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

Tumor elimination requires simultaneous expression of both class I and II neo-epitopes

The most tumors express mutant epitopes that could be detected by T cells. According to current paradigm, CD4+ T cells provides help to CD8+ T cells that in turn attack tumors. As tumor cells ordinarily express class I recognized by CD8+ T cells but not class II molecules recognized by CD4+ T cells, primary focus on CD8+ T cell epitopes made a lot of sense. But what about CD4+ T cell 'help' to CD8 T cells? 

Indeed, a new 'classically-done' immunology study from Robert Schreiber's lab clearly showed that irrespective class II expression, tumor cells must express both CD8+ and CD4+ T cell neo-epitopes to achieve efficient local tumor control following immunotherapy.

As a starting point, they used nonimmunogenic oncogene-driven KP9025 sarcoma cells (KP), which lack mutational neoantigens. Next they re-expressed in KP cells 2 mutant epitopes, one for class I, mLAMA4, and another for class II, mITGB1 (identified using a hidden Markov model (HMM)-based MHC binding predictor the authors claim is better than other available algorithms). 

 

A mutant but not wild-type version of ITGB1 was detected by CD4+ TILs.

  

Next, the authors showed that only KP tumors expressing both neo-epitopes but not single expressors, could be eliminated by T cells following immunotherapy.

  

As expected, presence of CD4+ T cell epitope enhanced CD8+ T cell response.

Interestingly, both class I and II  neo-epitopes must be expressed by the same tumor to mediate protection when used as immunized agents (mixing of single expressor tumors was not enough).

And notably, expression of both class I and II neo-epitopes were necessary to mediate efficient local tumor control (single expressor tumors were resistant against CD8+ or CD4+  T cells)

In summary, this is a simple, easy to follow experments that indicate the authors' thought process.  It shows that CD4+ T cells 'help' to CD8+ T cells are required both at priming and as well as at effector stage. It is not clear if it is simply a quantitative or rather qualitative issue. It is not known either whether CD4+ T cells do something directly against tumor beyond simply helping CD8+ T cells here. 

posted by David Usharauli

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


 

Do regulatory CD8+ T cells control autoreactive CD4+ T cells in the mouse model of human MS?

Recently journal Nature published a very thought-provoking study from Mark Davis' lab. In it, the authors have described the existence of a specialized population of CD8+ T cells that prevented auto-reactive CD4+ T cells from causing autoimmune brain inflammation (EAE) in mice, a laboratory model for human multiple sclerosis.

Let's analyze what the study shows. Both Fig. 1 and Fig. 2 are rather superfluous as they simply show either time kinetics of CD4+, CD8+ and γδ+ T cells responses in the blood or CNS following autoantigen immunization (Fig. 1) or frequency of TCR clonal  distribution based on TCR β or both γ and δ sequencing (Fig. 2). It is not clear what was the purpose of showing them within the paper itself.

    

Next, the authors tested the TCR specificities for expanded clones of CD4+ or CD8+ T cells. Four of these CD4+ TCRs expressed in human leukemia SKW αβ−/− cells yielded robust staining with a MOG35–55 I-Ab peptide–MHC tetramer. 

Curiously, out of nine TCRs from CD8+ T cell clones expressed in a mouse T cell hybridoma 58 αβ−/− cells, none of them get stimulated when co-cultured with bone-marrow-derived dendritic cells pulsed with myelin protein-derived peptides (total of 350 myelin peptides were tested). So, something else, besides myelin protein, was driving CD8+ T cell expansion.

To identify epitope specificity for TCRs from CD8+ T cells, the authors used H2-Db yeast-pMHC libraries. Six of the clonally expanded and one positive control CD8+ TCRs were used. Two, EAE6 and EAE7 TCRs showed robust tetramer staining but no matches were found in the mouse genome. They referred to these peptides identified in the peptide library screen the surrogate peptides (SPs).

Interestingly, the co-immunization of these SPs with myelin peptide inhibited the development of brain inflammation in mice. 

More importantly,  CD8+ T cells harvested from SPs-immunized mice but not control, naive mice, inhibited myelin-specific but not ovalbumin-specific CD4+  T cells in vitro. 

Moreover, only Ly49+ but not Ly49− fraction of CD8+CD44+CD122+ T cells from SPs-immunized donor mice showed inhibitory function both in vitro and in vivo. Of note, the application of the anti-Qa-1b antibody had no effect on the CD8 suppression of Myelin-specific CD4+ T cells. Here, the authors also tested CD8+ T cell reactivity to CFA (complete Freund’s adjuvant) or PTX (pertussis toxin) used in EAE immunization protocol and found that it did not increase Ly49+ fraction.

So, how can we summarize this paper? First, focus on γδ+ T cells here is extra and feels out of context. Second, no endogenous peptides were found that mimic SPs found in the library screen. Could it be microbiota-derived? The authors did not consider this possibility, it appears. Third, how SPs-specific Ly49+ CD8+ T cells inhibit myelin-specific CD4+ T cells? Not clear. It does appear highly specific to myelin-specific CD4+ T cells in the context of brain inflammation. But myelin-specific CD4+ T cells see myelin peptides but these CD8+ T cells do not seem to recognize them. Very confusing indeed. 

In general, the "memory-like" CD8+ T cells, such as Qa-1b-restricted population, has been known to inhibit immune response. This paper simply provides some new evidence in that direction. But it is not a novel idea or observation and without some novel mechanistic evidence I don't see how it could have landed in Nature's pages.  

posted by David Usharauli  

Pathological changes in the gut could initiate autoimmune diabetes microbiota-specific manner

The mouse strain, NOD, has been used to study the mechanism of type 1 diabetes (T1D). These mice spontaneously develop autoimmune diabetes though it is not clear how islet-specific T cells get activated. To track autoimmune T cell fate, TCR transgenic mice, BDC2.5XNOD, have been used. These mice harbor islet-specific TCR expressing T cells in high numbers that are easily monitored.

A new study in PNAS did some experiments on BDC2.5XNOD mice to understand the initiation of autoimmunity. First, they showed that the NOD background showed increased gut wall barrier permeability by measuring the FITC-dextran level in the blood after oral application (though they did not show the same permeability test for BDC2.5XNOD mice).

Interestingly, BDC2.5XNOD mice do not develop spontaneous autoimmune diabetes. However, oral application of low-dose dextran-sulfate sodium (DSS) activates T cells and initiates diabetes.

 

Diabetes, however, does not develop if DSS-treated BDC2.5XNOD mice are depleted of endogenous microbiota or if naive BDC2.5XNOD mice received DSS-modified microbiota.

In summary, this study showed that both gut inflammation and microbiota are necessary for the initiation of autoimmune diabetes in BDC2.5XNOD mice. The most likely scenario is that changes introduced by DSS allow endogenous microbiota to activate islet-specific T cells via cross-reactive antigens. DSS modifies both gut wall permeability and microbiota. Both of these phenomena have been observed by the authors. They conclude that "restoration of a healthy gut barrier through microbiota and diet modulation in diabetes-prone individuals could ultimately reduce intestinal activation of islet-reactive T cells and prevent T1D occurrence".

Others, in news/views for this article, even suggested using antibiotics to deplete endogenous microbiota, but in my opinion, this is a premature suggestion because the authors did not show that microbiota depletion after diabetes has already developed could stop it.

posted by David Usharauli

Select microbiota species provides protection against food allergy via RORγt+ Tregs

It is now undoubtedly acknowledged that body's microbiota plays a decisive role in protection against allergy, including food allergy. But how exactly microbiota does it is less clear.

A new study in mice published in Nature Medicine suggests the certain microbiota species signal subset of FOXP3+ Tregs called RORγt+ Tregs via adaptor MyD88 to exert its protective role against food allergy.

The most of the experiments reported here were done in genetically modified mice called Il4ra^F709 that shows a predisposition to allergy due to an alteration in IL-4 signaling. Here, germ-free Il4ra^F709 mice were colonized with microbiota consortium differently enriched between non-allergic versus allergic infants. Out of those, defined mix of Clostridiales and Bacteriodales but not Proteobacteria could reduce allergic reaction in Il4ra^F709 mice. (Note, you can click the image to expand it to see it more accurately).

In a separate set of experiments the authors noticed that Il4ra^F709 mice or mice specifically deficient for RORγt+ Tregs subset displayed similar phenotype in response to allergic challenge. They thought there could be a connection.

Indeed, Il4ra^F709 mice deficient for RORγt+ Tregs subset lost an ability to resist allergic reaction when colonized with defined mix of Clostridiales and Bacteriodales.

Finally, the authors attributed the loss of protection to loss of MyD88 adaptor signaling in Tregs because Il4ra^F709 mice deficient for MyD88 signaling in Tregs also showed loss of protection against allergic reaction when colonized with defined mix of Clostridiales and Bacteriodales (Note, oral short chain fatty acid (SCFA) therapy failed to protect Il4ra^F709 mice against allergic response) .

In summary, we could conclude based on this and other studies that RORγt+ Tregs do play a decisive role in protection against unwanted inflammatory response (Note, however, that allergic sensitization protocol employed here is not exactly "translational" approach).

One major drawback of this study is that the authors failed to examine why it is that Clostridiales and Bacteriodales but not Proteobacteria or other species could signal via MyD88 to provide protection against allergic response. In my view it is not a difference in innate signaling molecules that distinguishes protective versus non-protective microbiota species but rather their antigenic composition that provides epitopes to RORγt+ Tregs to keep them active and in a good functioning condition (MyD88 could be just necessary to keep such antigen-specific Tregs active due to its role in metabolic pathways).

posted by David Usharauli

A neonatal temporal window for thymic epitope-specific Foxp3+ Treg formation

The thymus-derived Foxp3+ Tregs are indisputably the most important immune cell type. Surprisingly, little has been done to found out their antigen specificity. One reason for this lack of interest to study it has to do with the fact most scientists thought Tregs inhibited unwanted T cell responses antigen non-specific manner. So, they reasoned why to bother with TCR specificity. More recently however they started to pay close attention to antigen-specificity of Tregs since it became clear that antigen-specific Tregs showed superior, maybe even exclusive, therapeutic effect in animal models.  

So any study that advances our understanding of the formation of antigen-specific Tregs is immensely valuable. Below I will review one such research published in Nature Immunology from Eric Huseby's lab at the University of Massachusetts Medical School, Worcester, MA, USA.

In this study, they cloned several hundred TCRs from Foxp3+ GFP+ Tregs and screened their specificity in an in vitro IL-2 bioassay using standard hybridoma technology and library of ~1,750 unique self-peptides (it is astounding that so few labs have used this readily available approach). About 17 peptides showed a positive response. They chose to focus on 2 peptides derived from peptidyl arginine deiminase type IV (Padi4_92–105) and Adducin 2 (Add2_606–621). TCR specificity for Padi4_92–105 or Add2_606–621 was confirmed with respective KO mice.

Curiously, they noticed that thymic development of  Padi4_92–105 specific Tregs was time restricted and their formation rapidly went down after 3 weeks post birth.

More importantly, specific antigen expression was primarily responsible for both initial Treg formation and later its reduction.

However, even if there was initially an age-related decline in the frequency of Padi4_92–105 specific Tregs both in the thymus and periphery, their absolute numbers were maintained at a constant level in the periphery afterward. This is important to highlight.

Notably, this age-related antigen-dependent Treg reduction could be reversed in chimera where only thymic stromal but not bone-marrow derived cells expressed specific epitope. It could mean dose effect or specialized antigen-presentation pathway contributes to age-related decline in Padi4_92–105 specific Tregs formation.

Furthermore, out of several Padi4_92–105 specific TCRs with different antigen response potency, only moderate potency responders were enriched in Tregs in the periphery (the highest potency T cells were lost in the thymus and the lowest potency T cells ended up in Tconv spleen pool). In my view, this is conveniently too clean to my liking.

Also, the authors found Treg formation best correlated with the TCR:self-MHC half-life (t_1/2).

In summary, this study identified several self epitopes that drive mouse Treg formation in the thymus and this process is restricted to a few weeks post birth. It is not clear why or how the cessation of Treg formation is happening in the thymus here. As absolute numbers of such epitope-specific Tregs that seeded the periphery stayed constant it could indicate that a saturation feedback loop may exist between periphery and thymus that adjust Treg numbers. Additionally, the authors suggest that Treg formation could be predicted based solely on TCR:self-MHC dwell half-life (t_1/2). However, dwell time cannot explain their own observation about the age-related decline of Treg formation. What has changed in 8-week versus 3-week thymus to upend dwell time so dramatically? Besides, this paper did not address a mechanism of bifurcation that determines deletion versus Treg formation at the single thymocyte level that has been shown to occur independently of TCR affinity. 

Of note, these results could explain why some CD4+ TCR transgenic mice don't show thymic Foxp3+ Treg formation but still harbor them in the periphery, for example, marilyn CD4+ TCR transgenic mouse. As such mice are ordinarily examined when they are adults (>8 weeks) it will miss the thymic phase.  

posted by David Usharauli

Allergy: A newborn's microbiota prevents hyper IgE antibody response to certain food antigens

Prevalence of allergy to food antigens is increasing in the world (alongside autoimmune diseases). Many believe it has to do with changes in microbiota composition due to environmental and processed food effects. But we still don't know how exactly microbiota prevents immune dysregulation characteristic of allergy or autoimmunity.     

For the past couple of years the team from South Korea has published several important papers addressing the role of microbiota and food antigens in modifying the gut immune system. This week they published yet another relevant paper in Science Advance. Below I present the highlights of the study.   

For this study the authors compared the level of IgE in sera from conventional mice fed regular diet, germ-free mice also fed regular sterile diet and germ-free mice fed with sterile antigen-free diet. As seen in the figure below GF mice develop, over time, hyper IgE condition. However, this effect was abolished in GF mice fed antigen-free diet. It indicated that antigens found in food interact with the immune system differently in the absence of microbiota.        

This was confirmed in reciprocal experiments where GF mice were introduced to the antigen-free diet or when antigen-free diet fed GF mice were introduced to a regular diet. In both conditions, a regular diet that contains antigens enhanced IgE level in the absence of microbiota.

Interestingly, the authors found that only certain food antigens, such as wheat gluten, could initiate hyper IgE response in absence of microbiota. Of note, the wheat gluten was shown to be digestion resistant.  

Another noteworthy observation was related to the age at which point mice were introduced to food antigens. Only young, but not older antigen-free fed mice, showed hyper IgE response when introduced to food antigens in the absence of microbiota. It indicated that there were some differences between young and older mice that made older mice resistant to hyper IgE production when responding to food antigens. 

So far we discussed how antigen-free fed mice respond to food antigens in the absence of microbiota. As expected, the introduction of microbiota to GF mice blocked hyper IgE response to food antigens.   

In summary, this study showed that in the absence of normal microbiota mice fed a regular diet that contains antigens will show hyper IgE response to certain food antigens. This response is abolished in GF mice fed antigen-free diet. The main question of how microbiota prevents IgE response or why certain food antigens are more immunogenic has not been addressed here. Also, the authors did not discuss it but it is important to mention here that hyper IgE response by itself does not mean pathological allergic response. The authors did not say that antigen-free mice fed regular diet became allergic to it (or to wheat gluten). It means that allergic sensitization requires additional mechanisms beyond hyper IgE response.

posted by David Usharauli     

CD80 sequesters PDL1 on DC's surface and promotes T cell activation

An excellent study by Japanese scientists in @sciencemagazine reveals how co-#stimulatory molecule #CD80 sequestered co-#inhibitory molecule #PDL1 on the same DC's surface and thus promotes #Tcell #activation. #immunology #tolerance #Immunotherapy https://t.co/RcArRXV9XT

— David Usharauli (@3DiMMUNE) April 23, 2019

1. Initially, the authors made an #observation that unlike #macrophages #dendritic cells did not stain with soluble #PD1 molecule even though DCs expressed high levels of #PDL1 or #PDL2 (PD1 ligands). However, it was noted that DCs expressed a higher level of #CD80 too. pic.twitter.com/hH1CbYxt7L

— David Usharauli (@3DiMMUNE) April 23, 2019

3. Co-expression of #PDL1 with #CD80 [but not with CD86 or PDL2 with CD80 or another co-#stimulatory molecule CD86], on the same cell surface had a #functional consequence for OVA-specific T cell activation. It allowed more vigorous #activation and IL-2 production. pic.twitter.com/X0miudAspA

— David Usharauli (@3DiMMUNE) April 23, 2019

5. Here too, the combination of #PDL1 and #CD80 molecules that cannot interact with each other allowed more vigorous #inhibition of IL-2 production by T cells. #Mechanistically, it showed that 'liberated' PDL1 on DCs cross-interacts with PD1 on T cell and #inhibits it. pic.twitter.com/YxM6oskg3q

— David Usharauli (@3DiMMUNE) April 23, 2019

7. Similarly, mice expressing mutant PDL1 or CD80 molecules showed reduced inflammation in the brain [in #EAE #model] after priming with brain-specific antigens though the effect seems to be minimal. pic.twitter.com/P2fyIusL1M

— David Usharauli (@3DiMMUNE) April 23, 2019

It indicates PDL1 #sequestration by CD80 on DCs is not a major path that controls immunity in #physiological condition. The authors did not show if it undermines protection against #pathogens or #tolerance to gut #commensals.

— David Usharauli (@3DiMMUNE) April 23, 2019

Crohn's disease-like phenotype could be initiated by one gut microbe in genetically susceptible mice

1. The authors used doubly deficient Nod2−/−Cybb−/− [NADPH oxidase] (DKO) mice (genes thought to be altered in #Crohns). When fostered with Taconic dams, but not Jax dams, these DKO pups get spontaneous gut #inflammation after weaning (3 weeks post birth). pic.twitter.com/LdoBA8Dfao

— David Usharauli (@3DiMMUNE) April 20, 2019

3. using the #microbiota analysis tool (linear discriminant analysis
(LDA) effect size (#LEfSe) methodology) the author found that one particular microbiota species, M. schaedleri, showed 1349-fold enrichment in DKO mice but not in single KOs. pic.twitter.com/BzMUB63cGf

— David Usharauli (@3DiMMUNE) April 20, 2019

5. Time analysis showed that DKO pups fostered with Tac dams harbor M. schaedleri-specific IgA and IgG but only before weaning. These #antibody titers quickly decline post weaning that correlated with the onset of #CrohnsDisease-like phenotype. pic.twitter.com/elMRXdMdv3

— David Usharauli (@3DiMMUNE) April 20, 2019

In summary, this study showed that #spontaneous gut #inflammation could be recapitulated in mice doubly deficient for two specific genes, #NOD2 and #NADPH oxidase and exposed to specific gut #microbiota, Mucispirillum schaedleri.

— David Usharauli (@3DiMMUNE) April 20, 2019

So, here we have a complete model of spontaneous gut inflammation initiated by a particular gut microbe in a genetically susceptible host. Is it how #CrohnsDisease develops too in humans?

— David Usharauli (@3DiMMUNE) April 20, 2019

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

A physiological T cells' "weaning reaction" to microbiota and solid foods in newborn pups requires FOXP3+ Tregs

1. "weaning reaction" is described as a #physiological CD4+ T cell response to to #microbiota+solid #foods in mouse pups that occurs between 2-4 weeks after birth measured by analysis of #TNF-alpha and #IFN-gamma levels. It does not occur in #germfree pups devoid of microbiota. pic.twitter.com/pSGWXOUBoC

— David Usharauli (@3DiMMUNE) March 21, 2019

3. More importantly, however, #microbiota exposure between 2-4 weeks after birth but not later could restore #physiological "#weaning reaction" in T cells in #germfree pups. #tolerance #immunology pic.twitter.com/9KUtk3MT7R

— David Usharauli (@3DiMMUNE) March 21, 2019

5. Interestingly, "weaning reaction" in #newborn pups were dependent on presence of gut residing RORgt+ FOXP3+ T regulatory cells (Tregs). #tolerance pic.twitter.com/s1nq2HPShK

— David Usharauli (@3DiMMUNE) March 21, 2019

2nd Correction (#6): giving #SCFAs to #antibiotic-treated newborn pups could prevent #inflammation later. It appears this effect of SCFAs requires #Tregs and #microbiota (it does not work in GF pups) that provides #antigens to Tregs to keeps them in #functioning state. pic.twitter.com/2ewMoDRobh

— David Usharauli (@3DiMMUNE) March 21, 2019

This suggests that the effect of SCFAs is mediated via Tregs and that artifically generated "weaning reaction" with SCFAs without proper Treg contribution (driven by microbiota and their antigens) is not optimal.

— David Usharauli (@3DiMMUNE) March 21, 2019

β-synuclein specific CD4+ T cells invade brain grey matter to cause array of MS-like symptoms

β-synuclein specific CD4+ T cells invade #brain grey matter and cause array of MS-like #symptoms in a rat model of human #MultipleSclerosis. Interestingly, the most useful distilled data points were in suppl materials. #immunology #autoimmunedisease https://t.co/NbIt5ijxuR

— David Usharauli (@3DiMMUNE) March 5, 2019

highlights from paper
2. Such exclusive invasion of brain grey matter were primarily correlated by pattern of #antigen (β-synuclein) expression. #autoimmunedisease #immunology pic.twitter.com/1r9ROgtTDV

— David Usharauli (@3DiMMUNE) March 5, 2019

Some thoughts: here the authors have used either WT or transgenic rat expressing β-synuclein-specific T cells. Diseases were induced either following direct immunization of transgenic rats or adoptive transfer of activated β-#synuclein-specific T cell blasts. #immunology

— David Usharauli (@3DiMMUNE) March 5, 2019

Con't: immunologists have fairly good idea about processes that represent immune response but at presently it is still unknown how immune response is #initiated naturally or how it is #stopped. This is Achilles heels of current experimental models in #immunology.

— David Usharauli (@3DiMMUNE) March 5, 2019

IL-17 response to airborne fungi are driven by gut commensal pathobiont C. albicans

A new paper in @CellCellPress from Bacher/Scheffold lab showed that IL-17+ T cell response to airborne #fungus such as #Aspergillus fumigatus found in individuals with #asthma,#COPD, and CF,are in fact expanded by gut commensal pathobiont #Candida #albicans via #crossreactivity.

— David Usharauli (@3DiMMUNE) February 23, 2019

1. majority of IL-17+ #Tcells found in healthy donors are specific C. albicans. #Immunology #TH17 #fungi pic.twitter.com/3WITidrcCn

— David Usharauli (@3DiMMUNE) February 23, 2019

3. In fact, when C. albicans-specific T cells were depleted from #Aspergillus fumigatus-specific T cell pool IL-17 response was gone too suggesting that most if not all of IL-17+ T cells specific for A. fumigatus were indeed #crossreactive to C. albicans. pic.twitter.com/cZEu50Uwep

— David Usharauli (@3DiMMUNE) February 23, 2019

5. similar expansion of Aspergillus fumigatus-specific T cells producing IL-17 were observed in individuals with #crohnsdisease too. pic.twitter.com/L0VbmCcFj5

— David Usharauli (@3DiMMUNE) February 23, 2019

As both #fungi are naturally available in the environment any #immunopathology driven by excess of IL-17 response must be the result of some other changes in the host that makes it susceptible to pathology. what???

— David Usharauli (@3DiMMUNE) February 23, 2019

No going back: conventional B cells (B2) could trans-differentiate into innate-like B cells (B1) but not vice versa

The study is about origin of B1vs.B2 cells. B1 cells are innate-like B cell lineage that reside in peritoneal & pleural cavities and express restricted BCRs specific for conserved molecules such as phosphatidylcholine. B2 cells are conventional B cells expressing diverse of BCR.

— David Usharauli (@3DiMMUNE) February 16, 2019

1. This is a model system the authors set up to do it. Normally, #transgenic B cells express either VH12 (B1) or B1-8 (B2) only. However, after cre #recombination, VH12 is #replaced with B1-8 and vice versa. So, B1 should start expressing B2 receptor and B2 --> B1 receptor. pic.twitter.com/L4J1nBJENh

— David Usharauli (@3DiMMUNE) February 16, 2019

3. The same outcome was observed in an in #vivo setting too. Conventional B2 cells could produce #functioning B1 cells after #BCR swap but not vice versa. pic.twitter.com/cXHJLWaHsv

— David Usharauli (@3DiMMUNE) February 16, 2019

#Summary: conventional B cells (a.k.a B2 cells) still harbor #potential for trans-differentiate into B1 cells while B1 cells lack such quality.

Why is that? B1 produce so called natural "housekeeping" antibodies specific for some self antigens exposed during cell #senescence.

— David Usharauli (@3DiMMUNE) February 16, 2019

B2 cells, on the other hand, if happened to express B1-like receptor it could end up in B1 pool. Then it would acquire B1 functioning and develop into regulatory B cell category joining other B1 cells. In other word, To be a B1 cells is to be a good B cells by definition 😎

— David Usharauli (@3DiMMUNE) February 16, 2019

An antigen from gut commensal Bacteroides thetaiotaomicron (B. theta) is recognized by Foxp3+ Tregs

1. B.theta-specific T cells when transferred into #antibiotic-treated and B.theta #colonized T cell-deficient mice (#RAG1-KO) develop into #Foxp3+ #Tregs as well as effector T cells. It is not clear if donor tg T cells were on RAG1-KO background as well. Host mice stay healthy. pic.twitter.com/TD0pnnxgAR

— David Usharauli (@3DiMMUNE) February 9, 2019

3. The authors were able to identify a #specific #epitope in B.theta recognized by B.theta-specific T cells. The #sequence, EEFNLPTTNGGHAT, were mapped in BT4295 predicted to be a SusE/SusF #lipoprotein in outer #membrane (OM). pic.twitter.com/rH5cQHQcVN

— David Usharauli (@3DiMMUNE) February 9, 2019

In #summary, this study showed that certain gut #commensal #microbiota species, in this case, #Bacteroides thetaiotaomicron (B. theta) are recognized by #Tregs and even induce #effector T cell differentiation though the host stays healthy because Tregs regulate effector T cells.

— David Usharauli (@3DiMMUNE) February 9, 2019

Another point is whether #microbiota-specific Tregs are #denovo induced or #amplified from existing ones. I personally think it is a latter. Most #Tregs develop from #CD25-negative #precursors poised for Foxp3 expression & such precursors are all #thymus derived, in my opinion.

— David Usharauli (@3DiMMUNE) February 9, 2019