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

Cross-reactivity to fungal antigen drives acquired IFN-γ auto-antibody mediated mycobacteria susceptibility

Inherited genetic deficiency in IFN-γ signaling underlies susceptibility to weakly virulent mycobacteria, such as bacille Calmette-Guérin (BCG) vaccines and nontuberculous mycobacteria. Here, new study in Nature Medicine reported group of patients with acquired susceptibility to mycobacteria due to presence of neutralizing anti-IFN-γ auto-antibodies that could have been results of its cross-reactivity to fungal Aspergillus antigens.

Molecular mimicry hypothesis suggests that if foreign [nonself] antigen shows antigenic similarity to self antigen, then immune response to such nonself antigen could lead to autoimmune diseases due to shared, cross-reactivity. Here, the authors showed that set of patients with mycobacteria infection expressed neutralizing anti-IFN-γ auto-antibodies.

Next, the authors found that conserved KRKR motif of IFN-γ, known to be crucial for the protein’s bioactivity, showed homology to amino acids 105–113 of the ribosome assembly protein Noc2 of Aspergillus terreus.

Indeed, sera from patients with neutralizing anti-IFN-γ auto-antibodies reacted with Noc2 antigen from Aspergillus.

In summary, this study suggested that immune response to Aspergillus in certain individuals carrying specific HLA polymorphism (HLA class II molecules HLA-DRB1*15:02–HLA-DQB1*05:01 and HLA-DRB1*16:02–HLA-DQB1*05:02) could lead to generation of cross-reactive neutralizing anti-IFN-γ auto-antibodies and acquisition of mycobacteria susceptibility.

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 

Tumor-induced T cell dysfunctions are antigen-specific and expressed early on

This week Immunity published new study showing that CD8 T cell dysfunctions frequently observed in tumors is induced (a) early on (between 1-3 weeks), (b) it is antigen-specific (driven by chronic antigenic stimulation) and (c) it is irreversible even when checkpoint inhibitors are applied.

For this paper the authors have used tamoxifen-inducible, autochthonous [indigenous] liver cancer model (ASTxCre-ER^T2; AST = Albumin-floxStop-SV40 large T antigen, Tag). In this model tamoxifen application induces Tag expression that inactivates two central tumor suppressor proteins, retinoblastoma protein and the p53. Analysis of Tag-specific CD8 T cells at weeks 1 or 4 showed that already by week 1 tumor antigen-specific CD8 T effector cells display signs of "exhaustion" phenotype, that was further exaggerated by week 4 (up-regulation of PD-1, 2B4, LAG3, TIM-3 and failure to express cytokines).

Furthermore, when such tumor "educated" CD8 T cells were transferred into tumor-free hosts and challenged with live Tag+ infection, only week 1 CD8 T cells could respond. Week 4 T cells were irreversibly dysfunctional. Week 4 tumor antigen-specific CD8 T cells could not be revived even with checkpoint anti-PD-1 antibody.   

Finally, the authors showed that tumor induced T cell dysfunction was antigen-specific because presence of tumor did not affect non-specific CD8 T cell functionality (Tag-specific vs. OVA-specific T cells).

In summary, this study indicates that tumors could imprint irreversible "epigenetic" effector dysfunction on tumor antigen-specific T cells early on. The results in this study have important implications for clinical immunotherapy. We need to recognize that even with recent progress in application of checkpoint inhibitors for tumor management success is not guaranteed.

One drawback of this study is that the authors did not determine relationship between "antigen dosage" and CD8 T cell dysfunction. Albumin could drive huge amount of tumor antigen expression and the speed of tumor formation in these mice is clear example for such rapid "non-physiological" model. Next step would be to design tumor models where tumor specific antigen expression [dosage] could be regulated as well.

David Usharauli

PD-1 expression on tumor-infiltrating T cells does not correlate with antigen-specific response

Patients selection [stratification] and then monitoring for immunotherapy effectiveness is part of precision medicine. In humans, especially, when determination of cancer neo-antigens is not always feasible, clinical diagnostic tests are focused on surrogate markers to tell whether patient has a tumor antigen-specific immune response. More recently, PD-1 has become one of such surrogate markers. However it is not clear whether expression of PD-1 could truly correlate with T cell antigen-specific response.

New paper in Journal Immunology provided data to suggest that at least in mice tumor models PD-1 expression does not correlate with T cell antigen-specific response within tumor tissue.

For example, they showed that OT-I CD8 T cells would express PD-1 irrespective whether tumor expresses or not specific OVA antigen. In contrast, Nur77 (part of TCR signaling) expression correlated with tumor neo-antigen expression. 

This study indicate that diagnostic tests measuring PD-1 expression on tumor-infiltrating T cells may over-estimate tumor antigen-specific immune response and lead to unpredictable outcomes during antibody immunotherapy.

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

IL-4R mutation drives IL-17 dominant asthma phenotype

Asthma represents a dysregulated immune response to antigens that normally do not produce clinically-detectable immune responses. Ordinarily asthma response was regarded as classical Th2 class response with dominant IL-4/IL-13/eosinophil axis. However, for past 10 years, since the discovery of Th17 cells, scientists started to find a separate subtype of asthma dominated by IL-17/neutrophil axis. 

New paper in Nature Medicine pinpointed one molecular mechanism that underlie Th2→Th17 switch in asthma. It showed that a single amino acid mutation in IL-4Rα introduces instability in regulatory T cell lineage leading to Th17 induction.

Initially, the authors observed that mice with a glutamine (Q)-to-arginine (R) substitution at amino acid residue 576 of IL-4Rα (Il4ra^R576 mice) develop more severe experimental asthma in response house dust mite allergen.

In vitro culture of conventional or induced regulatory T cells derived from mice on Il4ra^R576 background showed exaggerated IL-17 expression in response to TGFβ1 and IL-4.

Lineage tracing analysis revealed instability of regulatory T cell lineage and their differentiation into IL-17 producing cells (ex-Tregs).

Indeed, when Tregs were rendered incapable to differentiate into IL-17 producing cells in Foxp3^YFPCreRorc^Δ/Δ mice, severity of asthma was reduced.

In summary, this study showed that genetic mutation in IL-4Rα introduces instability in Treg lineage and leads to IL-17 dominant asthma phenotype. Such asthma conversion is expected to be sensitive to anti-IL6 therapy.

David Usharauli