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

Interleukin-17A links viral infection during pregnancy to autism spectrum disorder (ASD) in offspring

Pregnancy in mammalian females represents an unique challenge to their immune system. Essentially, from immune system's "point of view" growing fetus is a semi-allogeneic transplant that should be seen as a "foreign" and be rejected. But fetus, in general, is tolerated by maternal immune system. Such tolerance implies that maternal immune system takes some specific steps to avoid its attack on fetus. On the other hand, maternal immune system cannot let guard down completely because at least mother needs some level of defense.

Such conflicting "defense programs" operating in pregnant females occasionally damages fetus. For example, viral infection of women during pregnancy [and maternal immune response to it] could increase frequency of autism spectrum disorder (ASD) in the offspring. 

In this regard, new study in Science is of interest. In this paper the authors showed that injection of poly(I:C) to mimic viral infection produced ASD-like phenotypes in offspring via action of IL-17A.

First, the authors showed that poly(I:C) injection increased serum IL-17a in pregnant dams in a IL-6 dependent manner (this IL-17a induction was specific to placenta- and decidua [uterus]-associated mononuclear cells).

Second, poly(I:C) injection in pregnant dams produced abnormalities in layered structure of the developing fetal cortex that could be reversed by pre-treatment with IL-17a blocking antibody.

Similarly, poly(I:C)-induced malformation of the fetal brain was prevented in offspring from RORγt-KO mothers that lacked CD4 T cell-specific IL-17a expression.

The authors showed prophylactic pre-treatment with IL-17a blocking antibody of poly(I:C)-injected mothers could reverse ASD-like behavioral abnormalities in offspring such as increase in pup ultrasonic vocalization (USV) responses or repetitive/perseverative behaviors.

Finally, the authors found that therapeutic application of IL-17a blocking antibody after poly(I:C) injection could partially [but not fully] correct some of the ASD-like features in offspring.

In summary, these results suggest that in "susceptible" mothers immune response to virus infection could impair offspring's brain development via direct effect of maternal cytokine IL-17a on fetal neurons. Here the "susceptibility" in mothers could be defined as a tendency for hyperactive IL-6/IL-17 axis. In general, this finding is surprising since IL-17a isn't ordinarily associated with viral infection. But, IL-6/IL-17 axis is well accepted concept. 

Of note, there is FDA approved IL-17a antibody, Novartis Cosentyx (secukinumab) that could be used in a scenario described in this paper.

David Usharauli

Novel combinatorial antigen-sensing chimeric antigen receptor circuit permits tumor-selective therapeutic T cell activity

Few days ago journal Cell published new study on chimeric antigen receptor (CAR) T cells that produced lots of discussion in immuno-oncology field. I read the actual paper and here below I will provide my opinion about it. 

I will point out that overall this study is of high quality. Basically, the whole premise of this paper is based on what the authors had correctly pointed out in the introduction "Few antigens are absolutely tumor specific, and T cells targeted to antigens that are also found on normal bystander tissues can cause life-threatening adverse side effects."

So how this could be avoided? According the authors "combinatorial antigen recognition T cell circuits in which a synNotch receptor for one [tissue-selective] antigen drives the inducible expression of a CAR for a second [tumor-specific] antigen" could provide such a fail-safe mechanism. In essence, in this model CAR-T cells would behave very similarly to how NK cells are sensing healthy and "abnormal" cells or tissues.

In first set of experiments, the authors tested validity of this concept on Jurkat T cells, a human T cell line. Jurkat T cells were transduced with a-CD19 synNotch receptor and a-mesothelin CAR gene and then exposed to "K562 myelogenous leukemia cells with ectopic expression of CD19, mesothelin, or both antigens". The authors detected IL-2 production [as a readout of Jurkat T cell activation] only with K562 cells expressing both CD19 and mesothelin.

Similar results were obtained with primary human T cells transduced with a-GFP synNotch receptor and a-CD19 CAR and exposed to "K562 target cells expressing CD19 only, GFP only, or both GFP and CD19". 

Importantly, in vivo experiments also indicated that T cell response were restricted to tumor cells with dual antigen expression. Only dual GFP+/CD19+ Daudi B cell tumor, but not single CD19+ Daudi cells, could induce CAR-T cell activation [as measured by luciferase signal]. Of note, there was low but detectable level of CAR T cell engagement at the single CD19+ Daudi tumor site.

And even more importantly, even in mice simultaneously transplanted with both single or dual (GFP+/CD19+) expressing K562 tumor cells, only dual antigen-expressing tumor cells were targeted. This indicated tumor-selective nature of combinatorial CAR-T cells. It showed that in this model, combinatorial CAR-T cells do not "engage a tumor expressing the synNotch ligand (GFP), become primed by expressing the a-CD19 CAR, and then migrate elsewhere to then kill single antigen (CD19+ only) bystander tissues." (though, I would admit I was surprised with these results).

In summary, these results clearly showed some of the advantages of combinatorial CAR-T cells. By expressing tumor tissue-selective SynNotch "primer" receptor, these CAR-T cells are primed and engage tumor "specific" antigens within tumor tissue only (but not in other tissue). 

Now question is how far such tumor tissue "selectivity" goes. The authors have not done more direct experiment wherein tumor and actual healthy tissues share "common" antigen to see if combinatorial CAR-T cells would maintain their "bias" towards tumor tissue. Since the main premise of such combinatorial CAR-T cells is to prevent CAR-T cell's "side effects" against healthy tissue, we need model that reliably test this scenario.   

David Usharauli