/10.1111/sji.12940

 

 

 

Microbiota-Specific Foxp3+ Regulatory T Cells Could Control Pathological T Helper Responses

 

https://www.dl.begellhouse.com/journals/2ff21abf44b19838,40a32b197f7156bc,7988864072d2c7c6.html

 

 

   

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

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

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

   

MHC class II epitope presentation modulates microbiota and protects against autoimmunity

It is not clear how exactly microbiota modulates host's immune system. Evidence are largely based on empirical observations and nonspecific factors secreted by microbiota. Presently very little is known if microbiota-immune system interface is also controlled at the level of antigen-specific adaptive immune system.    

 

New study from Diane Mathis lab published in PNAS suggests it may be the case.

 

Her lab studies human type I diabetes (T1D) mouse model known as NOD. NOD mice lack MHC II allele, Eα. In this study they used NOD mice expressing Eα, referred as Eα16/NOD. In mating experiments, they noticed when Eα was expressed by female but not by male parent, baby mice with NOD genotype showed significant protection from developing T1D, suggesting protection was transmitted vertically from Eα16/NOD mother to NOD pups. Interestingly, this protection was lost when pregnant mothers (dams) were treated with antibiotics pointing towards role of microbiota.

Experiments with germ-free sterile mice confirmed this observation.

In summary, this study showed that MHC class II [epitope] presentation modulates composition of microbiota in such a way to harbor species protective against T1D. Again, the authors were unable to specifically pinpoint any specific mechanism of protection, though they reported increase in Foxp3+ Treg numbers in Eα16/NOD mice compared to NOD (but found no difference in microbiota bound to IgA between mouse strains). It is likely that epitope presentation at the level of adaptive CD4 T cells contributed to development of protective environment.

posted by David


    

 

Does TGF-β control T cell autoimmunity independent of Tregs?

When T cells attack body's own antigens its called autoimmunity. Each and every one with the adaptive immune system carry this potential. Mostly two mechanisms prevent autoimmunity: thymic deletion of overtly auto-reactive T cells (recessive tolerance) and Foxp3+ Tregs (dominant tolerance).

 

Within immune system, TGF-β plays important inhibitory role at T cell level. However, since TGF-β is involved in Treg biology, it is not clear if it has Treg-independent role in preventing autoimmunity.

 

New paper published in PNAS tried to answer this question.

 

The authors used OT-II RIP-mOva mice model (on RAG KO background) in which all CD4 T cells express OVA-specific T cell receptor and pancreas express OVA protein. These mice harbor OT-II Foxp3+ Tregs and they don't develop autoimmune diabetes.

To separate effect of Tregs versus TGF-β, the authors either compared TGF-βRII-KO mice vs. Foxp3KO (both on RAG1-KO OT-II RIP-mOva background) or adoptively transferred into RAG1-KO RIP-mOVA mice either Foxp3KO OT-II or OT-II T cells expressing TGF-βRII under the control of estrogen receptor. They noticed that OT-II T cell population lacking TGF-βRII but not Foxp3 could cause or accelerate autoimmune diabetes.

These two set of experiments are central for this paper. However, contrary to the authors' conclusions, these experiments do not fully answer Tregs versus TGF-β question. The main problem is that total TGF-βRII deficiency in all CD4 T cells affects both effector and Tregs (functionally at least if not number wise) while Foxp3 deficiency only affects Tregs. That is to say that if Tregs were TGF-βRII-sufficient and effector T cells TGF-βRII-deficient outcome could be different (WT Tregs might be able to stop effector OT-II cells). Another way to separate the role of Tregs versus TGF-β would be to specifically inactivate TGF-βRII in effector T cells leaving Tregs intact.

posted by David Usharauli


 

IBD converts tolerant antigens into immunogenic

Inflammatory bowel disease (IBD) is a pathological condition wherein body's immune cells wrongly attack its own or commensal microbiota-derived antigens that initiates a vicious cycles of permanent inflammation.

 

However, it is still not clear whether immune system attacks "new" antigens from microbiota or simply it loses adaptive tolerance to "old" ones. 

 

New study in Science Immunology tried to answer it to the extent it was possible to do in mouse model.

 

First, the authors generated IBD condition in mice by treating them with DSS + anti-IL10R. Keep in mind this is highly artificial model. Then, they transferred naïve T cells from previously established transgenic T cell lines specific for unknown commensal antigens that were known to drive Treg phenotype. Naïve T cells transferred into control WT mice generated Tregs while the naïve T cells transferred into IBD-conditioned mice preferably developed into effector T cells.

 

 

 

When analyzed for antigen specificity, the authors found that transgenic naïve T cells were reactive to antigens derived from Helicobacter species that have expanded during IBD-conditioning.

 

 

 

In vivo studies also confirmed that Helicobacter species could induce Treg generation from naïve transgenic T cells in "normal" condition.

 

 

 

Interestingly, transfer of T cells specific for other microbiota species that also underwent expansion during IBD-conditioning did not produce T cell expansion.

 

 

 

Finally, transfer of Treg-TCR transgenic naïve T cells into RAG-KO mice produced IBD when co-injected with Helicobacter species.

 

 

 

What these data indicate? In my view the authors made one correct and one wrong interpretation. First, they were correct to conclude that T cell response to IBD could be directed to "old" microbiota antigens rather than "new" never before seen microbiota-derived antigens. So basically in IBD we are losing tolerance rather than gaining immunity to microbiota antigens.

 

However, they made wrong conclusion that naïve T cells are converted into Tregs in vivo based on context (normal versus IBD). In their study loss of Treg generation is inhibited either during IBD-conditioning or in RAG KO hosts which could argue alternatively that such outcome has to do with failure of naïve T cells to interact with existing Tregs specific for the same or similar antigens in these scenarios (IBD or RAG-KO).

 

posted by David Usharauli

 

 

        

Identification of prostate-antigen specific natural Tregs (in mice)

Foxp3+ Tregs are central player in maintaining tolerance to self and other environmental antigens. However, till to this date we know little of their antigen specificity. It is because unlike conventional CD4+ T cells, Tregs do not secrete [upon antigen recognition] any cytokine that uniquely identifies them. The best marker is still Foxp3 molecule, an intracellular transcription factor.   

So it is always interesting to see new study that could identify Treg epitope, such as this new paper in Immunity that provided evidence that in mice peptide spanning residues 646–658 of prostate-specific TRPM8 channel-associated factor 3 protein (Tcaf3) is a natural epitope for thymic MJ23 TCR transgenic Treg development.

The authors showed that development of MJ23+ Tregs from adoptively transferred MJ23+ thymocytes (un-differentiated T cells) were only supported in hosts expressing intact Tcaf3 (and not in Tcaf3 KO mice).   

Next, using sensitive tetramer based antigen-specific T cell detection, the authors showed that WT mice also harbored Tcaf3[646–658]-tetramer specific T cells that were enriched in Tregs compared to other antigen-specific T cells (2W1S). Interestingly, Aire-KO mice which do not efficiently express peripheral antigens in the thymus harbored reduced numbers of Tcaf3[646–658]-tetramer specific Tregs.

Finally, the authors showed that prostate tissue from Aire KO mice harbored significantly more Tcaf3[646–658]-tetramer specific Tregs compared to prostate tissue from normal mice. I found these particular results problematic because should not normal mice prostate supposed to contain Tregs to prevent autoimmunity? Or are Tregs keeping autoreactive T cells in check in draining lymph nodes? 

In summary, this study showed that in mice prostate-specific Tcaf3[646–658] epitope is a natural ligand that selects Tregs in a Aire-dependent manner.

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

Fragility of FOXP3+ Treg phenotype

This week I am reviewing two papers dealing with different aspects of FOXP3+ Treg biology. Both papers were published in journal Cell. These are predominantly mouse works so no need to get too excited. 

1st paper was published by Dario Vignali's group (department of Immunology, University of Pittsburgh School of Medicine). They have analyzed role of Neuropilin-1 (Nrp1) in Treg phenotype in tumor context. Neuropilin-1 is a marker of thymic Tregs though this notion is not universally accepted. Interestingly, in mouse tumor model, mouse that expressed Nrp1 on half of its Tregs rejected tumor with the same vigor as mouse expressing Nrp1 on all of its Tregs. 

Furthermore, in the context of tumor challenge, Treg-specific Nrp1 deficiency drastically increased IFN-γ production in both KO and WT Tregs.

It appeared that WT Tregs required sensing of IFN-γ derived from Nrp1KO Tregs to acquire "Nrp1KO-like" phenotype. 

Finally, the authors showed that therapeutic effect of checkpoint inhibitor anti-PD1 antibody against tumor required IFN-γ sensing by Tregs.

In summary, this study showed that Nrp1 deficiency makes Tregs fragile by converting them into IFN-γ producer cells which in turn affect WT Tregs phenotype as well. The reason the authors are using term fragility rather than simply instability has to do, they claim, with the difference in FOXP3 expression between fragile and unstable Tregs. 

In a separate study, the authors showed that Treg depletion inhibits hair regrowth (after depilation). 

However, it is not clear why Tregs should be involved in such physiological process when any other innate cells could do the same.   

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

The role of LAG3 in Tregs and how infection modulates immune response to dietary antigens

Lymphocyte activation gene 3 (LAG3) is the most recent checkpoint inhibitor to be targeted in the clinic. LAG3 has been shown to dampen T cell activity. For instance, diabetic-prone mice deficient in LAG3 exhibit accelerated autoimmune diabetes with 100% of KO mice developing it. As Foxp3+ regulatory T cells express LAG3 it was thought that it played a role in Treg activity.

Surprisingly, however, according to new study mice with Treg-specific LAG3 deficiency showed improved rather than diminished protection against development of autoimmune diabetes. 

This unexpected outcome requires rethinking the role LAG3 within immune system. Systemic inhibition of LAG3 by blocking antibodies could enhance tumor progression, for example, by stimulating tumor-infiltrating Tregs.

Another paper, however, with ambitious title I read this week fell flat upon examining actual data. Its objective was to examine how viral infection could modify host's immune response to dietary antigens and lead to celiac disease. According to paper "celiac disease (CeD) is a complex immune disorder with an autoimmune component in which genetically susceptible individuals expressing the human leukocyte antigen (HLA) DQ2 or DQ8 molecules display an inflammatory T helper 1 (TH1) immune response against dietary gluten present in wheat".

To replicate hypothetical scenario that could lead to celiac disease, the authors infected mice with reovirus and simultaneously gave them nominal antigen, OVA, as a dietary antigen. As virus induced inflammation, it led to reduced numbers of Foxp3+ Tregs and parallel increase in Th1 transcription factor, T-bet+ T cells specific for dietary antigen.

The authors concluded that viral infection could modify body's response to dietary antigens. However, such conclusion is premature here as it is not obvious that those T cells that were destined to become Foxp3+ regulatory T cells were actually diverted into Th1 lineage. It is not clear either what would be the outcome of dietary antigen exposure once viral infection is cleared or how long the effect of viral infection modifies a physiological response to dietary antigens.

David Usharauli

   

Lactobacillus reuteri extends lifespan of FOXP3-deficient scurfy mouse via microbiota–inosine–A2A receptor axis

Scurfy mice harbor natural mutation in FOXP3 gene that clinically resembles FOXP3 deficiency. Both scurfy mice and genetically modified FOXP3-KO mice die prematurely within first month of life. In humans, clinically observed FOXP3 deficiencies do not seem to be as lethal as in mice though one could argue that scurfy mice lifespan could be extended if these mice were given human-like medical attention (for example, i.v. feeding, anti-inflammatory medication, so on). 

In this regard, a new paper in Journal of Experimental Medicine is of great interest. It showed that a member of gut microflora, Lactobacillus reuteri, a probiotic microbe, when given orally could drastically extend lifespan of scurfy mice (30d vs. >125d) via microbiota–inosine–A_2A receptor axis.

Morphologically, oral Lactobacillus reuteri significantly reduced tissue inflammation in scurfy mice.

Blood test showed that one molecule Lactobacillus reuteri could restore to WT level in scurfy mice was a purine metabolite inosine.

Indeed, oral inosine was able to recapitulate Lactobacillus reuteri effect on scurfy mice lifespan (and both Lactobacillus reuteri and inosine effects were specifically mediated via adenosine A_2A receptor).

In summary, this study revealed that purine metabolites, inosine or adenosine could protect scurfy mice from tissue immunopathology and drastically prolong their lifespan. It is quite rare to see that one molecule could produce such effect. It would be interesting to see how caffeine consumption affects immunopathologies in humans as it acts as a natural antagonist to adenosine A_2A receptor.

David Usharauli

TCR derived from gut Foxp3+ Tregs can generate two distinct T cell phenotypes

This is my first review of a paper published in new journal from Science family called Science Immunology.

This paper deals with the relationship between TCR and origin of peripheral [gut-residing] Foxp3+ regulatory T cells. Specifically, the authors have used method called somatic cell nuclear transfer (SCNT) that allowed generation a transnuclear (TN) mouse that carries a TCR cloned from gut tissue Foxp3+ Tregs. 

First surprising observation was that these Foxp3+ TCR transnuclear but otherwise unmanipulated mice (called pTreg TN/RKO mice, on RAG KO base) had no Foxp3+ Tregs neither in the thymus or the peripheral lymph nodes (mLNs).

Second, the authors found that transnuclear mice instead harbored specialized IFN-γ⁺CD8αα⁺ intraepithelial lymphocyte (IEL) subset.

Indeed, adoptive transfer Foxp3+ TCR transnuclear T cells into WT host revealed that donor T cells could differentiate either into CD8αα⁺ intraepithelial lymphocyte (IEL) subset or acquire Foxp3 marker as representative of peripheral Treg subset. Of note, development of both of these subsets dependent of gut microbiota.

Finally, the authors showed that donor Foxp3 transnuclear T cells transferred into T cell-deficient hosts did not induce gut inflammation even if they were derived from scurfy background (lacking ability to express functional Foxp3 protein), implying that functionality of CD8αα⁺ intraepithelial lymphocyte (IEL) derived from Foxp3+ TCR T cells did not cause any tissue pathology.

In summary, this study showed that depending on circumstances TCR derived from intestine Foxp3+ Tregs can drive development of two distinct subsets with immunoregulatory role: CD8αα⁺ intraepithelial lymphocyte (IEL) subset or peripheral Foxp3+ Treg subset.  

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 

PD-1 signaling assists regulatory T cells when Foxp3 is down

Foxp3⁺ regulatory T cells (Tregs) maintain peripheral tolerance to self. Several molecules expressed by Tregs, such as CTLA-4, play crucial role in maintaining this state of tolerance to self. PD-1 is another such molecule, though its role in Tregs function is less clear.

New paper in PNAS shed some light in PD-1 role in Tregs biology. It found that PD-1 keeps Tregs in shape when Foxp3 level is unstable.

This study arose from unexpected observation in new gene-modified mice where GFP was inserted in Foxp3 locus (mice carrying the IRES-GFPcre reporter KI at the 3′ untranslated region of the FoxP3 gene, FoxP3-GFPcreKI). When these mice were crossed with PD-1KO mice, it was found that male offspring of such cross showed early death, reminiscence of Foxp3KO mice. This was surprising since PD-1KO mice ordinarily do not show such phenotype.

Further experimentation found that GFP insertion affected Foxp3 stability thus resulted in reduced Foxp3 levels in FoxP3-GFPcreKI mice.

Indeed, FoxP3-GFPcreKI/PD-1KO male mice could be rescued with transfer of WT Tregs irrespective of their PD-1 expression indicating that PD-1 function were dispensable Tregs with normal level of Foxp3.

Finally, the authors showed that absence of PD-1 could further destabilize "Foxp3-low" Tregs function (conversion into ex-Foxp3 Tregs) resulting in lethal autoimmunity.

In summary, this study revealed that PD-1 could contribute to Tregs function in situations that affects Foxp3 stability.

David Usharauli

Runx3-dependent intraepithelial lymphocytes (CD4IELs) control gut tolerance to dietary antigens

Few days ago journal Science published another immunological study. This hard to read research paper showed that special subset of Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs) found within intestinal epithelium play distinct and dominant role in preventing gut inflammation to dietary antigens.

It appears that induction or maintenance of Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs) depends on antigens derived from either from gut flora [microbiome] or food.

The main finding of this paper was observation that antigen-specific T cell monoclonal mouse strain deficient for Foxp3 expression [but not Foxp3 wt] depleted of CD4IELs using anti-CD8α antibodies during antigen feeding showed severe intestinal inflammation and diarrhea. It suggests Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs) play distinct and separate [that from Foxp3wt T cells] role in protecting intestinal tissue against antigenic inflammation.

Why is this paper relevant? For one, it indicates that tolerance to food antigens in physiological situations, for example during feeding, could be mediated by Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs), and not by Foxp3+ CD4+ T cells, as scientists ordinarily assume. However, for now, we know relatively little about tissue-resident innate-like or adaptive-like cells, such as these Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs).

David Usharauli