The auto-reactive CD4+ T cells provide IL-2 to proto-Tregs in the thymus

The T cells expressing the transcription factor Foxp3 called regulatory T cells, abbreviated as Tregs, are the most important cell type in the immune system. Without them, the whole immune system goes haywire. As a result, the body simply dies in a very short time.

The Tregs develop in the thymus and require two things: TCR signaling and IL-2. The thymus expresses a very diverse set of epitopes including that from peripheral tissues such as the pancreas or prostate. The high-affinity interaction between TCR and epitope/MHC II makes proto-Treg sensitive to local IL-2, a necessary step to complete a Treg formation loop.

But what cell provides that crucial IL-2 to proto-Tregs? There hasn't been any consensus with this regard but a new paper in the Journal of Experimental Medicine from Sasha Rudensky's lab indicates that it is mature CD4+ T cells and CD25+Foxp3- CD4+ single-positive (SP) T cells that are the main source of thymic IL-2 required for Treg development.

For this study, they used an IL-2 reporter mouse wherein cells expressing or having a history of the expression of IL-2 are genetically labeled and analyzed. They found that IL-2 expression was restricted to TCRbeta expressing CD4+ population.

Out of CD4+ T cells, the most IL-2 was made by mature CD4 SP and CD25+Foxp3- CD4+ T cell population. Of note, CD25+Foxp3- T cell population contains proto-Tregs.

Interestingly, the authors also detected mature Tregs with the history of IL-2 expression. It implies that bifurcation between Tregs versus IL-2 producer is a stochastic process.

As expected, TCR signaling together with IL-2 was essential for Treg formation. A "bystander" effect on Foxp3 upregulation on antigen-independent proto-Tregs (Vbeta 8- T cells) could be explained by the fact that these T cells were likely TCR activated in vivo before harvesting for ex vivo experimentation.

Based on these data, the authors suggested the following model: among mature SP CD4 T cells, a small pool produces IL-2 that in the context of high-affinity TCR/epitope interaction and CD25 upregulation promotes Foxp3+ Treg formation either autocrine or paracrine manner. Since the thymus is expressing self epitopes we can conclude that those IL-2 producing T cells are auto-reactive T cells.

The following questions remain unanswered:

1. What determines Treg, IL-2-producer or deletion pathways? All three options are open for high-affinity TCR+ CD4 SP cells.

2. Do TCR specificity overlaps between Tregs and IL-2 producers?

3. What cells provide IL-2 to Tregs in the periphery?

4. Is IL-2 delivery TCR/epitope-specific or non-specific event?


We have recently published a new model, called SPIRAL, that provides answers to these questions. The SPIRAL is based on the principle of epitope cross-reactivity.


Shared TCR epitope cross-reactivity could permit dyads of Foxp3+ regulatory and IL-2-producing T cell precursors to escape thymic purge 


posted by David Usharauli


 

Innate help to Tregs in the gut

New study in @Nature indicates gut #ILC3 (type 3 innate lymphoid cells) produce IL-2 for #FOXP3+ Tregs. Is it relevant? lets discuss it. #Immunology #tolerance #microbiota https://t.co/iziPJb09Zy

— David Usharauli (@3DiMMUNE) April 3, 2019

2. Interestingly, the #frequency of IL-2-producing ILC3s was depended on the presence of #microbiota. Specifically, the authors showed that #cytokine IL-1 produced by #macrophages signaled ILC3 to produce IL-2. #MyD88 was involved (it is needed to produce IL-1 anyway). pic.twitter.com/dGb66Pqnwt

— David Usharauli (@3DiMMUNE) April 3, 2019

4. However, it is clear that IL-2 inactivation in NCR1-cre mouse is not clean and some ILC3 retain a capacity to secrete IL-2 (otherwise there should not be any IL-2+ ILC3 in this graph). pic.twitter.com/Zwbz2dDP3n

— David Usharauli (@3DiMMUNE) April 3, 2019

6. To complete the story the authors showed that individuals with #Crohn's disease harbor fewer ILC3 that produce IL-2 to feed Tregs in the small intestine suggesting that one reason why CDs could generate #inflammatory condition. pic.twitter.com/k2LN2LeMFe

— David Usharauli (@3DiMMUNE) April 3, 2019

in summary, this study showed that ILC3-produced IL-2 could support Treg functionality in the small intestine. This cooperation between ILC3 and Tregs appears to be antigen-nonspecific as mice with ILC3 deficient for MHCII does not show changes in Tregs. 1/3

— David Usharauli (@3DiMMUNE) April 3, 2019

I like to point out that out of so many figures only 1 figure shows a reduction of Treg numbers in the small intestine that supports the authors' claims of the role of ILC3-derived IL-2 in Treg homeostasis, the rest are shown as % (not optimal) or does not support claims. 3/3

— David Usharauli (@3DiMMUNE) April 3, 2019

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 control CD8 T cells, but not CD4 T cells, by IL-2 deprivation

This week Nature Immunology published another interesting study from Rudensky's lab done in collaboration with Jason Fontenot (who apparently moved from Biogen to Juno Therapeutics). So, if you are a fan of Tregs, below is my short scientific overview of that paper.

This study tries to answer what role IL-2 signaling plays in already established Foxp3+ Tregs. For such study mice with germline deficiency in IL-2 signaling cascade would have been impractical since IL-2's effect on thymocytes and etc. Instead, the authors went to already well established path of using mice lacking "molecule of interest" specifically in Tregs. In this case several Foxp3-cre mice were used, such as: l2rb^fl/flFoxp3^Cre, Il2ra^fl/flFoxp3^Cre, Stat5a^fl/flStat5b^fl/flFoxp3^Cre, Rosa26^Stat5bCAIl2rb^fl/flFoxp3^Cre and Rosa26^Stat5bCAIl2ra^fl/flFoxp3^Cre, Rosa26^Stat5bCAFoxp3^Cre–ERT2. All these gene-modified mice allows specific targeting of Foxp3+ Tregs.

Unsurprisingly, mice lacking IL-2Rβ or IL-2Rα or STAT5 signaling specifically in Tregs developed autoimmunity.

Surprisingly, however, while constitutive expression of STAT5 in Tregs lacking IL-2 signaling receptors (IL-2Rβ or IL-2Rα) could rescue mice from "CD4 effector phenotype" and early death, these mice still developed immunopathology later due to massive expansion of CD8 effector/memory cells, suggesting that sensing of IL-2 by Tregs, so called "IL-2 sink", was necessary to specifically control CD8 T cells, but not CD4 T cells.

In summary, this study suggests that Tregs are using distinct mechanisms to control CD4 and CD8 T cells. It kind of makes sense because MHC class-II restricted CD4+ Tregs cannot interact the same way with MHC class-I restricted CD8+ T cells as they could with conventional MHC class-II restricted conventional CD4+ T cells.

David Usharauli

IL-2 instructs allergen-specific tissue resident memory TH2 cell development

IL-2 is one of the first cytokines [interleukins] discovered and we still have no clear idea of the extent of its involvement and role within immune system. One reason has to do with the fact that IL-2 "in vitro" and IL-2 "in vivo" behave if completely 2 different cytokines.

So, if I see new paper that could tell us more about IL-2, I can't resist reading it. This week journal Immunity has published one such article. There, the authors showed that IL-2 signaling via its high-affinity receptor IL-2α (also known as CD25) directed development of allergen-specific tissue resident memory TH2 cells. I will discuss only those data that are relevant and unequivocal for the story.

For this study, the authors used class II tetramers and i.v. labeling techniques to identify and track house dust mite (HDM) allergen-specific tissue resident T cells. They showed that primary allergen exposure generates lung tissue resident HDM-specific tetramer-positive CD4 T cells.

These lung tissue resident tetramer-positive CD4 T cells expressed IL-13 upon HDM re-challenge (that identified them as TH2 cells).

Next, using parabiont mouse model the authors showed that tetramer-positive CD4 T cells found in allergen challenged lung tissue were bona fide non-circulatory resident-memory TH2 cells.

Finally, using WT:CD25KO mixed bone marrow chimera mice the authors showed that development of lung tissue resident memory TH2 cells required IL-2 signaling via its high-affinity receptor IL-2α.

In summary, this study suggests that IL-2 signaling via its high-affinity receptor IL-2α is mandatory for resident-memory TH2 cell development. This is addition to already known IL-2 roles in Foxp3⁺ Treg and memory CD8 T cell development. From therapeutic point of view, this is one big mess.

David Usharauli

Signal 1 = Signal 2 = Signal 3?

Occasionally one finds research articles published in the top journals, which rather than simplifying and clarifying the subject matters, make them more confusing. Frequently, even after reading and then re-reading them, one still cannot comprehend the "significance" of the finding.

I have one rule: if I cannot understand an immunology research paper with one read, I blame the editors. The author, of course, wants to publish his/her research in top journal, but it is a responsibility of the editors to be a gatekeepers and to make sure that the article is written in an easy to understand style.

I have another rule: the good data naturally make paper writing and reading easy.

The following paper from journal Science is such a disappointment. It starts strong with FucciRG mice model to track T cells acquiring a quiescent state after active cycling. The rest is confusing. The authors proposed that signal 1 (antigen), signal 2 (co-stimulation), and signal 3 (inflammatory milieu) play equal and quantitatively linear role in imprinting T cell division rate (they called it division destiny (DD), as if it would make it easier to understand). 

First of all, if one takes, for example cytokine IL-2, it not just quantitatively affects the T cell division rate but it qualitatively changes T cell fate and memory potential too. The authors suggestion that IL-2's physiological role is to maintain T cell division at a later stage of activation, at the tissue site, away from priming site (such as spleen or lymph nodes) does not account for IL-2's role in T cell effector class differentiation (CTL, IFN-gamma or IL-2 producers) or memory imprinting. These latter concepts could equally well explain the authors' observation with IL-2 receptor alpha deficient T cells.

I personally would suggest to Science editors to avoid publishing research papers filled with unnecessary models (especially mathematical models) or with one-sided explanations.

David Usharauli