A minor subset within CD11c+ dendritic cells is primarily responsible for peripheral Treg expansion

FOXP3+ Tregs play a major role in tolerance maintenance in the periphery. Most of FOXP3+ Tregs are thymus derived. Thymus is a specialized lymphoid tissue that generates novel T cells from their bone marrow precursors. The question whether naive T cells could convert into FOXP3+ Tregs in the periphery has not been settled. 

It is believed that a "steady-state" condition favors FOXP3+ Tregs conversion in the periphery. However, a concept of "steady-state" is an arbitrary one, defined as absence of deliberate immunization or experimentally observed infection. In fact, whether "steady-state" truly exist is an open question as well.

Why this matters? Almost everyone agrees that in absence of so called "steady-state" naive T cells would convert into effector T cells rather than into FOXP3+ Tregs following antigen recognition. For example, if one wants to generate new FOXP3+ Tregs specific for particular antigen to treat autoimmune diseases, this task would be almost impossible to achieve if condition of "steady-state" does not actually exist in the body [from T cells' "point of view"]. 

Also, what cell types are responsible for that supposed FOXP3+ Tregs conversion? A new study in Immunity clarified this question somewhat. It showed that even in "steady-state" condition only minor subset of DCs within CD11c+ population defined by DEC205/CD8 expression were responsible for FOXP3+ Tregs "conversion" in T cell-replete mice [which harbor endogenous FOXP3+ Tregs].

For this study, the authors have used chimeric anti-DEC205 Ab [or anti-CD11c chimeric Ab as a control] that incorporate antigen of interest [MOG or OVA]. When injected into mice chimeric anti-DEC205 Ab, but not control, could "convert" naive MOG or OVA-specific T cells into FOXP3+ Tregs.

It appeared that DEC205+ CD11c+ DCs were also primarily BTLA+ and its expression were required for FOXP3+ Tregs induction.

Interestingly, the authors proposed that BTLA to HVEM signaling in naive T cells up-regulated CD5 and permitted FOXP3+ Tregs conversion even in presence of inflammatory cytokines such as IL-4 and IL-6.  

However, in my view, such mechanism of FOXP3+ Tregs conversion even in presence of inflammation sounds counter-intuitive. Wouldn't it also induce FOXP3+ Tregs conversion from naive T cells specific for nonself antigen derived from pathogens during inflammation? Otherwise, how can system make sure that only self antigens are presented by DEC205+ DCs? The authors could only admit that this tolerance mechanism somehow only affects "self and tolerizing antigens". Also, what about endogenous FOXP3+ Tregs in these mice? Is it possible that endogenous thymus FOXP3+ Tregs are involved in assisting in FOXP3+ Tregs conversion, rather than DEC205+ DCs doing it alone from scratch? If so, implications are very different.  

David Usharauli

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 and allergic TH2 cells recognize mutually exclusive allergic proteins

This week journal Cell published new data about FOXP3+ Tregs that could significantly advance our understanding of immune regulation.  

One reason why it is so difficult to study human FOXP3+ Tregs is that we don't have a good surface marker that selectively identifies human FOXP3+ Tregs from that of conventional T cell memory. As a consequence, we can't easily study antigen-specific human FOXP3+ Tregs.

In this new paper, the authors proposed that expression of CD137 versus CD154 identifies FOXP3+ Tregs versus Tconv in antigen-specific stimulation assay. Using CD137 as a marker, the authors showed that presence of FOXP3+ Tregs specific for variety of nonself antigens, including allergens.

The vast majority of human antigen-specific FOXP3+ Tregs displayed memory CD45RO+ phenotype.

Interestingly, TCR Vbeta analysis revealed mutual exclusivity of FOXP3+ Tregs and Tconv memory.

Next, when analyzing T cells from allergic individuals the authors first confirmed that indeed allergic individuals harbor significantly more allergen-specific TH2 cells.   

However, surprisingly, the authors found that allergic individuals harbor normal numbers of allergen-specific FOXP3+ Tregs.  

To understand this finding, the authors conducted more stringent allergen-specific assay by culturing T cells with individual allergen protein rather than the whole allergen extract as customary (the whole extract contains several related proteins). Indeed, this modified assay showed mutual exclusivity of antigens recognized by human FOXP3+ Tregs and allergic TH2 cells.   

In summary, this paper showed that unlike conventional wisdom, failure of FOXP3+ Tregs to control TH2 cells is antigen-specific. In essence, allergic individuals have specific "holes" in FOXP3+ Tregs repertoire that do not allow them to prevent allergen-specific naive T cells differentiation into allergic TH2 cells. This is first checkpoint. However, simple absence of allergen-specific FOXP3+ Tregs is not sufficient to induce allergy. Second checkpoint for allergy development requires additional factors, not yet fully understood, that promote TH2 response.

David Usharauli 

Bifurcation of type 2 immunity

Type II immunity is referred to a Th2 dominant immune response to a wide array of proteases, venoms and mechanical irritants. Both innate ILC2 cells as well as adaptive Th2 cells are involved in this process. The relationship between innate and adaptive components of type II immunity is still being defined.

A new study from Richard Locksley's lab published in Nature Immunology showed that priming of Th2 cells in the lymph nodes and their subsequent type II effector functions in the peripheral tissues could be bifurcated.

For example, the authors showed that parasitic nematode Nippostrongylus brasiliensis (Nb)-infected mice triple deficient in sensing of TSLP, IL-25 and IL-33, the epithelial cytokines which have been linked to Th2 cell function, have normal lymph node IL-4+ Th2 differentiation and IgE production, but significantly diminished potential to secrete IL-13 and IL-5, effector Th2 cytokines, in the periphery. 

Importantly, such bifurcation of Th2 effector functionality was T cell intrinsic by sensing locally produced "release" cytokines, TSLP, IL-25 and IL-33.

In summary, this study indicate that even at the level of Th2 cells their effector functionality could be bifurcated depending on the local tissue micro-environment. This concept is important to better understand how to treat different types of allergies, for example, IL-4/IgE dominant systemic allergies versus IL-5/IL-13 dominant local tissue chronic allergies.     

David Usharauli

Antigen-specificity of human FOXP3+ Tregs

Foxp3+ regulatory T cells (Tregs) control immune response to prevent immunopathology. However, unlike conventional T cells, it is hard to determine antigen-specificity of Foxp3+ Tregs in a random T cell pool. Tregs do not secrete anything unique and they do not even proliferate when exposed to antigens in vitro, two functional readouts that are still used as a gold standard for determining antigen-specificity of conventional T cells. 

Hence, we have no clue as to antigen[epitope] specificity of vast majority of human Tregs. Specificity of T cells could be also determined by non-functional readout such as tetramer staining. This is what the authors in new PNAS paper have used to determine antigen-specificity of Tregs. 

They found that adult human peripheral blood contains FOXP3+ T cells that stain with tetramers specific for self as well as nonself peptides (Flu, melanoma protein, HIV epitopes).

Interestingly, frequency of antigen[epitope]-specific FOXP3+ Tregs varied among donors, but they were, on average, equally distributed among self and nonself [epitope]-specific Tregs, except Flu HA epitope.  

Finally, comparison of neonatal [cord blood] and adult blood revealed that actual number of [epitope]-specific Tregs / per ml of blood did not change much between newborn and adult indicating that most of Tregs tested in this study were generated already by the time of birth.

In summary, this study revealed that human peripheral blood contain Tregs specific for both self nonself antigens. Since tested donors were negative for some of the infection such as HSV, CMV or HIV, it begs the question what [cross-reactive?] antigens maintain CMV or HIV-specific Tregs in antigen-naive [antigen-unexposed] hosts?    

David Usharauli

Engineering T cells to cellular factories with synthetic Notch receptors

This week journal Cell published new article from Wendell Lim's lab at UCSF (also a founder of Cell Design Labs) that reads like a science fiction story. It was a continuation of previous work that focused on developing customized molecular architecture based on Notch core regulatory domain attached to synthetic extracellular recognition and intracellular transcriptional domains (SynNotch). By changing extracellular and intracellular domains one can design T cells producing molecule of interest upon engagement with specific ligand. 

For this new paper, the authors managed to transform T cells into cellular factories which upon specific SynNotch stimulation were making and expressing (a) cytokines (b) checkpoint inhibitors (c) bi-specific antibodies (d) CARs (e) transcriptional factors (f) lytic granules.

Basically, T cells are transfected with vector containing SynNotch module linked to promoter encoding molecule of interest. The most of the experiments were done in vitro. One in vivo experiment the authors put in paper was similar in overall design to one previously reported. In short, NSG mice were injected with K562 tumor expressing GFP + CD19 or only CD19. Afterwards T cells containing SynNotch module designed to recognize GFP were infused. Upon GFP recognition these T cells could start expressing soluble Blinatumomab, a-CD19/CD3 BiTE molecule that in turn can engage conventional TCR and produce T cell activation. Indeed, the authors showed that only tumor cells expressing both GFP and CD19 were efficiently controlled by engineered T cells (though its is not clear why soluble BiTE antibodies would not diffuse freely and engage single CD19+ tumors as well).    

While all these results look very impressive, it nonetheless lack some of the critical elements that are required to properly analyze beneficial effect of SynNotch. For example, in vivo experiments were done in mice that does not express tumor antigens naturally (anti-CD19 here was human in origin). So we can't tell how such T cells would behave in "human-like" environment. Similarly, in vitro experiments with SynNotch-anti-HER2 expressing T cells are of dubious value without testing it at least in humanized mouse models.

David Usharauli