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

Receptor-ligand specific labeling of immune cell interactions

This week journal Nature published new study in immunology that could be best described as a method paper. I personally don't understand the value of this paper been in Nature. Only positive characteristic I see in it is that experiments reported are done in a classical, cellular immunology "fashion" and very easy to follow and understand. Lets examine.

The new method, called LIPSTIC, which this study reported is about labeling receptor-ligand pair with naturally occurring enzyme, the Staphylococcus aureus transpeptidase sortase A (SrtA). SrtA appears to covalently transfers a substrate containing motif ‘LPXTG’ to a nearby oligoglycine (G5). Basically, receptor is genetically fused with SrtA and ligand is fused with G5 and when they interact, SrtA catalyses the transfer of the substrate onto the G5-tagged receptor. This transfer can be visualized by attaching to the substrate small labels such as biotin or a fluorophore.  

First the authors showed in vitro and ex vivo that the substrate transfer was specific to fused receptor-ligand pair and did not occur when enzyme was inactive or fused to unrelated receptor. Most of the reported experiments were done using CD40L/CD40 pair (CD40 signaling is relevant for DCs and CD8 T cell activation by CD40L expressing CD4 T cells).

Receptor-ligand specificity was maintained in vivo as well. Because CD40L upregulation on CD4 T cells depends on antigen-specific interactions, substrate transfer were restricted to those CD40+ DCs that were pulsed with cognate peptide (OVA).

However, this antigen-specific CD40L/CD40 interaction was maintained only for initial 10h-24h period. When OVA-specific T cells were left with DCs for longer period (48h) then even DCs pulsed with irrelevant peptide (LCMV peptide) got labeled with substrate. 

It is not clear what are the biological consequences of such non-specific T/DCs interactions. Are these two different DCs activated antigen or non-antigen specific manner somehow different with the regard of activation of CD8 T cells? We don't know. The problem with such model is that if activated CD4 T cells expressing CD40L can interact with CD40+ DCs and activate it (license it, to use polly matzinger's words) then we should expect that body should harbor only activated DCs because body constantly contains some number of CD40L+ activated CD4 T cells specific for all kind of antigens. It is possible high number of T cells transferred in these experiments created an artificial outcome.

In summary, this study showed new method how to label receptor-ligand pair in vivo. However, overall relevance of this method is not clear at present.

posted by David Usharauli

Newborns' resistance to pneumonia is driven by acquired microbiota

This week Science Translational Medicine published new study that showed that in newborn mice resistance to pneumonia is driven by neonatally acquired microbiota. It revealed how antibiotic therapy given to mothers near time of delivery could alter and weaken baby's defenses against airway pathogens.

Newborn mice derived from germ-free or from antibiotic-exposed pregnant mice display increased susceptibility to Streptococcus pneumoniae serotype 19A-induced pneumonia that could be reversed by microflora.

Application of epithelial-focused cytokine IL-22 had similar effect on reversing newborn mice susceptibility to pneumonia.

In the lungs of newborn mice, majority of IL-22 is made by RORgt+ group 3 innate lymphoid cells (ILC3).

Antibiotic therapy of pregnant mice reduced IL-22+ ILC3 population in the newborn lungs that could be reversed by microflora.

Moreover, depletion of endogenous ILC3 increased host's susceptibility to pneumonia that could be reversed by adoptive transfer of WT ILC3.

In summary, this study showed that antibiotic therapy of pregnant females at the time of delivery could profoundly affect newborns' ability to mount proper defense against airway pathogens by depleting microflora and disrupting microflora → ILC3 → IL-22 axis.


David Usharauli

PAMPs and DAMPs cooperate to drive vigorous adaptive immune response

This week Science published interesting article related to basic question of dendritic cell (DCs) activation and initiation of productive adaptive immune response. It showed that endogenously generated oxidized phospholipids (oxPAPC) cooperate with bacterial-derived LPS in a caspase 1/11 dependent manner to enhance viability and activation status of DCs enabling a better adaptive immune response.  

I would like to note here that title of this paper does not exactly captures the main idea behind this study. In fact, only reading the final portion of the paper one can get the sense and larger meaning of this study. I will try to explain it.   

This paper's main idea is to understand the difference between PAMPs and DAMPs with the regard of their impact on DCs. Every scientists in immunology is familiar with pathogen-associated molecular patterns (PAMPs, such as LPS) and damage-associated molecular patterns (DAMPs, such as ATP). Nonetheless, it is still unclear what role(s), for example, DAMPs play within immune system.

To understand it, the authors focused on one particular type of DAPMs, oxidized phospholipids (oxPAPCs). oxPAPCs are found in inflammatory milieu and can reach concentrations of 10-100 μM in damaged tissues. Initially, the authors showed that unlike LPS, oxPAPCs do not signal via TLR4.

However, similar to other DAMP molecules, oxPAPCs could induce IL-1β release from LPS-primed DCs. As expected, oxPAPC-induced IL-1β release from LPS-primed DCs required inflammasome activation, since such effect were absent in DCs from ASC knockout (KO), caspase-1 KO, caspase-1/caspase-11 double KO or NLRP3 KO mice, each of which are defective for inflammasome functions.

Pyroptosis is an inflammasome-dependent cell death characterized by loss of plasma membrane integrity. Interestingly, unlike LPS or LPS+ATP combination, oxPAPC-induced inflammasomes did not promote pyroptosis. Thus oxPAPC promoted IL-1β release from living DCs.

Finally, the authors showed that mice immunization with antigen in combination with oxPAPC+LPS could improve priming of adaptive T cells in a caspase 11-dependent manner.

In summary, this study showed that DAMP molecule, oxPAPC, promotes DC viability and IL-1β release when combined with PAMP molecule, LPS. Such combination of DAMP and PAMP promoted more productive adaptive immune response.

David Usharauli

DC vaccines produced with tumor cells undergoing immunogenic cell death

Dendritic cells are the most potent activators of naïve T cells. A lot of effort has been spent to develop DC-based cellular vaccine. We even had some success in mouse models. But, so far no DC based vaccine worked in humans to significantly prolong patients survival. It is not surprising that only company that developed FDA-approved DC vaccine prostate cancer went bankrupt. There are many reasons as to why it is so hard. For one, there are several types of dendritic cells. Secondly, just pulsing [tumor] antigens on dendritic cells were found not to be sufficient.

Few weeks ago, Science Translational Medicine published study wherein the authors had tested next-generation tumor vaccine produced by pulsing DCs with tumor cells undergoing immunogenic cell death.

Basically, the authors had used a high-grade glioma (HGG) cell lines as a source of tumor. To generate immunogenic cell death, tumor cells were exposed to hypericin-based photodynamic therapy (Hyp-PDT). This treatment induces expression/release of several damage-associated molecular patterns (DAMPs) acting as potent danger signals for DCs. When mice were injected with DCs pulsed with Hyp-PDT treated glioma cells, they showed resistance to subsequent live glioma brain challenge.

Mechanistically, tumor protection provided by Hyp-PDT DCs depended on (1) DAMPs, (2) DC-specific MyD88, (3) CD8 T cells (T cells data are shown here).

Importantly, Hyp-PDT DC vaccines improved survival of brain tumor-bearing mice when applied therapeutically and in combination with standard-of-care therapy such as temozolomide (TMZ).

In summary, this study showed that hypericin-based photodynamic therapy could generate immunogenic tumor cells required for proper activation of DC for vaccination purpose. Conceptually, such mode of DCs preparation makes a lot of sense, but there are several technical challenges before this system could be translated into clinic, as discussed by the authors (such as, how to avoid presence of "surviving" live tumor cells within DC vaccine, route of DC vaccine injection [the authors have used intra-peritoneal injection, for some reason], what type of DC is the best for this purpose). 

David Usharauli

IL-13/CCL17 axis drives Th2 memory response to allergen

Allergic response is immunological "mystery". In allergy, first step is sensitization when immune system detects new entity [hapten/antigen] and marks it for IgE mediated destruction. This step is more confusing because all kind of bio/chemical entities could be marked for IgE path by immune system, for no apparent reason.

No universal hypothesis exists that could explain and predict allergic response. Right now, we are just left to treat clinical "effector stage" [that usually happens during re-challenge with the same entity].

The following paper from Nature Immunology is one of those type of research articles. Here, the authors showed that memory Th2 response to allergen require engagement of IL-13 producing innate lymphoid cell type 2 (ILC2), followed by CCL17 producing DCs.

The authors used papain allergy model. In this model eosinophils are rapidly recruited in response to papain re-challenge (at day 15).

Next, the authors used gene-modified mouse strain (ICOS-T mouse) where ILC2 could be selectively depleted. Papain re-challenge of ICOS-T mice depleted of ILC2 reduced Th2 accumulation in the lung.

Next, the authors found that IL-13 and CCL17 were rapidly produced in the lung upon papain re-challenge.

They found that ILC2 were primary producers of IL-13 upon papain re-challenge.

They also found that neutralization of either IL-13 or CCL17 reduced Th2 accumulation in the lung upon papain re-challenge.

Next, the authors found that IL-13 receptor expression on DCs were necessary for lung CCL17 production and Th2 accumulation in response to papain re-challenge.

Finally, the authors showed that similar mechanism of Th2 accumulation was operational in papain re-challenged skin as well.

In summary, this study indicates that effector stage of allergic response were promoted by IL-13⁺ ILC2 / CCL17⁺ DCs axis. This knowledge provides additional therapeutic targets for allergy management.
 
David Usharauli

Perforin deficiency in dendritic cells leads to overweight and metabolic syndrome

Dendritic cells (DCs) are both necessary for immunity and tolerance. While immunity part is more or less clear, DC's role in tolerance is not clear at all.

Here is another weird tolerogenic DC paper from Immunity. Here, the authors found that in BM chimera mice that lack perforin specifically in CD11^hi DCs develop a metabolic syndrome (obesity, insulin resistance and low glucose tolerance).

To tell you truth, this study should not be in Immunity. Journal of Immunology would have been better place. The problem is that the authors did not even bother to check any role of gut micirobiota in all these. But if you mention metabolic syndrome and did not check for gut microbiota, this is a cardinal sin for today's immunology.

In brief, the authors have generated mixed BM chimera mice wherein only DCs in mouse would be derived from BM cells lacking perforin (though having CD11c-driven Cre model would have been better alternative). Unexpectedly, the authors found that starting at 3 month these BM chimera mice become overweight.

The authors found that these mice with CD11c-specific perforin deficiency developed large adipose cells.

Finally, they showed that metabolic syndrome in these mice could be reversed by T cell depletion (but the authors have no data to explain why or how presence of T cells induces metabolic syndrome).

In summary, according to authors, this study suggests that when DCs lack perforin and are not able to eliminate T cells, this leads to accumulation of T cells that promote metabolic syndrome. What or how is not known. No words about gut microbiome.

David Usharauli  

Melanoma-intrinsic β-catenin signaling prevents local DCs activation and anti-tumor T cell-priming

A high-profile scientific literature in immunology for the past 2 years is dominated by papers related to tumor immunotherapy. This probably has to do with the clinical successes of various immunotherapeutic approaches seen in recent years and the shift in funding for cancer research.

Here is new paper from journal Nature discussing immune response to tumor. The authors showed that melanoma-intrinsic β-catenin signaling prevents DC-mediated anti-tumor T cell priming.

By profiling melanoma tumor samples based on CD8 T cells numbers the authors observed that melanoma samples with low T cell numbers were enriched for mutations in genes involved in β-catenin pathway (gain-of-function or loss-of-function mutations).  

Using mouse model of spontaneous tumor induction, the authors confirmed that presence of active β-catenin signaling prevented T cell recruitment to the tumor tissue.

Experiments with adoptive transfer of nominal antigen (SIY)-specific T cells indicated that active β-catenin signaling in tumors prevented T cell priming.

Next, the authors showed that β-catenin signaling in tumor prevented recruitment of CD8α⁺/CD103⁺ DCs, a DC subset responsible for cross-priming. This suggested that lack of infiltration of tumors by T cells may have to do with lack of T cell priming by DCs.

Indeed, the authors showed that intra-tumoral injection of in vitro generated wild-type DCs activated with Poly(I:C) could restore T cell tumor infiltration.

Finally, the authors showed that adoptive transfer of wild-type of DCs in combination with antibodies against CLTA4/PD-L1 restored anti-tumor effect against β-catenin active tumor.

In summary, these results indicate that melanoma with constantly active β-catenin pathway inhibits anti-tumor T cell priming. Of note, β-catenin pathway plays a role in DCs maturation. It is not clear whether β-catenin pathway in DC per se was a contributing factor in this mouse model (separately from tumor).

David Usharauli