INTESTINAL STEM CELL DAMAGE IN GRAFT-VERSUS-HOST DISEASE IS PRIMARILY CAUSED BY T-CELL DERIVED INTERFERON-GAMMA
Author(s): ,
Suze Anne Jansen
Affiliations:
Paediatrics,University Medical Center Utrecht,Utrecht,Netherlands;Medicine,Memorial Sloan Kettering Cancer Center,New York,United States
,
Shuichiro Takashima
Affiliations:
Medicine,Memorial Sloan Kettering Cancer Center,New York,United States
,
Maria Laura Martin
Affiliations:
Molecular Pharmacology,Memorial Sloan Kettering Cancer Center,New York,United States
,
Ya-Yuan Fu
Affiliations:
Medicine,Memorial Sloan Kettering Cancer Center,New York,United States
,
Joris Bos
Affiliations:
Paediatrics,University Medical Center Utrecht,Utrecht,Netherlands
,
Richard Kolesnick
Affiliations:
Molecular Pharmacology,Memorial Sloan Kettering Cancer Center,New York,United States
,
Caroline A. Lindemans
Affiliations:
Paediatrics,University Medical Center Utrecht,Utrecht,Netherlands;Princess Máxima Center for pediatric oncology,Utrecht,Netherlands
Alan M. Hanash
Affiliations:
Medicine,Memorial Sloan Kettering Cancer Center,New York,United States;Medicine,Weill Cornell Medical College,New York,United States
EHA Library. Jansen S. Jun 15, 2019; 267475; S892
Suze Jansen
Suze Jansen
Contributions
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Abstract

Abstract: S892

Type: Oral Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 16:30 - 16:45

Location: Hall 3A

Background

Graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) is a cause of profound clinical gastro-intestinal disease, which is associated with severe intestinal crypt damage. Although loss of the Lgr5+ intestinal stem cell (ISC) compartment is an established component of GVHD, the mechanisms for this immune mediated damage are unknown.

Aims
To examine the specific cellular interactions and molecular mechanisms underlying ISC loss in GVHD intestinal damage.

Methods

We used clinically relevant major (B6 into BDF1) and minor (LP into B6) mismatched experimental allo-BMT models to evaluate the ISC compartment after immune-mediated damage. Crypts were harvested early post-BMT to assess intestinal organoid forming potential as read-out for ISC function ex vivo. To investigate possible damage mechanisms we established both mouse and human co-culture systems of activated T-cells with intestinal organoids and used both neutralizing antibodies and knock-out T-cells. Several mouse models (Lgr5-lacZ, lgr5-gfp-ires-CreERT2, Ifngr-/-, Infg-/-) were used in in vivo and ex vivo experiments to validate results.

Results

We found that Lgr5+ ISCs are rapidly depleted after pre-transplant conditioning, but recover in recipients of minor mismatched T-cell-depleted (TCD) BMT by day 10 post-BMT. In GVHD mice ISCs failed to recover. In line with these results, crypts from TCD recipients were able to form organoids ex vivo by day 10 post-BMT, while crypts from GVHD mice remained impaired, indicating immune-mediated injury to the ISC compartment (Fig. 1A). Activated allo-T-cells significantly reduced ISC function ex vivo in both human and mouse co-culture systems as evidenced by reduced organoid growth (Fig. 1B). Screening numerous effector mechanisms, we found that IFNγ blockade significantly protected organoid formation (Fig. 1C), confirmed by IFNγ resistance of IFNγ receptor knockout (IFNγR-/-) organoids. Furthermore, culture of mouse or human organoids with IFNγ in the absence of T-cells indicated that IFNγ was sufficient for organoid suppression. We tested if ISC damage is indirectly caused by damage to its supporting Paneth cell (PC) niche. However, using organoid cultures from PC deficient mice and from PC and/or ISC-specific IFNγR-/- KO mice, we determined it is the ISCs themselves that are sensitive to IFNγ toxicity. Ruxolitinib, a selective JAK1/2 inhibitor in clinical investigation, protected intestinal organoids from allo-T-cells as well as from direct IFNγ toxicity (Fig. 1D). We validated our findings in vivo and found that both anti-IFNγ and Ruxolitinib treatment protected ISC from elimination on day 10 post-BMT (Fig. 1E). When using IFNγ-/- T-cells in allo-BMT to eliminate T-cell-derived IFNγ effects, we found significantly less damage to the ISC compartment than in the GvHD control with WT T-cells (Fig. 1F). Gene expression analysis revealed the initiation of an apoptotic program in ISCs upon IFNγ exposure, with decreased anti-apoptotic Bcl2 and Bcl2l1 and increased apoptotic Bak1 expression. In addition, we found increased apoptosis by Annexin V analysis and activity and presence of cleaved caspase 3 after IFNγ stimulation.

Conclusion

Our findings reveal that damage to the ISC compartment is a common feature of GvHD, and T-cell derived IFNγ plays a central role in ISC damage and elimination, instituting a gene expression program resulting in ISC apoptosis. Jak/STAT signaling inhibition can protect target tissue stem cells and as such may provide a promising approach for protecting epithelial tissues from T-cell mediated damage in GVHD.

Session topic: 21. Stem cell transplantation - Experimental

Keyword(s): Graft-versus-host disease (GVHD), Stem cell, T cell

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