Thursday, June 18, 2015

Mechanism of high fat diet (HFD)-induced skin inflammation

High fat diet (HFD) has been implicated in a metabolic syndrome that combines several set of diseases such as Type II diabetes, obesity, etc. Frequently, metabolic syndrome is associated with skin inflammation (acne, dermatitis, etc). Mechanism is unknown.

A new paper in journal Immunity provided results that indicate that epidermal fatty acid binding protein, E-FABP, is a direct link between skin inflammation and HFD.

This is a simple, observation-type of research. I would say that the authors were lucky to get it published in Immunity. Though, I liked the idea that the authors developed the concept for the paper through serendipitous observation.

Initially, the authors showed in their mouse facility, B6 mouse strain fed HFD for 3-6 months developed skin lesions that correlated with fat content in the diet.

Next, the authors found that lesion skin from HFD-fed mice contained higher proportion of macrophages.

These macrophages from lesion skin tend to produce high levels of IL-1β and IL-18.

Analysis of E-FABP expression (fatty acid chaperons) showed its up-regulation in lesion skin and macrophages from HFD-fed mice.

Finally, using E-FABP knockout mice, the authors showed that absence of E-FABP prevented development of skin lesions in HFD-fed mice (with no effect on weight gain).

In summary, these results indicate that excess of dietary fat (saturated fat) induces skin inflammation via epidermal fatty acid binding protein. The authors think it has to do with activation of NALP3 pathway in macrophages by excess saturated fat (leading to IL-1β and IL-18 production) and recruitment of T cells secreting IFN-γ and IL-17 (though those are correlative results and not particularly persuasive). However, effect of E-FABP on skin lesion development is striking.

David Usharauli

Friday, June 12, 2015

Systemic distribution of common origin T cell clones after skin immunization

Rapid memory to antigenic challenge is a fundamental feature of adaptive immune system. Accurate detection of antigen-specific T cells would play a major role in uncovering the mechanism leading to memory formation.

Thus far the most widely used technique to detect and track antigen-specific T cells was MHC-(or HLA) tetramer technology, developed in late 90s. However, this method is only optimal in tracking a limited number of antigen-specific T cells.

More recently new method of antigen-specific T cells tracking has started to be rapidly adopted by both academic and industry researchers. This new method is using HTS sequencing of TCR Vβ CDR3 regions that is unique to each clone. This method allows massive parallel tracking of T cell clones involved in immune response to tumors or immunization.

In this regard, this new paper in Nature Medicine has provided results based on tracking of T cell clones after skin immunization using TCR Vβ CDR3 sequencing data developed by Adaptive Biotechnologies. Of note, many recent high profile research articles published in top journals have used services and technologies developed by teams at Adaptive Biotechnologies. Here, the authors showed that clonal composition of skin and LN-derived T cells expanded after skin immunization were very similar, implying common origin.    

First, the authors showed that skin immunization induced accumulation of identical TCR Vβ CDR3 T cell clones in both local and distant skin tissue (TRM), as well as in draining and distal LNs (TCM). T cell frequency is measured as # of Vβ CDR3 copies per 400ng of genomic DNA.

Next, the authors showed that repeated immunization dramatically increased the frequency of TRM clones in the skin relative to its abundance in LNs. However, LNs still contained larger reservoir of different T cell clones that expanded after skin immunization.

Finally, using parabiosis model, the authors showed that skin-associated TRM (sedentary) cells provided rapid response to antigenic challenge compared to LN-associated TCM (migratory T cells).  However, for some reason, the magnitude of secondary response of TRM in distal skin tissue of sensitized mouse was less compared to its response in local sensitized tissue. This contradict their earlier data in Fig 1 where both local and distal tissues contain equal frequency of specific T cell clones expanded after skin immunization.

In summary, this new method may allow more efficient tracking of T cells expanded in response to antigen of interest. One caveat of this approach, of course, is the fact that there is no way to know for sure whether expanded T cell clones are indeed antigen-specific. Another important finding in this paper is the fact that T cell clones sampled from the blood after immunization showed no clonotypic resembles to clones found in skin or LN. This may indicate that experiments on blood (PBMC)-derived T cells may not provide an accurate picture of antigen-specific T cell response to antigens after skin immunization.

David Usharauli

Sunday, June 7, 2015

Not all CAR-T cells are created equal

CAR-T cell-based adoptive immunotherapy against B cell-derived malignancies shows remarkable effectiveness in clinical trials. But, for some reason, other CAR-T cells, specific for diverse tumor-associated antigens, are not as effective as α-CD19 CAR-T cells. So why is that?

New paper in Nature Medicine may provide some clues to this mystery. The authors, led by Crystal Mackall, showed that unlike α-CD19 CAR-T cells, several CAR-T constructs displayed antigen-independent basal CD3zeta signaling leading to early exhaustion and lack of anti-tumor in vivo effectiveness.   

When comparing two CAR constructs α-CD19 and α-GD2 (sarcoma antigen), the authors showed that while both CAR-T cells displayed similar in vitro cytotoxicity, their in vivo anti-tumor activity against sarcoma cell line double-positive for CD19 and GD2 were vastly different: only α-CD19 CAR-T cells could control tumor growth.  

These results suggested that CAR construct, rather than tumor targets, was responsible for this difference. Indeed, T cells double-transduced with both α-CD19 and α-GD2 CAR constructs displayed diminished in vivo anti-tumor effectiveness against CD19+sarcoma (compared to CD19-only CAR-T cells).  

Further analysis showed that unlike CD19 CAR-T cells, GD2 CAR-T cells showed spontaneous, basal CD3zeta activity. Such basal CD3zeta activity were absent in GD2 CAR-T cells with mutations in CD28/CD3 signaling domains indicating that GD2 CAR was responsible for this spontaneous signaling.

Furthermore, the authors showed that spontaneous clustering of GD2 CAR construct (oligomerization of scFv fragments) probably was responsible for basal signaling in GD2 CAR-T cells (and not vice versa).

Finally, the authors showed that substitution of CD28 domain with 4-1BB (CD137) domain in CAR-T construct could partially rescue GD2 CAR-T cells exhaustion phenotype and improve its in vivo anti-tumor effectiveness.

In summary, data provided in this paper indicate that biochemical property of CAR constructs impart their anti-tumor effectiveness. This suggests that design of effective CAR-T construct would take more than just specificity. In my view the issue is that scFv are of BCR origin, but precise nature of BCR signaling is not clear even today. One model suggests that BCR signaling requires their oligomerization and other model suggests exactly opposite: BCR signaling requires disruption of BCR oligomers. But since assembly of TCR singaling complexes may differ from that of BCR signaling complex assembly, simply transplanting BCR domain into T cells may not be as successful as initially thought.

David Usharauli        

Tuesday, June 2, 2015

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