Graft-Vs-Host Reactivity Against the Bone Marrow Is Directed Against the Hematopoietic and Non-Hematopoietic Compartments in Mice
Authors: Wong Hui Chyn , Isringhausen Stephan , Manz Markus G. , Nombela-Arrieta Cesar , Mueller Antonia MS 
Published: October 2019

Abstract
Hematopoietic dysfunction is frequently observed following allogeneic hematopoietic cell transplantation (allo-HCT) and contributes to substantial morbidity. Various factors, e.g. viral infections, poor graft-function, and certain drugs can cause cytopenias, but usually, graft-vs-host disease (GVHD) of the bone marrow (BM) is not taken into account as a major cause of hematopoietic dysfunction, particularly when no other signs of GVHD in classical target organs are present.
Here, we studied in an MHC-matched, minor antigen mismatched HCT model (C3H.SWàC57BL/6 (B6)) the effects of lethal irradiation and allo-HCT of HSC (cKIT+Sca1+Lin-) alone or in combination with T cells (TC) on the hematopoietic compartment (HC) and non-hematopoietic compartment (NHC) of the BM. FACS analysis was used to assess relative and absolute cell counts of hematopoietic and non-hematopoietic populations. Confocal microscopy was used to establish 3D images of structural integrity vs. disruption, and qRT-PCR was used to examine functional alterations of gene expression within NHC populations.
In this HCT model mice did not develop overt signs of acute GVHD. In recipients of HSC+TC recovery of hemoglobin (Hb) and neutrophils (Nph) was significantly delayed beyond d+56, while Hb and Nph were normalized by d+21 in recipients of pure HSC. Recovery of BM cell counts was also faster in HSC recipients, whereas addition of TC to the graft delayed reconstitution of total BM counts, BM B cells and BM neutrophils significantly. Not only the HC was affected by alloreactive TC, but also non-hematopoietic populations, including CXCL12-abundant reticular (CAR) cells (CD45-Ter119-CD31-CD140b+) and endothelial cells (EC; CD45-Ter119-CD31+) declined post-HCT, and showed a significantly slower recovery in mice given allogeneic TC within their HCT. 3D-confocal microscopy confirmed the alloreactive damage to the NHC, as imaging revealed rapid recovery of CAR cells in B6.CXCL12-GFP recipients of pure HSC, whereas regeneration of CAR cells was impaired in mice given HSC+TC. Likewise, the extracellular matrix and sinusoidal vascular structures recovered promptly in the HSC group, whereas HSC+TC recipients displayed severe disruption of the structural integrity with impaired recovery of the BM microvessels. Increased microvascular permeability was shown in all transplanted (and irradiated) mice on d+7 post-HCT by Evans blue dye extravasation assay. Recovery from leakiness was faster in mice transplanted with HSC alone compared with those given alloreactive TC. Of note, confocal microscopy revealed that in recipients of alloreactive TC hematopoietic cell content was replaced by an abundance of adipocytes for a prolonged period of time (Figure 1A). To further elucidate functional alterations of the NHC in the context of HCT with/without TC we FACS-isolated CAR cells and EC from HSC and HSC+TC recipients on d+14 and d+28 post-HCT and performed qRT-PCR of candidate genes in these populations. In HSC+TC recipients both CAR cells and EC showed significantly lower relative expression levels of SCF (stem cell factor, ligand to cKIT), CXCL12 (C-X-C-motif chemokine 12, = SDF-1, a cytokine with various functions in hematopoiesis and lymphopoiesis), and IL-7 (a growth factor that stimulates the differentiation of pluripotent HSC into lymphoid progenitors) compared with mice transplanted with HSC only. Rather, in CAR cells of HSC+TC recipients adiponectin and PPARγ (peroxisome proliferator-activated receptor γ, stimulates lipid uptake and adipogenesis) were significantly higher compared with HSC only recipients (Figure 1B). These findings suggest that alloreactive TC promote adipogenesis while at the same time suppressing hematopoiesis via deprivation of essential cytokines and growth factors required for HSC function and maturation.
Delayed hematopoietic regeneration post allo-HCT represents a major problem in clinical patient care. GVH reactions of the BM have been poorly characterized. Our experiments help to further characterize "marrow GVHD" and its impact on hematopoietic output. We illustrate the damage that alloreactive TC can have on both hematopoiesis directly but also on the nonhematopoietic compartment and its critical functions in stimulating and supporting hematopoietic regeneration.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.