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

Protozoa-enabled non-genetic colitis after T cell transfer in immune-deficient mice

Adoptive transfer of naive T cells into T-cell deficient host mice has been used as a colitis (gut inflammation) model that led to discovery of FOXP3+ T regulatory cells which when co-transferred with naive T cells prevented gut immunopathology.

A new study in Journal of Experimental Medicine, however, provided evidence to show that there is a limit how much FOXP3+ T regs could do. This study found that adoptive transfer of naive T cells into Rip2−/−Rag1−/− mice (RIP2 is an essential signaling adapter molecule downstream of both NOD1 and NOD2) led to protozoa, Tritrichomonas muris-enabled dominant colitis that could not be prevented by FOXP3+ T regs.

Initially, the authors observed in non-littermates that naive T cell transfer led to severe gut inflammation in  Rip2−/−Rag1−/− host as compared to just Rag1−/− mice [though it is strange that in their mouse facility Rag1−/− host did not show weight loss after T cell transfer].

Interestingly, colitis in Rip2−/−Rag1−/− hosts could not be prevented by co-transfer of FOXP3+ T regs.

Co-housing and littermate control control experiments confirmed that a non-genetic factor was responsible for colitis development in Rip2−/−Rag1−/− hosts and that factor could be transferred between mice when co-housed together.

Fecal matter analysis showed that Rip2−/−Rag1−/− hosts were selectively enriched with protozoa Tritrichomonas muris, and that its transfer to other mouse accelerated T cell-mediated colitis.

In summary, this study shows that Rip2−/−Rag1−/− double deficient hosts harbor protozoa Tritrichomonas muris that by itself or through modulation of gut microbiota establishes a dominant colitogenic gut ecosystem that is even transferable to genetically non-related mice.  

David Usharauli

  

Carbon-derived environmental pollutants induce atopic dermatitis-associated skin itch via Artemin

It has been recognized that various organic [carbon-derived] pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). AhR initiate the expression of genes encoding detoxification enzymes in response to xenobiotics (foreign chemicals, not ordinarily found in the body). Such response, if overwhelmed, could lead to pathology. As an example, studies have revealed a relationship between air pollution and the prevalence and exacerbation of atopic dermatitis (AD), a chronic, itchy skin inflammation. 

New study in Nature Immunology showed that AhR signaling in response to organic pollutants initiates skin itch by producing neurotrophic factor, Artemin.

Initially the authors created mice that would constitutively express Ahr in skin epithelial cells (AhR-CA mice). These mice developed more itchy skin and their skin contained more of inflammatory cytokines and cells.

In addition, skin of Ahr-CA mice contained more abundant network of neurons associated with itching behavior (TRPV1+ neurons).

The authors found that neurothropic factor Artemin was highly expressed in skin of Ahr-CA mice.

Depletion of Artemin via antibody injection reduced itching behavior and skin epithelial neuronal density.

Finally, mice lacking Ahr in skin epithelial cells (approximately 70% efficiency for the deletion of Ahr specifically in the epidermis) expressed reduced level of Artemin and display less skin inflammation in response to organic pollutants (though skin-specific Artemin deficiency would have been more valuable here).

In summary, this study suggests that air pollution by carbon derivatives, such as diesel exhaust particles, could initiate manifestation of atopic dermatitis by hyper-activation of the skin Ahr function.

David Usharauli

Part of AIRE KO phenotype (APECED in humans) is dictated by gamma-delta T Cells

AIRE deficiency in mice (human equivalent of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, APECED) leads to chronic tissue immunopathology. Due to role of AIRE in generation of thymic FOXP3+ T regulatory cells, it is believed that underlying cause for tissue pathology on AIRE KO background is a lack of Tregs specific for tissue specific antigens.

However, the new data in journal Immunity from the research group which pioneered the study of AIRE suggest that part of AIRE KO phenotype is dictated by changes in γδ T cells secreting cytokine IL-17.

The authors observed that while total γδ T cells numbers were not different WT and AIRE KO littermates, γδ T cells expressing IL-17 were up-regulated in AIRE KO mice.

Interestingly, AIRE KO mice also deficient for γδ T cells (double deficient mice) showed marked resistance to immunopathology to such tissues as eyes and lungs (though other target tissues were still susceptible).

At the cellular level, there was a significant increase in IL-23r-GFP+ γδ T cells (marker for IL-17+ γδ+ T cells) in retinal tissue in AIRE KO mice at early age, even before tissue pathology was apparent. Of note, the fact that IL-17+ γδ+ T cells are present in retinal tissue in WT mice as well points to its role in physiological processes, not just in pathology).

In summary, this study indicates that subset of γδ T cells could play a leading role in initiating certain tissue pathology on AIRE KO background.

It is not clear, however, how changes in γδ T cell compartment relates to changes in Treg compartment on AIRE KO mice. Also, the role of tissue microbiota and its changes in KOs should be considered to fully understand immunopathologies. In this paper the authors provided the answers to neither to these two relevant topics.

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

Tetra-punch against solid tumors

Checkpoint blockade therapies has become a gold standard for cancer immunotherapy. However, only in minority of cancer patients did these antibody therapies show significant benefits. Many think that a multi-pronged approach to cancer therapy could tip the balance in favor of anti-tumor therapy.

For sure, data from mouse studies support this line of thinking. For example, this week Nature Medicine published a mouse study showing dramatic benefits of immunotherapy when four different approaches were combined: 

1. Anti-cancer Antibody (A)

2. Long-lived IL-2 (I)

3. Checkpoint PD1 inhibitor (P)

4. Cancer Vaccine (V)

Referred as AIPV this experimental tetra-pronged immunotherapy could clear an established solid tumors (melanoma, breast cancer, adenocarcinoma) in 75%-80% of mice.

Success of AIPV therapy depended mostly on CD8 T cell and NK cells.

Of note, however, frequency of IFN-γ⁺ CD8 T cells did not correlate with anti-tumor effectiveness.

Interestingly, through AIPV could induced endogenous anti-cancer antibodies that transferred protection in naive hosts against intravenous tumor inoculum, B cell deficient mice were still protected against tumors when immunized with AIPV.

Finally, AIPV protected against autochthonous [endogenously developed] tumor in Braf^CA Pten^loxPTyr::Cre^ERT2 mice.

In summary, this mouse study shows that multi pronged immunotherapeutic approach could significantly improve survival rate during cancer therapy. The authors claimed that "AIPV therapy was associated with minimal systemic toxicity, as mice did not show weight loss or substantial elevation in the amounts of liver enzymes in the blood".

Of course, it is difficult to compare outcome in mouse study versus human study. In humans, even single approach with anti-PD1 antibody frequently leads to lung or liver toxicity. Now imagine injecting cancer patients with 4 different immunotherapeutics. So, we have a long way to go before immuntherapy will show the same acceptable-level effectiveness in humans as it does in lab mice.  

David Usharauli 

   

FOXP3+ Tregs and allergic TH2 cells recognize mutually exclusive allergic proteins

This week journal Cell published new data about FOXP3+ Tregs that could significantly advance our understanding of immune regulation.  

One reason why it is so difficult to study human FOXP3+ Tregs is that we don't have a good surface marker that selectively identifies human FOXP3+ Tregs from that of conventional T cell memory. As a consequence, we can't easily study antigen-specific human FOXP3+ Tregs.

In this new paper, the authors proposed that expression of CD137 versus CD154 identifies FOXP3+ Tregs versus Tconv in antigen-specific stimulation assay. Using CD137 as a marker, the authors showed that presence of FOXP3+ Tregs specific for variety of nonself antigens, including allergens.

The vast majority of human antigen-specific FOXP3+ Tregs displayed memory CD45RO+ phenotype.

Interestingly, TCR Vbeta analysis revealed mutual exclusivity of FOXP3+ Tregs and Tconv memory.

Next, when analyzing T cells from allergic individuals the authors first confirmed that indeed allergic individuals harbor significantly more allergen-specific TH2 cells.   

However, surprisingly, the authors found that allergic individuals harbor normal numbers of allergen-specific FOXP3+ Tregs.  

To understand this finding, the authors conducted more stringent allergen-specific assay by culturing T cells with individual allergen protein rather than the whole allergen extract as customary (the whole extract contains several related proteins). Indeed, this modified assay showed mutual exclusivity of antigens recognized by human FOXP3+ Tregs and allergic TH2 cells.   

In summary, this paper showed that unlike conventional wisdom, failure of FOXP3+ Tregs to control TH2 cells is antigen-specific. In essence, allergic individuals have specific "holes" in FOXP3+ Tregs repertoire that do not allow them to prevent allergen-specific naive T cells differentiation into allergic TH2 cells. This is first checkpoint. However, simple absence of allergen-specific FOXP3+ Tregs is not sufficient to induce allergy. Second checkpoint for allergy development requires additional factors, not yet fully understood, that promote TH2 response.

David Usharauli 

Bifurcation of type 2 immunity

Type II immunity is referred to a Th2 dominant immune response to a wide array of proteases, venoms and mechanical irritants. Both innate ILC2 cells as well as adaptive Th2 cells are involved in this process. The relationship between innate and adaptive components of type II immunity is still being defined.

A new study from Richard Locksley's lab published in Nature Immunology showed that priming of Th2 cells in the lymph nodes and their subsequent type II effector functions in the peripheral tissues could be bifurcated.

For example, the authors showed that parasitic nematode Nippostrongylus brasiliensis (Nb)-infected mice triple deficient in sensing of TSLP, IL-25 and IL-33, the epithelial cytokines which have been linked to Th2 cell function, have normal lymph node IL-4+ Th2 differentiation and IgE production, but significantly diminished potential to secrete IL-13 and IL-5, effector Th2 cytokines, in the periphery. 

Importantly, such bifurcation of Th2 effector functionality was T cell intrinsic by sensing locally produced "release" cytokines, TSLP, IL-25 and IL-33.

In summary, this study indicate that even at the level of Th2 cells their effector functionality could be bifurcated depending on the local tissue micro-environment. This concept is important to better understand how to treat different types of allergies, for example, IL-4/IgE dominant systemic allergies versus IL-5/IL-13 dominant local tissue chronic allergies.     

David Usharauli

Antigen-specificity of human FOXP3+ Tregs

Foxp3+ regulatory T cells (Tregs) control immune response to prevent immunopathology. However, unlike conventional T cells, it is hard to determine antigen-specificity of Foxp3+ Tregs in a random T cell pool. Tregs do not secrete anything unique and they do not even proliferate when exposed to antigens in vitro, two functional readouts that are still used as a gold standard for determining antigen-specificity of conventional T cells. 

Hence, we have no clue as to antigen[epitope] specificity of vast majority of human Tregs. Specificity of T cells could be also determined by non-functional readout such as tetramer staining. This is what the authors in new PNAS paper have used to determine antigen-specificity of Tregs. 

They found that adult human peripheral blood contains FOXP3+ T cells that stain with tetramers specific for self as well as nonself peptides (Flu, melanoma protein, HIV epitopes).

Interestingly, frequency of antigen[epitope]-specific FOXP3+ Tregs varied among donors, but they were, on average, equally distributed among self and nonself [epitope]-specific Tregs, except Flu HA epitope.  

Finally, comparison of neonatal [cord blood] and adult blood revealed that actual number of [epitope]-specific Tregs / per ml of blood did not change much between newborn and adult indicating that most of Tregs tested in this study were generated already by the time of birth.

In summary, this study revealed that human peripheral blood contain Tregs specific for both self nonself antigens. Since tested donors were negative for some of the infection such as HSV, CMV or HIV, it begs the question what [cross-reactive?] antigens maintain CMV or HIV-specific Tregs in antigen-naive [antigen-unexposed] hosts?    

David Usharauli

Engineering T cells to cellular factories with synthetic Notch receptors

This week journal Cell published new article from Wendell Lim's lab at UCSF (also a founder of Cell Design Labs) that reads like a science fiction story. It was a continuation of previous work that focused on developing customized molecular architecture based on Notch core regulatory domain attached to synthetic extracellular recognition and intracellular transcriptional domains (SynNotch). By changing extracellular and intracellular domains one can design T cells producing molecule of interest upon engagement with specific ligand. 

For this new paper, the authors managed to transform T cells into cellular factories which upon specific SynNotch stimulation were making and expressing (a) cytokines (b) checkpoint inhibitors (c) bi-specific antibodies (d) CARs (e) transcriptional factors (f) lytic granules.

Basically, T cells are transfected with vector containing SynNotch module linked to promoter encoding molecule of interest. The most of the experiments were done in vitro. One in vivo experiment the authors put in paper was similar in overall design to one previously reported. In short, NSG mice were injected with K562 tumor expressing GFP + CD19 or only CD19. Afterwards T cells containing SynNotch module designed to recognize GFP were infused. Upon GFP recognition these T cells could start expressing soluble Blinatumomab, a-CD19/CD3 BiTE molecule that in turn can engage conventional TCR and produce T cell activation. Indeed, the authors showed that only tumor cells expressing both GFP and CD19 were efficiently controlled by engineered T cells (though its is not clear why soluble BiTE antibodies would not diffuse freely and engage single CD19+ tumors as well).    

While all these results look very impressive, it nonetheless lack some of the critical elements that are required to properly analyze beneficial effect of SynNotch. For example, in vivo experiments were done in mice that does not express tumor antigens naturally (anti-CD19 here was human in origin). So we can't tell how such T cells would behave in "human-like" environment. Similarly, in vitro experiments with SynNotch-anti-HER2 expressing T cells are of dubious value without testing it at least in humanized mouse models.

David Usharauli

Wiskott-Aldrich syndrome protein (WASp) deficient FOXP3+ Tregs fail to control IgE mediated allergies

The inherited immunodeficiencies are frequently characterized with dysregulated Th2 responses, atopy, and elevated IgE levels. Mutations in Foxp3, STAT3, DOCK8, PGM3, LAT, ZAP70, or RAG result in hyper IgE phenotypes. Mechanism is unclear.

This time the scientists found that patients with another inherited immunodeficiency disorder, called Wiskott-Aldrich syndrome (WAS), also show elevated IgE signature and increased frequency of sensitization to food allergens. In mouse model, selective deficiency of WAS protein in FOXP3+ Tregs could recapitulate hyper IgE signature.

For this study the researchers analyzed "the overall burden of clinical food allergy within a cohort of 25 patients with mutations in the WAS gene" and found that individuals with WAS mutations were more likely to demonstrate serum sensitization to peanut, milk, and egg (compared to the general population).

Lab mice deficient for WASp also display elevated IgE (and IgG1) antibody levels to components of their chow food (even on germ-free background).

Since WASp is expressed in different cell types, the authors tested mice selectively deficient for WASp in B cells, DCs or FOXP3+ Tregs. Out of these gene-modified mice, only Was^fl/fl Foxp3-Cre mice showed deviation to Th2 phenotype and development of IgE to chow food.

In vitro T cell stimulation confirmed that total T cells from Was^fl/fl Foxp3-Cre mice showed selective enhancement in Th2 cytokines.

In sum, these results indicate that mice with selective deficiency of WASp in FOXP3+ Tregs display excess in Th2 subsets. It is possible that absence of WASp destabilizes FOXP3+ Tregs and this somehow drives their de-differentiation into Th2-like cells (and not into Th1 or Th17, for instance).

David Usharauli

   

Genomic alterations in IFN-γ pathway underlie resistance to anti-CTLA-4 (Yervoy) therapy

This week journal Cell published a short "reverse translational" study conducted on small cohort of patients non-responsive to immunotherapy with anti-CTLA-4 antibody (ipilimumab, Yervoy) that  showed that genomic alterations in IFN-γ pathway in non-responder patients could underlie their resistance to immunotherapy.  

For this study the authors compared 12 patients who did not respond to ipilimumab therapy (non-responders) and 4 patients who did respond to ipilimumab therapy (responders). They found that "tumor samples from non-responders were found to have significantly more somatic mutations, including copy-number alterations (CNAs) and single-nucleotide variants (SNVs) of the IFN-γ pathway genes".

Next, the authors showed that primary melanoma cell lines derived from anti-CTLA-4 responder or non-responder patients could be differentiated based on their in vitro sensitivity to IFN-γ.

Next, they showed quite bizarre experiment. By knocking down IFN-γ receptor in mouse B16 melanoma cells the authors showed that these cell line became less sensitive to IFN-γ in vitro. Not sure about logic behind these experiment.  

Finally, the authors showed that B16 melanoma cells deficient for IFN-γ signaling and transplanted into WT mice were less sensitive to anti-CTLA-4 therapy.

In summary, this small cohort study suggests that screening of melanoma patients for genomic alterations in IFN-γ pathway could be useful in better predicting therapeutic outcome for this checkpoint inhibitor therapy.

David Usharauli

Fusobacteria, a gut commensal, contributes to autoimmune type I diabetes in mice

Initiation of autoimmune disease is still an immunological mystery. Some forms of autoimmune diseases are results of genuine genetic defects in signaling molecules within immune system. Other forms show strong linkage to certain HLA haplotypes that present antigenic epitopes. More recently scientists focused on the role of gut commensals in autoimmune diseases.

A new study in Jounral of Experimental Medicine showed that cross-reactivity at the epitope level between gut commensal Fusobacteria-derived magnesium transporter and β islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP) contributed in autoimmune diabetes development in IGRP-specific CD8 T cell transgenic, CD8+ TCR NY8.3 NOD mice.

Initially, the authors observed that unlike MyD88KO NOD mice, MyD88KO CD8+ TCR NY8.3 transgenic NOD mice showed accelerated diabetes development (though unlike the authors, I don't find this surprising).

Interestingly, when co-housed with WT NOD mice, MyD88KO TCR NY8.3 transgenic NOD mice showed enhanced protection against diabetes, suggesting dominant role of fecal bacteria present in WT NOD mice in providing this protection.

Since it is known that NOD mice susceptibility to diabetes is commensal-dependent, the authors sequenced fecal microbiome in MyD88KO NY8.3 NOD mice to determine its composition. Not surprising, certain families of commensals underwent changes on MyD88KO background.

When the authors compared the IGRP_206–214 peptide sequence against bacterial protein sequences in the nonredundant protein sequence database, they found several hits shared strong homology with IGRP_206–214 peptid, the native autoantigen detected by NY8.3 CD8+ T cells. One such peptide, W15944, was derived from L. goodfellowii, a member of the phylum Fusobacteria (gram-negative anaerobe), a human and NOD mouse oral commensal.

Indeed, W15944 stimulated NY8.3 CD8+ T cells could transfer diabetes in NOD mice.

Finally, introduction of L. goodfellowii into WT NY8.3 NOD mice accelerated diabetes development, further suggesting role of L. goodfellowii in this process (while in general, this paper is of high quality, it lacks some of the crucial experiments such as, for example, (1) introduction of L. goodfellowii into NY8.3 NOD mice on germ-free background and (2) experiments with L. goodfellowii lacking cross-reactive W15944 peptide).

In summary, the author showed that in this artificially constructed NOD mice, IGRP-specific CD8 T cells could be activated by commensal-derived cross-reactive peptide and initiate autoimmune destruction of  β cells (it is remains to be seen why Foxp3+ regulatory T cells are incapable of preventing such T cell attack).

David Usharauli

Eye-specific autoimmunity in AIRE/Lyn double deficient animal

AIRE protein in the thymus directs expression of tissue-specific antigens that on one hand reduces number of auto-antigen specific T cell clones and on the other hand leads to generation of auto-antigen specific Foxp3+ regulatory T cells. However relationship between between central tolerance and its peripheral counterpart is not fully understood.

New study in Journal of Clinical Investigation examined relationship between hypomorphic AIRE function on Lyn^-/- background. The authors found that such combination selectively accentuated eye-specific autoimmunity.  

First, the authors observed that mice double deficient for AIRE^GW/+/Lyn^-/- functions (but not single deficient) developed spontaneous eye inflammation.

These AIRE^GW/+/Lyn^-/- mice showed expansion of eye-protein specific CD4 T cells and autoantibodies.

These auto-antigen specific T cells could infiltrate eye tissue causing inflammation.

The authors found that selective absence of Lyn in CD11c+ dendritic cells (Aire^GW/+CD11c-Cre Lyn^fl/fl) were sufficient to recapitulate this eye immunopathology.

Finally, the authors found that auto-antigen uptake by Lyn^-/- DCs in eye draining lymph nodes contributed to eye inflammation.

In summary, this study indicates that some of the clinically relevant autoimmune phenotypes could be linked to failure to establish both central and peripheral tolerance.

David Usharauli