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Donor macrophages promote human acute GvHD in allogeneic BMT

Jul 30, 2020

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 situmicroscopy 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 Investigation 1examined 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, 13304)
  • 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, 28100 days) post transplant
  • The healthy control group included patients undergoing mammoplasty or abdominoplasty
  • 515 mm 2skin shaves were biopsied from 13304 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 identities 1

BMT, bone marrow transplant; CD, cluster of differentiation; FXIII, factor XIII

Marker

Cell type

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 monocytemacrophages and resident dermal macrophages, and least similar to dendritic cell lineages
    • Genotype analysis using XY fluorescent in situhybridization (FISH) in sex-mismatched transplants determined that cells were 98100% 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 macrophages 1

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

Class of Genes

Genes

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 PBMCs 1

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

 

Genes

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 macrophages 1

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

 

Genes

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.

  1. Jardine L, Cytlak U, Gunawan M, et al. Donor monocyte-derived macrophages promote human acute graft-versus-host disease.  J Clin Invest. 2020. Online ahead of print.  DOI: 10.1172/jci133909