Tregs new motto: united we stand

Few days ago journal Nature has published thought-provoking study dealing with fundamental aspect of immune regulation by Foxp3⁺ Treg cells. 

Tregs are a major players within immune system but Tregs-mediated "negative" regulation of antigen-specific immune response has a fundamental problem at the theoretical, mechanistic level. No major, widely accepted immune theories such as Burnet's clonal selection theory, 2-signal model, Janeway-Medzhitov's PAMP model, or Matzinger's Danger theory could satisfactorily explain the role of Tregs within immune system. 

Actually, all these major immune theories excludes or simply prohibit the existence of any antigen-specific T cells that are of inhibitory nature. Antigen cannot be the basis of inhibition, according to these models, because T cell has no way of "knowing" the difference between self or nonself antigens. Moreover, at the innate immune system level, simply eliminating "irritants" that caused initiation of immune response in first place would automatically return immune system into quiet state without need of any negative regulation.

So why Tregs exist and how they regulate immune response? So this new paper tried to unlock the first door for this theoretical labyrinth. The authors showed that fraction of Tregs within lymph nodes are constantly active and constantly inhibiting proto-effector T cells. This suggests that Tregs function is more complex that typically assumed "off"/"on" model.

Initially, the authors led by Ron Germain at NIH, showed that lymph nodes contains functionally active Tregs clusters responding to local IL-2 in real time (phospho-STAT5).

Interestingly, the authors found no difference between active Treg clusters in wild-type and germ-free mice, suggesting that commensal bacteria derived non-self antigens were not responsible for Treg cluster formation (i.e. clusters were formed in response to self-antigens).

Next, the authors showed that when TCR signaling was interrupted in Tregs, these Treg clusters became small or disbanded.

Actual quantification of active, pSTAT5⁺ Tregs revealed both Treg cluster number and density of T regs within pSTAT5⁺ clusters were reduced in absence of TCR signaling, implying active role of antigen recognition for Treg cluster formation. Interestingly, total number of pSTAT5⁺ Tregs was not affected in absence of TCR signalling suggesting wider availability of free IL-2 secreted by proto-effector T cells in absence of suppressive Treg clusters (this could be compensatory increase in functionally impotent single Tregs).

This conclusion was supported by the fact that when IL-2 was blocked by anti-IL-2 antibody (that indirectly would have "inactivated" Tregs within clusters), there was an increase in IL-2 production by proto-effector CD4 T cells.

Finally, using more refined antigen-specific adoptive transfer experiment, the authors showed that when proto-effector CD4 T cells lacked IL-2 (and hence could not signal Tregs in clusters, these IL-2-deficient proto-effector CD4 T cells were able to form long-lasting interaction with dendritic cells, potentially increasing their chance for effector differentiation.     

In summary, this study suggests that Tregs are constantly scanning, responding and provide feedback to self-reactive proto-effector T cells in "steady state" within specialized "suppressor" clusters. But what happens to clusters during typical immune response?

In general the model proposed here is more like a network and I clearly see its basic similarity with Niels Jerne's Network model, a theory that was very popular during 80s but later was abandoned. Who knows, may be this paper is a new beginning for Network theory.

For Treg fans, Nature also published another, less impressive study from Rudensky's lab where the authors explored the role epigenetic modification of Foxp3 intronic enhancer, CNS3, in Treg TCR  repertoire variability and its role in preventing autoimmune inflammation.

David Usharauli          

Autologous Foxp3+ polyTreg adoptive transfer immunotherapy: phase I study

2nd paper in this week's Science Translational Medicine is more directly related to Foxp3⁺ T_regs. Here, the authors has conducted phase I, dose-escalation study in recent onset T1D patients (16 participants) to determine safety of adoptive transfer of ex-vivo expanded autologous polyclonal Foxp3⁺ T_regs cells.

Unlike CAR-T therapy with its bulk PBMCs as its main ex-vivo target population, working with flow sorted polyT_regs cells is more demanding and few institutions are equipped to conduct such GMP-grade clinical trials. As shown in Table 3, there is variability in ex-vivo expansion potential between polyT_regs cells derived from various donors [since no "healthy" control subjects were included in this trial, it is no clear whether this level of expansion of polyT_regs cells is specific for T1D patients].

The authors conducted several functional/marker analysis on 14-day expanded polyT_regs cells. These results revealed that expanded polyT_regs cells retain the same degree of (a) demethylation at Foxp3 locus, (b) they showed improved phosphorylation of STAT5 in response to IL-2, (c) displayed improved suppressive potential in an in vitro proliferation assay.

After adoptive transfer of expanded polyT_regs cells patients were monitored for any [metabolic] abnormalities. There were few adverse events related to T1D metabolic events that probably were not caused by polyT_regs cell transfer per se. However, due to small sample size, adoptive transfer of polyT_regs cells did not reveal any substantial improvement of T1D in recipients either [though transferred polyT_regs cells labeled with [6,6-²H₂]glucose were detected up to 1 year in cohort 3 and 4].

In summary, this phase I study of polyT_regs cells confirms feasibility of adoptive transfer immunotherapy with Foxp3⁺ polyT_regs cells. In general, it is my opinion that only by understanding Foxp3⁺ T_regs cells can we truly unlock the full benefits of immunotherapy for cancer, allergy and autoimmune diseases.

David Usharauli

Immunotoxins show anti-cancer synergy with immune checkpoint inhibitors

This week Science Translational Medicine published 2 papers directly or indirectly related to Foxp3⁺ T cells [this is in in addition of two Foxp3⁺ T cell papers in Nature]. Since I am very curious about the role of Foxp3⁺ T cells in immune regulation I decided to review them.

First paper I review here is not a typical Foxp3⁺ T cell paper. It is actually a study of tumor immunotherapy. The authors showed that when combined with checkpoint inhibitors CTLA-4/PD-1, direct tumor immunotoxins, such as T-DM1 [trastuzumab emtansine], significantly augment anti-cancer immune response and immune memory.

Trastuzumab [herceptin] is a humanized antibody that targets HER2 antigen over-expressed in breast cancer cells. Initially, the authors showed that anti-cancer effect of ado-trastuzumab conjugated to cancer toxin, T-DM1 (Kadcyla®), was depended on T cells [as revealed by T cell depletion]. This suggested potential synergy with checkpoint inhibitors.

Indeed, a combined application of T-DM1 and CTLA-4/PD-1 antibodies showed close to  100% protection against breast cancer in mice.

Surprisingly, the authors noticed that combo therapy, that fully protected mice against tumors, actually increased infiltration of tumor tissue by functioning Foxp3⁺ T cells [in addition to conventional T cells]. This was counter-intuitive.

However, T cell depletion confirmed that when CD4 T cells [that includes Foxp3⁺ T cells] were removed, more than 50% of mice on combo therapy [100% tumor-free] developed severe autoimmune inflammation [results with Treg-specific depletion would have been more valuable here, of course].

In summary, this study points to 2 distinct results: first, direct cancer cytotoxocity by immunotoxins could synergize with checkpoint inhibitors, probably via efficient antigen uptake and DC maturation [antigen processing stage]. Second, tumor infiltrating Foxp3⁺ T cell could play not yet understood tissue protective role during such combo immunotherapy [in effector stage].

David Usharauli

CD5-like likes to go easy with Th17

Two new studies about Th17 cells were published in journal Cell. Using single cell RNA-seq, these papers found additional molecular markers of Th17 pathogenicity. One of those new molecules was CD5L, a lipid scavenger receptor.

The authors, led by Vijay Kuchroo, found that CD5L expression segregated with non-pathogenic Th17 cells [induced in vitro with TGF-β1 and IL-6].

In addition, CD5L expression segregated with non-pathogenic Th17 cells in an in vivo brain inflammation model as well (compare CNS vs. spleen or intestine).

This observation was confirmed in CD5L-KO mice which showed exaggerated brain inflammation.

Adoptive transfer of in vitro [TGF-β1 and IL-6]-differentiated CD5L-KO Th17 cells indicated that absence of CD5L in T cells were sufficient to increase their pathogenicity in brain inflammation model.

Mechanistically, the authors showed that exaggerated brain inflammation with CD5L-KO T cells were related to increased production of IL-17 in Th17 effector stage [but not during naive T cell differentiation to Th17].

Since CD5L was previously implicated in lipid metabolism, the authors tested whether Th17-specific CD5L expression affected their cytokine profile via lipids. Indeed, this was a case. Specifically, it appeared that CD5L was antagonizing RORγt activity and its sensitivity to endogenous lipid ligands [such as oxysterols, a cholesterol metabolites].

In addition to CD5L, second paper in Cell showed Gpr65, Plzp, Toso were individually contributing to Th17 pathogenicity.  

In summary, involvement of cholesterol metabolites in Th17 pathogenicity is clinically highly relevant.

David Usharauli

Fat-loving Tregs control insulin sensitivity during ageing

I have noticed in the past couple of weeks that there is a surge of Foxp3⁺ T cell papers published in top journals. Just yesterday Nature published another paper where the authors claim that they have discovered ageing-related [separate] pathway of insulin resistance driven by fat-associated Tregs.

Lets review their claims. First, the authors showed that unlike "classical" obesity-driven insulin resistance, ageing-related insulin resistance is characterized by enrichment of Foxp3⁺ T cells in adipose tissue (visceral adipose tissue, VAT).

Next, the authors showed that selective depletion of fat Tregs in ageing Foxp3^cre PPARγ ^fl/fl mice promoted shift into "lean body" metabolism[selective decrease in fat mass].

This shift to lean body metabolism in fat Treg depleted mice [i.e. insulin sensitivity ↑] occurred in ageing mice, not in young or obese mice.

Finally, since most of fat Tregs were positive for surface ST2, a receptor for IL-33, their depletion in ageing mice with anti-ST2 antibody [not very efficient by the way] could still improve insulin sensitivity by 25% over control [without any overt inflammation].

In summary, this study uncovered a novel pathway of ageing-associated insulin resistance driven by ST2⁺ fat Tregs. This pathway differs from obesity-driven insulin resistance. However, it is important to remember here that B6 mice [used in this experiments] have strong tendency for obesity during ageing and it remains seen whether this phenomenon with fat Tregs will be applicable for other strains or for humans.

David Usharauli

Time dictates tolerance to skin microbiota

Yesterday journal Immunity published a very interesting study showing that early wave of thymic Foxp3-positive T cells specifically populate neonatal skin between days 7-13 and contribute to tolerance establishment to skin microbiota.   

To study relationship between skin microbiota and skin immune response, the authors modified human skin commensal microbe, Staphylococcus Epidermidis, by introducing into it a nominal peptide antigen 2W [Epi-2W]. This allowed tracking of 2W-tetramer⁺ CD4 T cells. Primary application of Epi-2W to adult mouse skin for the first time induced recruitment of 2W-tetramer⁺ CD4 T cells in skin draining lymph nodes without inducing any skin inflammation [proof-of-principle experiment].

Next, the authors tested how adult mice immune system would respond to secondary skin challenge with Epi-2W. They showed that adult mice exposed to skin Epi-2W do not develop tolerance to it upon secondary challenge. These mice displayed (a) skin inflammation

(b) no accumulation of 2W-tetramer⁺ Foxp3⁺ CD4 T cells, implying failure of tolerance.

However, if skin of neonatal day 7 mice were exposed to Epi-2W and later challenged again, these mice showed (a) minimal skin inflammation 

(b) and robust accumulation of 2W-tetramer⁺ Foxp3⁺ CD4 T cells, implying active tolerance to Epi-2W.

Followup experiments found that between neonatal day 6-13 there was a sudden increase of thymic Foxp3⁺ CD4 T cell numbers specifically in skin.

Finally, the authors showed that when this increase of neonatal skin Foxp3⁺ CD4 T cells were blocked, it resulted in tolerance failure towards Epi-2W challenge and skin inflammation.

In summary, these results indicate that timely [neonatal] exposure to skin commensals is necessary to establish antigen-specific Foxp3⁺ CD4 T cell-mediated immune tolerance to skin microbiota and to prevent pathological skin inflammation later on. It is possible that in near future human neonates will be artificially exposed to the defined sets of human commensals via skin application, nasal inhalations and oral application to specifically train their neonatal immune system for tolerance towards commensals and hence better distinguish between beneficial and pathogenic microorganims.

David Usharauli

Microbiota connects type II immune system to lean body metabolism

There is a renewed interest in gut microbiota research. Initially, this revival of microbiota studies came from observations that obese and lean people have different gut microbiota. It is not clear how exactly microbiota modulates energy metabolism and the research is ongoing in this direction.

For example, journal Nature Medicine has just published another paper where the authors showed that microbiota modulates energy metabolism through its action on a specialized adipose tissue called brown [beige] adipose tissue.

A peculiar aspect of this study [and main reason why it was published in this journal in the first place] has to do with sophisticated methodologies the authors used to study energy metabolism in mice (such as glucose uptake assays with 2-[¹⁴C]-deoxyglucose, [¹⁸F]fluorodeoxyglucose, 2-[1-³H]deoxyglucose, micro-PET-CT, etc).

Basically, the authors showed that when mice are treated with broad spectrum antibiotics, inguinal subcutaneous and perigonadal visceral adipocytes undergo modification (browning and cell size reduction) resembling adipose tissue in Germ-free mice.

Antibiotic treatment led to the increase in Ucp-1⁺ cells in inguinal adipose tissue confirming "browning" of adipose tissue.

In addition, the authors observed "browning" of inguinal and perigonadal adipose tissues in antibiotic treated mice even at thermoneutral conditions (at 30℃ for mice).

Next, the authors showed that microbiota depletion improved "lean body" metabolism both in obese-prone [ob/ob] and high-fat diet fed [HFD] mice.

Finally, the authors showed that antibiotic treatment was associated with type II cytokine profile in "browning" inguinal adipose tissue, including presence of tyrosine hydroxylase (TH) expressing M2 MΦ.

In summary, these results [re]confirmed that microbiota played an important role in "lean body" metabolism associated with development of "brown" adipose tissue. Interestingly, two pathways known for brown adipose tissue development [exposure to low temperature and microbiota depletion] required type II immune system. Type II immune system is mostly known for its involvement in allergy and more recently tyrosine hydroxylase (TH) expressing M2 MΦ has been implicated in neuro-inflammation. It remains to be discovered whether "lean body" metabolism and allergy are interconnected or whether they represent two independent outcome of type II immunity 

David Usharauli

Gut-vascular barrier locks intestinal bacteria within its lymph tissue

This week journal Science published short study from research group from Italy showing the existence of gut-vascular barrier that restricts access of intestinal gut flora to body's internal tissues.

The authors showed that gut endothelial-specific constitutively active (Cre⁺) Wnt/β-catenin pathway prevented blood dissemination of S. typhimurium ΔAroA (aromatic amino acid-dependent mutant). However, translocation of S. typhimurium ΔAroA into intestinal payer patches (PP) or mesenteric lymph nodes (MLN) were not affected by Wnt/β-catenin pathway.

Conversely, S. typhimurium deficient for virulence factor Spi2 (S. typhimurium ΔssaV) was unable to penetrate gut-vascular barrier and to disseminate to liver and spleen.

In vitro experiment confirmed that WT S. typhimurium targets Wnt/β-catenin pathway gene Axin2 in endothelial cells.

Finally, the authors showed celiac disease patients with increased liver damage (ALT^hi) expressed higher level of PV1, a marker of gut endothelial cell damage.

In summary, this study revealed that gut endothelial cells represent additional barrier that locks live bacteria within the borders of gut tissue. This knowledge could be exploited for therapeutic use.

David Usharauli

Without thymic assistance peripheral tolerance to self is easily breached

Yesterday journal Immunity published an important paper about mechanisms of immune tolerance. This study showed that peripheral immune tolerance, as we knew it, doesn't really exist. Here is my analysis of main findings from this study.

The authors have used 4 types of mouse strains for this study, each of them expressing nominal neo-self protein, Cre recombinase, under the guidance of ubiquitous or tissue-specific promoters (UBC-Cre (everywhere), RIP-Cre (insulin producing cells), CC10-Cre (lung tissue), Vil-Cre (intestine tissue). To track endogenous Cre:I-A^bspecific CD4 T cells the authors designed CD4 T cell Cre [pp61-71]:I-A^b tetramers. Staining for Cre:I-A^b tetramer positive CD4 T cells revealed that all strains, except UBC-Cre mice, harbored equal number of Cre:I-A^b specific CD4 T cells.

To test the functionality of Cre:I-A^b tetramer positive CD4 T cells mice were immunized with Cre peptide/CFA. Both WT and RIP-Cre mice showed similar expansion of Cre:I-A^b tetramer positive CD4 T cells. UBC-Cre mice showed minimal expansion as expected [due to low frequency]. Interestingly, expansion of Cre:I-A^b tetramer positive CD4 T cells from CC10-Cre and Vil-Cre were minimal too, even though these mice harbored similar frequency of Cre:I-A^b tetramer positive CD4 T cells as WT or RIP-Cre mice.

These results suggested that in CC10-Cre and Vil-Cre mice Cre:I-A^b tetramer positive CD4 T cells were actively "contained". Indeed, CC10-Cre and Vil-Cre mice harbored large proportion of Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells

and depletion of Foxp3⁺ CD4 T cells in these mice [but not in WT, RIP-Cre or UBC-Cre mice] allowed further expansion of Foxp3-negative Cre:I-A^b tetramer positive CD4 T cells upon Cre peptide/CFA immunization.

Additional experiments revealed that development of Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells were not affected in Foxp3^ΔCNS1 BM chimera mice, implying that Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells were mostly thymic Foxp3⁺ CD4 T cells.

However, peripheral antigen expression promoted tonic signaling and active cycling of Cre:I-A^b tetramer positive Foxp3⁺ CD4 T cells in CC10-Cre and Vil-Cre mice.

Finally, the authors showed that multiple immunization [Lm-Cre infection followed by Cre/CFA immunization] was able to overcome Foxp3⁺ CD4 T cell-mediated non-deletional tolerance in RIP-Cre, CC10-Cre and Vil-Cre mice [but not in UBC-Cre mice] that resulted in antigen-specific tissue retention of Cre:I-A^b tetramer positive CD4 T cells and could even induce a transient increase in blood glucose level in RIP-Cre mice [though none of these mice showed any overt sustained autoimmunity].

In summary, these results indicate that (a) peripheral tolerance is primarily maintained by non-deletional action of thymus derived Foxp3⁺ CD4 T cells, and (b) without ectopic expression of peripheral antigen in the thymus [via Aire or Fezl], such non-deletional tolerance could be easily breached by recurrent infection resulting in sustained autoimmunity.

David Usharauli

Gut commensal Bifidobacterium promotes anti-cancer efficacy of anti-PD-L1 immunotherapy. Part II

This is a second paper in this series (microbiota and immunotherapy). This study came from Thomas Gajewski's lab. It showed that cancer growth or effectiveness of anti-PD-L1 anti-cancer immunotherapy were influenced by composition of gut microflora, more specifically by presence of Bifidobacterium. 

Now this paper starts with experimental results that became classical experiments in gut immunology [since the discovery of TH17 subset]. Namely, the authors showed that B6 black mouse colonies derived from JAX lab or Taconic showed different susceptibility to cancer growth.

This difference between JAX and Taconic mice disappeared when mice were co-housed, implying the role of gut microflora. 

This hypothesis was confirmed in fecal cross-feeding experiments between JAX and Tac mice. It turned out that gut microbiota from JAX mice could enhance anti-tumor immunity when transplanted into Tac mice [but not vise versa].

Furthermore, JAX mouse microbiota could enhance anti-cancer effectiveness of anti-PD-L1 immunotherapy.

Finally, the authors found that a single commensal species, called Bifidobacterium, was largely responsible for microbiota's effect on cancer immunity.

In summary, the authors proposed that live Bifidobacterium could enhance anti-cancer immunity by supporting "generic" dendritic cell maturation and improving T cells antigen-sensitivity. 

This model, however, lacks data that could meaningfully explain why and how only Bifidobacterium has such influence on anti-cancer immunity. Also, it would have been more relevant to test FDA approved anti-PD1 antibody here rather than not-yet-approved anti-PD-L1 antibody therapy. 

David Usharauli