Tumor protein encoding [negatively charged] RNA-nanoparticles induced robust anti-tumor response

This week journal Nature published study that showed that negatively charged [cancer antigen encoding] RNA-nanoparticles are selectively take up by antigen-presenting cells in lymphoid tissues and induced robust type I IFN driven anti-tumor immune response to all tumors tested.

In earlier studies for in vivo delivery of RNA/DNA nucleic acids into antigen-presenting cells scientists have used positively charged [cationic] nanoparticles. However, such particles were mostly trapped within lungs rather than lymphoid tissues where most antigen-presenting cells, such as dendrtic cells, reside. Surprisingly, here the authors showed that negatively charged RNA-nanoparticles selectively accumulated in spleen within antigen-presenting cells after in vivo injection.

Strangely, accumulation of RNA-nanoparticles were reduced when mice were depleted of conventional DCs (cDCs) after diphtheria toxin injection (CD11-DRT BM chimera mice), even though these mice retain both plasmacytoid DCs (pDCs) and macrophages which can themselves capture RNA-nanoparticles.

RNA-nanoparticle injection was associated with rapid IFN-α secretion in a manner that depended on TLR7 expression.

In addition, RNA-nanoparticles induced robust priming of antigen-specific adaptive immune response.

More importantly, injection of tumor protein encoding RNA-nanoparticles delivered incredibly robust prophylactic and therapeutic anti-tumor effect (article has some preliminary  results from phase I study showing T cell priming in cancer patient after RNA-nanoparticle delivery).

In summary, the authors of this study believe that they found universal antigen delivery RNA-nanoparticle construct that induces robust adaptive immune response.

There is one thing that is puzzling about this study which the authors did not tried explain: in mice depletion of cDCs eliminated RNA-nanoparticle uptake and T cell priming. However, pDCs are not depleted in CD11c-DTR chimera mice and the data showed that pDCs are themselves could capture RNA-nanoparticles and are the main source of type I IFNs. So, the question then is why cDCs depletion has such disproportional effect?

David Usharauli

New mouse model to mimic immune response to Flu virus in elderly

This week Science published several research papers in immunology. One of these papers is the study from Yale School of Medicine showing that Tlr7^–/– × Mavs^–/– double KO mice expressing human anti-viral molecule Mx1 display susceptibility to influenza A virus in a caspase 1/11-dependent manner.

It is known that elderly individuals show susceptibility to influenza A virus. One reason for this susceptibility is a reduced ability of elderly immune system to produce type I IFNs in response to influenza A virus.

In contrast, aging mice do not show such susceptibility to influenza A virus. Mouse does not express Mx1, a dynamin-like guanosine triphosphatase that blocks primary transcription of influenza in humans. To make mouse model "usable" for  studying human response influenza A virus, the authors created Mx1⁺ mice. Mx1⁺ mice were resistant to experimental influenza A virus infection and this resistance was dependent on Tlr7 and Mavs (both molecules are involved in anti-viral response).

Interestingly, while Mx1⁺ mice double deficient for Tlr7^–/– × Mavs^–/– were susceptible to influenza A virus infection,  Mx1⁺ mice triple deficient for Tlr7^–/– × Mavs^–/–× Casp1/11^–/– were resistant to influenza A virus infection even though both showed similar viral burden (Of note, Tlr7^–/– × Mavs^–/–× Casp1/11^–/– mice were eventually cleared the virus by 30 days after infection).

Moreover, Mx1⁺ mice triple deficient for Tlr7^–/– × Mavs^–/–× Casp1/11^–/– were resistant to influenza A virus infection even though they too showed similar secondary bacterial "bloom" in their airways.

These data suggested that caspase 1/11 signaling reduced "tissue tolerance" [rather than increased anti-viral response] to influenza A virus infection and secondary bacterial "bloom" in Mx1⁺ mice deficient for anti-viral innate signaling via Tlr7^–/– × Mavs^–/–.

In summary, this study showed that intact caspase 1/11 signaling compromises tissue tolerance to acute influenza A virus infection and secondary bacterial "bloom" in host with a weakened anti-viral signaling.

David Usharauli

CD4 T cells provide universal "help" to CD8 T cells via pathogen-tailored DCs

Ordinarily [but not always] naive CD8 T cells require "help" from CD4 T cells to undergo full differentiation and to develop into memory. Such CD4 T cell help is provided via so called "licensed" antigen-presenting cells, DCs. In a simple scenario, when pathogen invades tissue, local DCs will pick up its antigens and present them to both CD4 and CD8 T cells. In turn, activated antigen-specific CD4 T cell "licenses" the same DCs to up-regulate or secrete necessary molecules to complete priming of naive CD8 T cells (I am going to use terms "help and "license" interchangeably).

This simple model is complicated by fact those "licensing" molecules for CD8 T cells differ depending on pathogens. The most well described "helps" include IL-12, IL-15 or type I IFNs. So, how CD4 T cells are able to deliver so many different licensing signals?  

Apparently, CD4 T cells don't. According to new paper published in Cell Reports, CD4 T cells simply amplifies pre-existing pathogen-tailored signals within DCs. Lets see if data are convincing (note, this paper was under review process for > 2 years)

Initially, the authors confirmed that CD8 T cell priming/expansion during viral infection, HSV-1, required presence of CD4 T cells, MHC II, CD40L or CD40.

Next, the authors showed that CD8 T cells priming/expansion during HSV-1 infection required signaling via either IFNαR or IL-15.

Experiments with BM chimeras, IL-15^KO:CD11c^DTR and IFNαR^KO:CD11c^DTR, revealed that DCs-specific expression of IFNαR and IL-15 were required for CD8 T cell priming during HSV-1 infection.

However, production of IL-15 by DCs in response to IFNα also required presence of CD4 T cells.

In fact, ex vivo stimulation of CD8α⁺ DCs with IFNα and αCD40-mimetic (as a surrogate for CD4 T cell help) showed that CD4 T cell "help" amplified IL-15 induced by innate [viral-induced] IFNα (since αCD40-mimetic alone had no effect). However, it is not clear whether αCD40-mimetic could fully recapitulate CD4 T cell function. So, this requires additional tests.

Dominant role of innate signaling in determining the nature of CD4 T cell "help" was revealed in experiments in which mice were challenged with cell-associated OVA in combination with LPS or Poly(I:C). In presence of LPS, "help" was IL-12 dependent, while in presence of Poly(I:C), "help" was IL-15 dependent.

In summary, the conclusion of this study, according to the authors, is that CD4 T cells simply amplify pathogen-tailored innate signals already generated within DCs, rather than proving unique maturation signals. My interpretation of these results is not very different from earlier models. I don't think that anyone claimed that CD4 T cell "help" and innate signals were completely interchangeable. For me, "licensing" and in this case "amplification" are very same concepts. For me, more important question is how those CD4 T cells that deliver "help" are getting activated in first place (basically, who primes the "primers").

David Usharauli

Type I IFNs produced during viral infection promotes bystander regulatory T cells

Foxp3⁺ regulatory T cells are one of my favorite topics. Foxp3⁺ T cells are the most powerful regulatory subset within immune system. It is unbelievable but true that as of today only sure thing we know about Foxp3⁺ T cells is the fact that their absence or ablation leads to severe and total autoimmune organ disorders and death. No other T or B cells or innate subsets produce such a dramatic effect on the body.

At the same time, we are still have no clear answer whether Foxp3⁺ regulatory T cells operate in an antigen-specific manner or whether they non-specifically suppress "overly" excessive immune response (both innate or adaptive driven).

There are several hypothetical questions regarding Foxp3⁺ T cells that would require experimental proof before we can make any significant breakthrough. 

For example, 

"if Foxp3⁺ T cells are so potent, how immune response is initiated in the first place?" 

"If initial inflammatory stimuli temporary inactivates Foxp3⁺ T cells to allow initiation of immune response, how long such inactivation lasts?" 

"if Foxp3⁺ T cells regulate excessive immune response (excessive inflammation), how they can sense what is excessive?"

In this regard new paper in Nature Immunology from Steven Ziegler's lab is interesting to read. Here, the authors showed that pre-exposure of naive antigen-inexperienced CD4 T cell to type I IFNs initiates their differentiation towards regulatory pathway rather than effector. Such scenario possibly prevents excessive bystander activation of naive T cells and reduces overall tissue damage.

In vitro studies showed that CD4 T cells initially pre-exposed in vivo to polyI:C (as a source of type I IFN) and then co-cultured with antigen-pulsed DCs and TGF-β tended to preferentially develop into Foxp3⁺ T cells.

Such preference for Foxp3⁺ T cell development were abolished with CD4 T cells from IFNαR1^KO mice.

RIP-mOva × Rag2KO hosts receiving naive OVA-specific T cells pre-exposed to polyI:C do not develop diabetes (c), while RIP-mOva × Rag2KO hosts receiving control naive OVA-specific T cells together with polyI:C rapidly developed diabetes. Importantly, secondary exposure to polyI:C of RIP-mOva × Rag2KO hosts transferred with naive OVA-specific T cells pre-exposed to polyI:C still did not break tolerance (d).

These experiments indicated that simultaneous exposure of naive T cells to antigen and type I IFNs drove effector differentiation, but if they were first pre-exposed to type I IFNs and then to antigens it drove their Foxp3⁺ T cell differentiation. Indeed, this observations were confirmed in subsequent experiments that revealed that exposure to type I IFNs 2-3 days before antigenic exposure provided the most optimal condition for Foxp3⁺ T cell development.

In summary, this study suggests the following scenario: type I IFNs released during viral infection conditioned bystander, nonspecific naive T cells for Foxp3⁺ T cell development. This mechanism potentially prevents excessive activation and effector differentiation of naive T cells encountering antigens later during immune response (including tissue-derived self-antigens).

David Usharauli

Collapse of cholesterol biosythesis triggers universal anti-viral response

New study in journal Cell may provide mechanistic clue how obesity [and hyper-cholesterolemia] affect immunity. The authors showed that limiting cholesterol biosynthesis automatically triggers universal anti-viral defense state, mimicking natural response to viruses. 

Initially, the authors studied effect of type I IFNs [and its triggers] on lipid metabolism in macrophages. These pilot experiments reveal that de novo synthesis of cholesterol was inhibited by type I IFNs and its triggers, such as virus or PolyI:C. Interestingly, total cholesterol level or its import were not affected.

To decouple direct involvement of host defense response in lipid metabolism, the authors used LysM-Cre-Scap ^fl/fl mice that show limited lipid biosynthesis in myeloid cells. Indeed, Scap^-/- macrophages displayed the same decrease in cholesterol synthesis as type I IFN treated control cells.  

Reduction of cholesterol synthesis in Scap^-/- macrophages was accompanied with increased resistant to viral infection [similar resistance were observed in MKV (mevalonate kinase) and  HMG-CoA reductase deficient cells that have limitation in cholesterol biosynthesis].

This resistance in Scap^-/- macrophages was transferable to control cells via soluble component in conditioned medium (type I IFNs).

Notably, the authors showed that anti-viral resistance in Scap^-/- macrophages could be inhibited by exogenous free cholesterol.

Finally, the authors showed that DNA sensing STING-cGAS pathway was responsible for increased anti-viral state that accompanied collapse of cholesterol synthesis, and here too, free cholesterol supplementation was able to inhibit cGAS activity by its ligand [mechanism is unknown].

In summary, this study raises some of the important questions related to host metabolic shift observed during infections. Inhibition of anti-viral state by free cholesterol could explain diminished immune state observed in obese individuals. In addition, there are studies showing how cholesterol pathways, such as geranylgeranylation could influence TH17 and Foxp3⁺ Treg balance.

David Usharauli

Mitochondrial DNA abnormality activates cytosolic type I IFN system

Mitochondria is a cell's energy hub. It is of a prokaryotic (simple bacterial) origin. It has its own DNA. However, prokaryotic DNA can activate innate immune system. So, what would happen if our mtDNA undergo misfolding stress?

This is exactly what the new Nature paper tried to uncover. This study led by Gerald Shadel at the Yale School of Medicine, has examined the outcome of mitochondrial DNA (mtDNA) packaging abnormality by studying mouse model heterozygous for mitochondrial transcription factor A (TFAM^-/+), a protein responsible for mtDNA higher-order nucleoid organization.

Gene expression profiling revealed that cells in TFAM^-/+ mice showed enrichment in IFN-stimulated pathway genes.

The authors showed that this type I IFN response was significantly abrogated in cGAS, STING, IRF3 and TBK1 deficient backgrounds indicating that in TFAM^-/+ mice aberrant mtDNA released into cytosol activates cGAS-STING-IRF3 pathway leading to type IFN production.

Additional experiments showed that such heightened type I IFN response in TFAM^-/+ mice reduced viral cellular burden.

Finally, the authors showed that viruses from herpesviridae family could induce mtDNA misfolding (by TFAM depletion via viral-encoded UL12 M185 molecule) and mtDNA dependent type I IFN response in wild type mice, mimicking TFAM^-/+ mice, suggesting physiological function of mtDNA stress response.

In summary, these results highlight how mitochondrial DNA stress could activate innate immune system and cause inflammation (in human disorders such as systemic lupus erythematosus, cancer, multiple sclerosis, etc).

This study provides additional support for recently described immunological function of mtDNA and its detection by cGAS-STING-IRF3-type I IFN pathway. It is of note that many cellular abnormalities analysed from immunological point of view are converging on type I IFN response.

David Usharauli