Donor macrophages promote human acute GvHD in allogeneic BMT

Acute graft-versus-host disease (GvHD) affects up to 50% of patients receiving allogeneic bone marrow transplant (BMT) and is the leading cause of morbidity and mortality from BMT. While animal models suggest that donor myeloid cells play a role in the enhancement of GvHD, technical limitations of in situ microscopy prevent the precise identification of which cells infiltrate GvHD lesions. Splenic T cells are used to induce GvHD in animal models, but human BMT recipients are depleted of T cells prior to transplant to decrease graft rejection, so these may not be the sole mediators of GvHD pathology.

A study published by Laura Jardine and colleagues in the Journal of Clinical Investigation¹ examined acute GvHD skin lesions, to identify which cells were present in skin explants and whether these cells were donor or recipient in origin.

Study design and patient characteristics

• This study was conducted at Northern Centre for Bone Marrow Transplantation at Newcastle upon Tyne Hospitals NHS Foundation Trust
• Recruitment occurred for a 3-year period between 2013 and 2016
• Patients in the BMT with acute GvHD arm included those with immunosuppression-withdrawal acute GvHD, acute GvHD following donor lymphocyte infusion, or classical acute GvHD, and had a skin biopsy at the onset of rash at a median of 53 days (range, 13–304)
• Patients with clinical or histological features of chronic GvHD were excluded
• The BMT control group included BMT recipients without GvHD skin lesions, and these patients were assessed at median Day 83 (range, 28–100 days) post transplant
• The healthy control group included patients undergoing mammoplasty or abdominoplasty
• 5–15 mm² skin shaves were biopsied from 13–304 days post BMT and analyzed within 24 hours
• Histological grading of skin shaves was conducted by independent pathologists
• Peripheral blood mononuclear cells (PBMCs) were collected and used to generate macrophages

Results

• Skin biopsies were stained with antibodies (Table 1), showing that:
  • A greater number of CD3⁺ T cells and CD11c⁺ myeloid cells were detected in GvHD skin biopsies compared to controls
  • CD11c⁺ myeloid cells were distinct from FX111A⁺ tissue resident macrophages, indicating that they were donor myeloid cells

Table 1. Cell surface markers used to determine cell identities¹

Marker	Cell type
BMT, bone marrow transplant; CD, cluster of differentiation; FXIII, factor XIII
CD11c	Myeloid cells
CD3	T cells
CD163	Macrophages (not monocytes)
FXIII	Tissue resident macrophages (BMT recipient macrophages)

• Single cell suspensions were prepared from skin biopsies:
  • No overall increase in the proportion of myeloid cells was detected in biopsies of GvHD patients compared to control biopsies
  • However, the CD11c⁺CD14⁺ subset of myeloid cells was ten-fold greater in GvHD biopsies than control biopsies
• Morphology analysis was performed on CD11c⁺CD14⁺ cells:
  • Cells were small macrophages with dense nuclei, cytoplasmic vacuoles, and granules 
  • These cells were distinct from CD11c^–CD14⁺ tissue resident macrophages, which are larger with dense melanin, and were relatively depleted in GvHD biopsies
• Genomic profiling of CD11c⁺CD14⁺ cells from GvHD biopsies was performed:
  • Nanostring analysis of RNA and DNA examining 609 immunology-related genes found that these cells were most similar to steady-state monocyte–macrophages and resident dermal macrophages, and least similar to dendritic cell lineages
  • Genotype analysis using XY fluorescent in situ hybridization (FISH) in sex-mismatched transplants determined that cells were 98–100% donor origin
• Mixed leukocyte reactions (MLRs) were used to study the ability of GvHD macrophages to stimulate T cells from healthy donors:
  • While steady state CD14⁺ macrophages do not potently stimulate T cells, macrophages from GvHD biopsies induced T cell proliferation and expression of activation markers on T cells
  • Gene expression profiling identified genes associated with T cell activation, and induction of pro-inflammatory cytokines was upregulated in GvHD macrophages (Table 2)

Table 2. Genes upregulated in GvHD macrophages¹

Class of Genes	Genes
CCL, C-C chemokine ligand; CD, cluster of differentiation; GvHD, graft-versus-host disease; HLA, human leukocyte antigen; IL, interleukin; RANTES, regulated upon activation, normal T cell expressed, and presumably secreted; SELPLG, selectin P ligand; SPP, secreted phosphoprotein; TAP, transporter associated with antigen processing; TNF, tumor necrosis factor
Antigen presentation	HLA TAP1
Cell recruitment	CCL24
Lymphocyte stimulation	CD82
Pro-inflammatory cytokine stimulation	SPP1
Leukocyte extravasation	SELPLG
Cytokines and chemokines	CCL5/RANTES, CXCL10, IL-8, TNFβ, IL-10

• Gene expression of PBMCs, which are found circulating and are not localized at the site of GvHD, were analyzed in GvHD patients and compared to controls (Table 3):
  • Classical monocytes were enriched in GvHD patients
  • Genes associated with dendritic cell differentiation were downregulated

Table 3. Gene expression in GvHD PBMCs¹

	Genes
CCR, C-C chemokine receptor; CIITA, Class II major histocompatibility complex transactivator; FCER, Fc epsilon receptor; FCGR, Fc gamma receptor; GBP, guanylate-binding proteins; GNLY, granulysin; GvHD, graft-versus-host disease; IFITM, interferon-induced transmembrane; IRF, interferon regulatory factor; MRC, macrophage mannose receptor; PBMC, peripheral blood mononuclear cell; ZBTB, zinc finger and BTB domain
Upregulated	CCR5, MRC1, FCGR3A/B, GNLY, IFITM1, GBP1
Downregulated	FCER1A, IRF4, ZBTB46, CIITA

• Macrophages derived from the MLRs were compared to matched unstimulated monocytes (Table 4):
  • 118 differentially expressed transcripts were identified
  • Genes identified to be upregulated in GvHD macrophages were also upregulated in MLR macrophages
  • MLR macrophages also expressed many cytotoxic molecules

Table 4. Genes expressed in GvHD macrophages and MLR macrophages¹

	Genes
CCR, C-C chemokine receptor; GNLY, granulysin; GvHD, graft-versus-host disease; MLR, mixed leukocyte reaction; MRC, macrophage mannose receptor; PPBP, pro-platelet basic protein; TRAIL, TNF-related apoptosis-inducing ligand
Upregulated in GvHD and MLR macrophages	CCR5 MRC1 PPBP
Upregulated in MLR macrophages	Perforin Granzyme A GNLY TRAIL

• The ability of MLR macrophages to kill epidermal cells was tested:
  • MLR macrophages killed keratinocytes in cell culture
  • MLR macrophages also induced GvHD pathology in a more physiologically relevant skin explant model
  • T cells and macrophages equally mediated tissue damage in the skin explant model

Conclusion

The authors found that skin biopsies from GvHD patients contain donor derived CD11c⁺CD14⁺ macrophages that potently activate T cells, and highlight that this interaction between donor macrophages and T cells has the potential to mediate pathology in the GvHD skin lesion. The authors conclude that understanding the function of these macrophages may enhance GvHD treatment and prevention options.