Characterizing Graft-Vs-Host Disease of the Bone Marrow Post Allogeneic Hematopoietic Cell Transplantation in Mice

Graft-vs-Host Disease (GVHD) is a common complication post allogeneic hematopoietic cell transplantation (allo-HCT). In contrast to classical target organs alloreactivity directed against hematopoietic and non-hematopoietic bone marrow (BM) structures has been poorly characterized, even though hematopoietic dysfunction following allo-HCT is frequently observed.

Here, we studied in MHC-matched, minor antigen mismatched HCT models (C3H-SW > C57BL/6 (B6)) the effects of lethal total body irradiation and HCT of HSC (cKIT⁺Sca1⁺Lin^-) alone or in combination with Tc / Tc subsets (CD4+, CD8+ or total (CD4+CD8) T cells (Tc)) on the hematopoietic compartment (HC) and non-hematopoietic compartment (NHC) of the BM. Up to 8 weeks (w) post-HCT bones and marrow were analyzed by flow cytometry and 3D-confocal microscopy imaging.

Post allo-HCT there was a transient weight loss and decrease of total BM cell counts in all experimental groups, but no signs of overt GVHD. At 2w mice given HSC had significantly higher absolute BM counts compared with HSC+Tc/Tc subset recipients. Most pronouncedly affected were B-cells that recovered promptly in HSC recipients but were severely delayed beyond w8 in the HSC+Tc groups (particularly in CD4 recipients). Likewise, BM granulocyte recovery during the first 4w was significantly better in HSC vs. HSC+Tc recipients. Moreover, alloreactive (but not congenic) Tc severely disrupted the NHC: at 2w post allo-HCT HSC recipients showed prompt recovery of BM endothelial cells (EC; CD45^-Ter119^-CD31⁺), and CXCL12-abundant reticular cells (CAR; CD45^-Ter119^-CD31^-CD140B⁺). In contrast, HSC+Tc recipients had significantly lower EC and CAR cells counts and their recovery was significantly delayed. 3D-confocal microscopy confirmed these observations and revealed rapid recovery of extracellular matrix and sinusoidal vascular structures, with simultaneous disappearance of adipocytes at 2w post-HCT in the HSC group, whereas HSC+Tc recipients displayed severe disruption of the structural integrity with impaired recovery of the BM microvessels but rather occupation of space by adipocytes. To visualize CAR cells by 3D imaging we used B6.CXCL12-GFP recipients and found, again, impaired CAR cell regeneration in HSC+Tc, but not pure HSC recipients. We next addressed the question whether alloreactive Tc exert direct GVH-related damage to NHC structures, or alternatively, only the recipient's HC is attacked whereas suppression of EC and CAR cells results from the inflammatory milieu. By transplanting C3H.SW BM into lethally irradiated B6 recipients chimeras with a C3H.SW HC and B6 NHC were generated. In a 2^nd HCT HSC +/- Tc were transplanted from (i) C3H.SW (allo to NHC; syngeneic to HC) or (ii) B6 (congenic to NHC, allo to HC) donors into these chimeras. (i) At 2w post-2^nd HCT BM cellularity, EC and CAR cell numbers were decreased when donor Tc were allogeneic to the NHC, but syngeneic to the HC. NHC populations were normal in number when pure allogeneic HSC (without Tc) were given. (ii) When grafts for 2^nd HCT were from B6 donors (allo to HC, congenic to NHC), donor Tc had no effect on EC numbers. CAR cells were reduced in numbers, but less severely compared with after transfer of allogeneic HSC+Tc. Together, these findings suggest, that EC can be directly targeted by alloreactive Tc; CAR cells are affected by both, alloreactive Tc, and inflammatory conditions generated by attack of the HC. Finally, to test the influence of GVH-related cytokines on the NHC we used B6.TNFaR-/- and B6.IFNyR-/- mice as recipients of C3H.SW HSC +/- Tc. Recovery of HC and NHC was identical in WT and TNFaR-/- recipients of C3H.SW grafts. In contrast, IFNyR-/- recipients of allogeneic HSC+CD4 grafts had significantly higher CAR cell counts compared with WT.B6 recipients (but lower than recipients of pure HSC). These findings support the hypothesis that CAR cells are also susceptible to inflammatory damage.

In clinical HCT delayed hematopoietic reconstitution presents a major problem contributing to increased morbidity and mortality. GVH reactions of the BM have been poorly characterized. Our data show that not only host hematopoiesis, but also non-hematopoietic stromal and vascular compartments of the BM are affected by both alloreactivity of donor Tc and their inflammatory microenvironment, even in the absence of clinical GVHD.