Abstract

Background

   Graft-versus host disease (GVHD) is a complication of stem cell
   transplantation associated with significant morbidity and mortality.
   Non-specific immune-suppression, the mainstay of treatment, may result
   in immune-surveillance dysfunction and disease recurrence.

Methods

   We created humanised mice model for chronic GVHD (cGVHD) by injecting
   cord blood (CB)-derived human CD34^+CD38^−CD45RA^− haematopoietic
   stem/progenitor cells (HSPCs) into hIL-6 transgenic NOD/SCID/Il2rgKO
   (NSG) newborns, and compared GVHD progression with NSG newborns
   receiving CB CD34^− cells mimicking acute GVHD. We characterised human
   immune cell subsets, target organ infiltration, T-cell repertoire (TCR)
   and transcriptome in the humanised mice.

Findings

   In cGVHD humanised mice, we found activation of T cells in the spleen,
   lung, liver, and skin, activation of macrophages in lung and liver, and
   loss of appendages in skin, obstruction of bronchioles in lung and
   portal fibrosis in liver recapitulating cGVHD. Acute GVHD humanised
   mice showed activation of T cells with skewed TCR repertoire without
   significant macrophage activation.

Interpretation

   Using humanised mouse models, we demonstrated distinct immune
   mechanisms contributing acute and chronic GVHD. In cGVHD model,
   co-activation of human HSPC-derived macrophages and T cells educated in
   the recipient thymus contributed to delayed onset, multi-organ disease.
   In acute GVHD model, mature human T cells contained in the graft
   resulted in rapid disease progression. These humanised mouse models may
   facilitate future development of new molecular medicine targeting GVHD.

   Keywords: Acute GVHD, Chronic GVHD, IL-6, Humanised mouse

Research in context

Evidence before this study

   GVHD is an important complication of haematopoietic stem cell
   transplantation. Mouse models and clinical experience suggested
   important roles of activated T cells both in acute and chronic GVHD.
   GWAS studies implicated IL-6 in pathogenesis of chronic GVHD and
   anti-IL6R Ab has been reported to ameliorate disease status in patients
   with chronic GVHD.

Added value of this study

   We created a chronic GVHD humanised mouse model by transplanting human
   HSPCs into hIL-6 TG NSG newborns. In this model, activation of both
   human T cells and macrophages was associated with tissue damage in
   skin, lung, and liver. We also created acute GVHD humanised mice by
   transplanting mature human leukocyte and myeloid cells to determine
   cellular and molecular distinction between acute and chronic GVHD.
   Through the comparison, we found that co-activation of
   fibrotic/sclerotic changes associated with T lymphocytes and
   macrophages in affected organs and higher expression of genes
   associated with TGF-β and SMAD signaling are characteristic of chronic
   GVHD.

Implications of all the available evidence

   Our study revealed an important role of IL-6 and subsequent
   macrophage/T cell co-activation in the development of chronic GVHD. The
   preclinical humanised mouse model may serve as a beneficial tool for
   the investigation of chronic GVHD biology and new treatment strategy.

1. Introduction

   Graft-versus host disease (GVHD) is characterised by an attack of host
   organs by donor-derived lymphocytes, and remains as one of the serious
   complications following haematopoietic stem/progenitor cell
   transplantation (HSCT). GVHD is classified into acute (aGVHD) and
   chronic (cGVHD) forms according to the time of disease onset. Recently,
   distinct mechanisms of pathogenesis between these two forms of GVHD
   have been reported [[65][1], [66][2], [67][3]]. Nevertheless, the
   pathophysiology of cGVHD at a molecular level is not well-understood,
   and studies that directly compared cGVHD with aGVHD using the same
   donor cell source are few.

   Allogeneic mouse models have been developed to investigate acute and
   chronic GVHD [[68][4], [69][5], [70][6], [71][7], [72][8], [73][9],
   [74][10]]. These models supported clarification of the critical roles
   of T cells as well as donor- and host-derived antigen presenting cells
   in GVHD. Furthermore, Lockridge et al. described cGVHD in a bone
   marrow-liver-thymus (BLT) model of humanised NOD/SCID/Il2rgKO (NSG)
   mice engrafted with human fetal thymic and liver tissue under the renal
   capsule and injected with HSPCs [[75]11]. Sonntag et al. reported that
   two out of 15 NSG recipients of human CD34^+ haematopoietic
   stem/progenitor cells (HSPCs) showed cGVHD-like changes at 6–7 months
   post-transplantation [[76]12]. While these xenotransplantation models
   are important milestones in the development of humanised mouse models
   of in vivo cGVHD, they lacked appropriate humanised microenvironment in
   the recipient mice. In particular, we focused on human IL-6 signaling,
   since IL-6 gene polymorphism is associated with the development of
   cGVHD and anti-human IL-6 receptor monoclonal antibody (Tocilizumab)
   has shown some benefit in steroid-refractory cGVHD patients
   [[77]13,[78]14].

   Here we present a humanised mouse model for cGVHD through
   transplantation of human cord blood (CB)-derived HSPCs without
   implantation of human fetal tissues in human IL-6 expressing NSG mice
   (hIL-6 Tg NSG). During long-term observation, these cGVHD humanised
   mice showed cGVHD-like changes in multiple target organs including
   skin, lung and liver. In addition, we created a humanised mouse model
   for aGVHD by transplantation of human CB-derived CD34^− mature blood
   and immune cells in NSG mice. Comparison of these two humanised mouse
   models demonstrated the unique role of macrophages in cGVHD
   pathogenesis. Moreover, we identified differentially expressed genes in
   human T cells associated with cGVHD and aGVHD humanised mice. These
   findings suggest that treatment strategies directed against macrophages
   as well as specific target molecules associated with pathogenic T cells
   should be considered as a future therapy for steroid-refractory GVHD.

2. Materials and methods

2.1. Human samples

   CB samples were obtained from the Tokai and the Chubu Cord Blood Bank
   under written informed consent. Peripheral blood (PB) samples were
   obtained from patients with cGVHD and non-GVHD HSCT recipient at
   Toranomon Hospital under written informed consent. All experiments were
   performed with authorisation from the Institutional Review Board for
   Human Research at RIKEN and were conducted according to the principles
   expressed in the Declaration of Helsinki.

2.2. Mice

   Human IL-6 Transgenic NSG mice (hIL-6 Tg NSG) were generated by
   pronuclear microinjection of BAC clone CTD-2594 N23 (GRCh37/hg19
   chromosome7 22,724,723–22,964,038; BAC1), or RP11-692 K8 (GRCh37/hg19
   chromosome7 22,320,340–22,505,348; BAC2) followed by backcrossing of
   the transgene >5 generations using a marker-assisted selection protocol
   from the original C57BL/6 strain onto NOD.Cg-Prkdc^scidIL2rg^tm1Wjl
   (NSG) mice [[79]15]. The copy numbers of the BAC transgene were
   estimated by quantitative PCR of chloramphenicol-resistance gene in a
   BAC vector using a mouse endogenous gene (RAVER2) as an internal
   copy-number reference. All mice were bred and maintained under SPF
   conditions at the animal facility of RIKEN IMS according to guidelines
   established and approved by the Institutional Animal Committees at
   RIKEN.

2.3. Purification and transplantation of human cord blood cells

   Human CD34^+ cells enriched using immune-magnetic beads (130–046-703,
   Miltenyi Biotec) were used to isolate 7AAD^−Lin^−CD34^+CD38^− or
   7AAD^−Lin^−CD34^+CD38^−CD45RA^− human HSPCs using FACSAria or FACSAria
   III (BD Biosciences). CB CD34^− cells prepared by immune-magnetic
   microbeads (130–050-1014, Miltenyi Biotec) were used to isolate CB
   CD34^−CD3^+ cells using FACSAria. Non-Tg NSG and hIL-6 Tg NSG newborns
   were sublethally irradiated at 150 cGy total body irradiation using a
   ^137Cs-source irradiator followed by intravenous injection via the
   facial vein. To create cGVHD humanised mice, 1.7 × 10^3 to 4.5 × 10^4
   HSPCs were transplanted. To create aGVHD humanised mice, 10^5 to 10^6
   CD34^− or CD34^−CD3^+ cells were transplanted. To create non-Tg NSG
   humanised mice, 7.2 × 10^3 to 4.2 × 10^5 HSPCs were transplanted. When
   recipients became moribund (reduced activity or tachypnea), these mice
   were euthanised and analysed. Non-Tg NSG humanised mice were euthanised
   concomitantly as controls. Antibodies used for flow cytometric analysis
   and cell sorting are listed in Supplementary materials and methods.

2.4. RNA-seq analysis

   Total RNA was extracted from human T cells and mouse keratinocytes
   obtained from hIL-6 Tg NSG humanised mice (n = 5), acute GVHD humanised
   mice (n = 2), non-Tg NSG humanised mice (n = 2), and non-transplanted
   non-Tg NSG control mice (n = 3) using Trizol (ThermoFisher Scientific).
   RNA sequencing data are deposited in the National Bioscience Database
   Center. RNA-seq libraries were prepared using an NEBNext Ultra RNA
   Library Prep Kit for Illumina (New England Biolabs) according to the
   manufacturer's protocol and were sequenced using a HiSeq1500 DNA
   sequencer (Illumina single read, 50 bp). The sequence reads were mapped
   to mouse genome (NCBI version 37) or human genome (NCBI version 19)
   using TopHat2 (version 2.0.8) and bowtie2 (version 2.1.0) with default
   parameters and gene annotation was provided by the NCBI RefSeq. The
   transcript abundances were estimated using Cufflinks (version 2.1.1).
   Cufflinks was run with the same reference annotation with TopHat2 to
   generate FPKM (fragments per kilobase per million mapped reads) values
   for known gene models.

2.5. Transcription factor (TF) enrichment analysis

   Enrichment analysis to evaluate effect of a TF on their binding target
   genes was performed as described previously [[80]16]. Briefly, we
   compared the mean fold changes of gene expression of target genes of a
   TF against that of the background (whole genes). To consider
   heterogeneous frequencies of TF binding among target genes, we used a
   weighted t-test procedure into a parametric gene set enrichment
   analysis. The frequency of binding of a TF to their target genes was
   evaluated based on the numerous high-throughput chromatin
   immunoprecipitation (ChIP) experiments obtained from the gene
   expression omnibus database (GEO, [81]www.ncbi.nlm.nih.gov/geo/). We
   used the weighted t-statistic as an enrichment score indicating the
   relative activity of a TF.

2.6. TCR sequencing

   Total RNA was extracted from human T cells in hIL-6 Tg NSG humanised
   mice and acute GVHD humanised mice using Trizol (ThermoFisher
   Scientific). TCR-seq libraries were prepared using a SMARTer Human TCR
   a/b Profiling Kit (Clontech Laboratories, Inc.) according to the
   manufacturer's protocols and were sequenced on a Miseq DNA sequencer
   (Illumina). The human TCR repertoires were analysed using MiXCR
   [[82]17].

2.7. Measurement of human cytokine /chemokine levels

   Plasma cytokine/chemokine levels in humanised mice and patient samples
   were measured using the Bio-Plex system (Bio-Rad) with Bio-Plex Human
   Cytokine 27-Plex and 21-Plex Assay kits (Bio-Rad).

2.8. Statistical analysis

   The numerical data are presented as means ± SEM. Statistical analyses
   (two-tailed t-tests and one-way ANOVA with Bonferroni post-test) were
   performed using Microsoft Excel. The differences were determined by
   two-tailed t-tests unless otherwise indicated. P value <.05 was
   considered statistically significant. Statistics for Kaplan-Meier
   analysis were obtained using EZR (Saitama Medical Center, Jichi Medical
   University, Saitama, Japan), which is a graphical user interface for R
   (The R Foundation for Statistical Computing, Vienna, Austria) [[83]18].

3. Results

3.1. hIL-6 Tg NSG humanised mice transplanted with human HSPCs develop
cGVHD-like changes

   We created human IL6 transgenic NSG mice (hIL-6 Tg NSG) by
   microinjecting a bacterial artificial chromosome (BAC) containing the
   human IL6 gene (clone: 2594 N23 or 692 K8) into C57BL/6 mice and
   backcrossing onto the NSG background. The BAC transgene was stably
   propagated in a Mendelian inheritance mode and their copy numbers in
   mouse clones BAC3 and BAC32 were estimated to be 2.0 copies and 2.9
   copies per haploid genome on average of triplicated measurements,
   respectively. Plasma hIL-6 levels in hIL-6 Tg NSG mice were elevated at
   baseline (IL-6, n = 2: IL-6#1 63.4 pg/ml, IL-6#2 60.7 pg/ml) while
   non-transgenic NSG mice showed background levels of human IL-6 in
   plasma (NSG, n = 2: NSG#1 0.5 pg/ml, NSG#2 0.6 pg/ml).

   To create cGVHD humanised mouse, we transplanted human CB-derived HPSCs
   into sublethally-irradiated hIL-6 Tg NSG newborns. At 20 to 43 weeks
   post-transplantation, all hIL-6 Tg NSG humanised mice developed signs
   of weakness including ruffled fur, anemia and reduced mobility
   (n = 58). In 22 out of 58 these mice, we found macroscopic skin lesions
   consistent with skin cGVHD. We examined human immuno-haematopoietic
   reconstitution in 18 out of 22 and found long-term multi-lineage
   engraftment of human CD19^+ B cells, CD33^+ myeloid cells, CD3^+ T
   cells, and CD56^+ natural killer cells in the bone marrow (BM) and
   spleen of hIL-6 Tg NSG mice (Representative flow cytometry plots shown
   in [84]Fig. 1a, [85]Table S1). Engrafted human T cells were
   predominantly TCRαβ^+ T cells. Interestingly, the frequency of Foxp3^+
   Tregs was lower in hIL-6 Tg NSG humanised mice (hIL-6 Tg, n = 14:
   5.7 ± 0.9%, non-Tg NSG, n = 11: 10.5 ± 0.6%, p = .0004; [86]Fig. 1b).
   Analysis of Foxp3+ Treg subsets CD45RA^+Foxp3^lo naïve Tregs (nTregs),
   CD45RA^−Foxp3^hi effector Tregs (eTregs) and CD45RA^−Foxp3^lo non-Tregs
   (non-Tregs) (Representative flow cytometry plots in [87]Fig. 1b)
   [[88]19] showed reduction of both naïve (p = 0.02) and effector
   (p = 0.03) fractions in the spleen of hIL-6 Tg NSG humanised mice
   (non-Tg NSG, n = 11: nTregs 0.5 ± 0.1%, eTregs 3.9 ± 0.6%; IL-6, n = 7,
   nTregs 0.1 ± 0.04%, eTregs 1.8 ± 0.6%; [89]Fig. 1c).

Fig. 1.

   [90]Fig. 1
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   Reconstitution of human immunity in hIL-6 Tg NSG mice.

   (a) Representative flow cytometry plots of a hIL-6 Tg NSG humanised
   mouse (IL6#1–1) demonstrating the engraftment of human CD19^+ B cells,
   CD33^+ myeloid cells, CD3^+ T cells, and CD56^+ NK cells in BM and
   spleen. (b) Representative flow cytometry plots showing splenic Treg
   subsets in a hIL-6 Tg NSG humanised mouse (IL6#1–1). Foxp3^+ Tregs are
   classified into CD45RA^+Foxp3^lo naïve Tregs (I), CD45RA^−Foxp3^hi
   effector Tregs (II), and CD45RA^−Foxp3^lo non-Tregs (III). (c)
   Frequency of Foxp3^+ Tregs in the spleen are lower in hIL-6 Tg NSG
   humanised mice (NSG: n = 11, IL-6: n = 14), with significant reduction
   in naïve Tregs and effector Tregs (NSG: n = 11, IL-6: n = 7)
   (*p = 0.0004, **p = 0.02, ***p = 0.03). Error bars represent
   mean ± SEM. (d) T cell-dominant engraftment was found in the skin of
   hIL-6 Tg NSG humanised mouse as shown by representative flow cytometry
   plot (IL6#1-1).

   We performed histological examination of the skin, lung and liver in
   hIL-6 NSG humanised mice with and without macroscopic skin lesions. We
   found infiltration of human T cells in the macroscopically-apparent
   skin lesions in all 14 mice examined (Representative flow cytometry
   plots shown in [92]Fig. 1d). We performed histological examination of
   the skin in 27 mice (with macroscopic skin lesions: n = 14; no
   macroscopic skin lesions: n = 13). In 21 of 27, we found epidermal
   thickening, interface dermatitis (vacuolar changes beneath basal
   keratinocytes), reduced numbers of adipocytes, reduced numbers of hair
   follicles and infiltration of human CD4^+ and CD8^+ T cells in
   epidermis and dermis ([93]Fig. 2a). These abnormalities were present
   even in 7 of 13 recipients with no macroscopic skin lesions. In three
   out of 14, sclerotic changes were present in the dermis. Altered
   expression of cytokeratins may trigger or exacerbate inflammatory skin
   diseases [[94]20]. We found ectopic expression of cytokeratin 17 and
   reduced expression of cytokeratin 13 in skin sections of hIL-6 Tg NSG
   humanised mice, suggesting possible role of these cytoskeletal
   filaments in skin pathology [[95]21,[96]22] (Fig. S1). In 19 of 27
   examined, hIL-6 Tg NSG humanised mice lungs showed infiltration of
   human T cells in peri-vascular areas, obstruction of bronchioles, and
   peri-bronchiolar areas with fibrotic changes ([97]Fig. 2b). In 13 of 21
   mice examined, we found reduction of cytokeratin 19-positive small bile
   ducts in the liver and obstruction of portal triads presumably as a
   consequence of chronic inflammation ([98]Fig. 2c). These
   histopathological findings are consistent with cGVHD in skin, lung and
   liver of mice with macroscopically normal skin as well as those with
   macroscopically appreciable skin lesions. Notably, cGVHD-like changes
   were found in the majority of mice examined whether or not macroscopic
   skin lesions were present. These changes were not found in non-Tg NSG
   humanised mice and untransplanted NSG and hIL-6 Tg mice ([99]Fig. 2 and
   Fig. S2) [[100]23].

Fig. 2.

   [101]Fig. 2
   [102]Open in a new tab

   Histological analysis of cGVHD humanised mice.

   Histological analysis of (a) skin, (b) lung and (c) liver from hIL-6 Tg
   NSG humanised mice with a hIL-6 non-Tg NSG humanised mouse as control.
   (a) H&E and immunohistochemical staining is consistent with interface
   dermatitis with epidermal thickening associated with infiltration of
   CD4^+ and CD8^+ T cells in cGVHD humanised mice. White arrow shows
   sclerotic and thickened upper dermis (IL6#2-1). (b) Pulmonary sections
   of the same recipients show infiltration of CD4^+ T and CD8^+ T cells.
   (c) Infiltration of human CD45^+ leucocytes including CD3^+ T cells is
   detected in the liver of cGVHD humanised mice. In contrast, a non-Tg
   NSG humanised mouse (NSG#12-1) shows normal skin, lung and liver
   histology. Scale bars: Low magnification 100 μm; high magnification
   50 μm.

3.2. Both cGVHD and aGVHD humanised mice show T cell activation

   Although damage to host organs mediated by activated T cells is found
   in both aGVHD and cGVHD, distinct mechanisms underlying the activation
   of donor T cells in these two clinical entities are not fully
   understood. To evaluate the differences between aGVHD and cGVHD, we
   created an aGVHD humanised mouse model by transplanting human
   CB-derived CD34-negative mature haematopoietic cells (n = 16) or
   CD34^−CD3^+ T cells (n = 10) into non-Tg NSG newborns ([103]Table S2).
   CB-derived CD34-negative cells contain T cells, B cells, myeloid cells
   and NK cells (CD34- cells, n = 10: CD3^+ cells 26.8 ± 3.6%; CD19^+
   cells 22.3 ± 5.4%; CD33^+ cells 33.4 ± 5.9%; CD56^+ cells 14.3 ± 3.4%,
   Fig. S3). Unlike hIL-6 Tg NSG cGVHD humanised mice, onset of disease
   was rapid in aGVHD humanised mice with 23 of 26 recipients becoming
   moribund within 73 days of transplantation. Early onset of GVHD in
   these recipients led to reduced survival compared with cGVHD humanised
   mice ([104]Fig. 3a). Vast majority of human CD45^+ leukocytes in the
   PB, spleen, liver, lung, gastrointestinal tract and skin of the aGVHD
   humanised mice were CD3^+ T cells ([105]Table S2 and Fig. S4a). Nine of
   26 recipients developed macroscopically-apparent skin lesions.
   Histological analysis of the skin showed epidermal thickening with
   human T cell infiltration and ectopic cytokeratin 17 expression in the
   epidermis [[106]21,[107]22] ([108]Fig. 3b, Fig. S1). Infiltration of
   human T cells was also observed in the liver and small intestine, major
   target organs of clinical aGVHD (Fig. S4a, [109]Fig. 3b).
   Histologically, sclerotic changes and reduction of bile ducts were
   absent in aGVHD humanised mice, in contrast to hIL-6 Tg NSG cGVHD
   humanised mice ([110]Fig. 3b and Fig. S4b).

Fig. 3.

   [111]Fig. 3
   [112]Open in a new tab

   Comparison between hIL-6 Tg NSG cGVHD humanised mice and human aGVHD
   humanised mice.

   (a) Kaplan-Meier plot showing reduced survival of both cGVHD humanised
   mice (n = 58, black) and aGVHD humanised mice (n = 26, red) compared
   with non-Tg NSG humanised mice (n = 15, blue) (cGVHD vs. non-Tg,
   p = 0.000225; aGVHD vs. non-Tg, p = 1.68 × 10^−7; cGVHD vs. aGVHD,
   p = 2.11 × 10^−22 by log-rank test). (b) Histological analyses of skin,
   liver and small intestine of an aGVHD humanised mouse (acute#A-1).
   Bars: (far left) 100 μm; (the others) 50 μm. (c) Representative flow
   cytometry plots of a cGVHD humanised mouse (IL6#2-1). (d) Frequency of
   IFN-γ and IL-17A producing splenic T cells in non-Tg NSG humanised mice
   (NSG-IFN-γ+, n = 11, NSG-IL-17A+, n = 9), aGVHD humanised mice (aGVHD:
   n = 12), and cGVHD humanised mice (IL6-IFN-γ+, n = 11, IL6-IL-17A+,
   n = 5). *P = 0.005, **P = 0.004, ***P < 0.0001 by 1-way ANOVA with
   Bonferroni post-test. Error bars represent mean ± SEM. (e) Plasma
   cytokine/chemokine concentration of cGVHD humanised mice (Black: IL-6,
   n = 8), aGVHD humanised mice (Red: aGVHD, n = 6) and non-Tg NSG
   humanised mice (Blue: NSG, n = 5). (For interpretation of the
   references to colour in this figure legend, the reader is referred to