Cytotoxic Therapy Impairs the Stromal Niche Supporting ILC Development in the Bone Marrow

Background. Innate lymphoid cells (ILC) include ILC1, ILC2 and ILC3. In adults, common lymphoid progenitors (CLP) in the bone marrow serve as common precursor for B- and T-lymphoid lineage cells, as well as all major subsets of ILCs. Upon upregulation of ID2, TCF-1, NFIL1, and TOX (Reviewed by Cherrier et al., 2018), CLP differentiate into EILPs and ChILP cells. More restricted ChILP cells are precursors for type-specific progenitors that express signature transcription factors. This group includes ILC2Ps, which express GATA3 and give rise to ILC2 cells in adult bone marrow.

Although recent works shed light on a regulation of the ILC2 progenitor commitment and specification on a transcriptional level, the cellular composition of bone marrow niches that provide environmental cues (cytokines, chemokines and contact interactions) that play instructive and survival roles is yet to be fully characterized.

In addition to hematopoietic progenitors, stromal compartment, particularly LeptinR+ cells, Nestin+ cells, and osteoblast population in the bone marrow were impaired after irradiation and chemotherapy (Severe et al., 2019, Tikhonova et al., 2019). Moreover, in the context of transplantation cytotoxic effect of infiltrating allogeneic T-cells can further exacerbate this damage. Here, we show a long-term loss of ILC2 precursors in the bone morrow after syngeneic transplantation. We also observed a decreased numbers of CXCL12-abundant reticular cells (CAR) and DLL1-expressing vascular endothelial cells that together provide IL-7 and Notch signaling critical for development of ILC2P in the bone marrow.

Interventions targeted towards protection of these stromal population during transplantation may help to restore development of ILC2.

Select Methods. For CLP co-culture, Lin-c-Kit^intSca-1^intCD127+Flt3+ bone marrow cells were sorted and plated with 3 sets of feeder cells WT OP9 cells, OP9-DLL1 and OP9-DLL4 cells. We plated 100 CLP cells/well and cultured them for 14 days changing the media every other day. Harvested cells were analyzed using flow cytometry.

Results. We observed significant decrease in frequency and absolute numbers of ChILP cells (Lin-Flt3- CD127+α4β7+CD244+CD25-) and ILC2P cells (Lin-Flt3- CD127+α4β7+CD244-CD25+) in the bone marrow of mice 40 days after syngeneic bone marrow transplantation compared to control group (ChILP: 9.77±0.53 x10³ vs. 4.17±0.27 x 10³, p<0.01; ILC2P: 4.91±0.42 x 10⁴ vs. 0.41±0.04 x 10⁴, p<0.01). Next, we screened for cytokines and chemokines critical for generation of ILC2 from CLP cells using OP9 co-culture system with WT feeder cells and cells overexpressing DLL1 or DLL4. IL-7 and Notch-signaling provided by DLL1 but not DLL4 OP9 cells was critical to support ILC2 generation and resulted in significantly higher counts of Lin-CD25+ST2+ cells (82±0.42 vs 5,226±1,008, p<0.01).

Recent single-cell profiling of the bone marrow demonstrated that vascular endothelial compartment expresses high level of DLL1 and DLL4 (Tikhonova et al., 2019), while CAR cells are one of the major producers of IL-7 in the bone marrow. Therefore, we sought to examine changes in the CAR cells and vascular endothelial cells in mice expressing dsRed-labeled CXCL12 and in Dll1-mCherry (or Dll4-mCherry) animals. Imaging analysis of femurs of mice at 45 days after syngeneic transplantation revealed a significant reduction of CXCL12-expressing cell population in the bone marrow compared to controls (Mean fluorescence intensity: 30.5±4.7 vs. 10.9±1.2, p<0.01). Flow cytometry analysis of these animals confirmed 2-2.5-fold reduction in the number of Lin-CD31-CXCL12+ cells in the bone marrow as early as 15 days. We have also observed a dramatic reduction of DLL1 and DLL4-expressing cells in CD45-Lin-CD31+ compartment following syngeneic bone marrow transplantation. The most prominent 10-fold decrease in absolute counts of was detected in Podoplanin+Sca-1int sinusoidal endothelial cells expressing DLL1.

Conclusions. These studies demonstrate that BM development of ILC2 is impaired in a context of syngeneic transplantation in mice. Our ex vivo and in vivo data suggest that CAR and vascular endothelial cells of the bone marrow producing critical factors such as IL-7 and DLL1, respectively, are significantly reduced 45 days after transplantation by cytotoxic therapy. Future efforts aimed at protecting these bone marrow niches may restore generation of ILC2s.

Bruce:Dr. Bruce: Patents & Royalties: provisional patent for the use of ILC2 cells to treat or prevent GvHD. Serody:STING activation: Patents & Royalties: provisional patent to enhance CAR therapy for solid tumors/ provisional patent for the use of ILC2 cells to treat or prevent GvHD.