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. 2019 Mar:41:584-596.
doi: 10.1016/j.ebiom.2019.02.001. Epub 2019 Feb 13.

Co-activation of macrophages and T cells contribute to chronic GVHD in human IL-6 transgenic humanised mouse model

Rintaro Ono  1 , Takashi Watanabe  2 , Eiryo Kawakami  3 , Makoto Iwasaki  1 , Mariko Tomizawa-Murasawa  1 , Masashi Matsuda  4 , Yuho Najima  1 , Shinsuke Takagi  5 , Saera Fujiki  1 , Rumi Sato  4 , Yoshiki Mochizuki  2 , Hisahiro Yoshida  4 , Kaoru Sato  6 , Hiromasa Yabe  6 , Shunichi Kato  6 , Yoriko Saito  1 , Shuichi Taniguchi  5 , Leonard D Shultz  7 , Osamu Ohara  8 , Masayuki Amagai  9 , Haruhiko Koseki  4 , Fumihiko Ishikawa  10
Affiliations

"V体育安卓版" Co-activation of macrophages and T cells contribute to chronic GVHD in human IL-6 transgenic humanised mouse model

Rintaro Ono (VSports手机版) et al. EBioMedicine. 2019 Mar.

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. VSports手机版.

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 V体育安卓版. 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 V体育ios版. 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 VSports最新版本. 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; Humanised mouse; IL-6. V体育平台登录.

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"V体育官网入口" Figures

Fig. 1
Fig. 1
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+Foxp3lo naïve Tregs (I), CD45RA−Foxp3hi effector Tregs (II), and CD45RA−Foxp3lo 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).
Fig. 2
Fig. 2
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.
Fig. 3
Fig. 3
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 the web version of this article.)
Fig. 4
Fig. 4
Infiltration of human immune cells and histopathology characteristic of acute and chronic GVHD humanised mice. (a-c) Histological sections of cGVHD humanised mice (IL-6: n = 3), an aGVHD humanised mouse (acute: n = 1), and non-Tg NSG humanised mouse (NSG: n = 1). (a) HE and hCD3 stained sections of skin dermo-epidermal junction (top two panels) and skin appendages (bottom two panels) are shown. (b) HE and CD68 stained sections of peri-bronchiolar (top two panels) and peri-vascular (bottom two panels) regions are shown. (c) hCD45 and CD68 stained sections of portal triad (top two panels) and peri-central vein (bottom two panels) regions are shown.
Fig. 5
Fig. 5
Gene expression signatures of T cells and keratinocytes in cGVHD and aGVHD humanised mice. (a) Venn diagrams indicating differentially expressed genes in splenic T cells among cGVHD and aGVHD humanised mice. (b) Differentially expressed genes among skin and spleen T cells in acute and chronic GVHD mice and non-Tg NSG humanised mice. (c) KEGG pathway enrichment analysis and (d) transcription factor (TF) enrichment analysis of skin T cells. (e) Venn diagrams indicating differentially expressed genes in skin keratinocytes from cGVHD and aGVHD humanised mice. (f) Dimensionality reduction plot using tSNE. (g) KEGG pathway enrichment analysis and (h) transcription factor (TF) enrichment analysis of skin keratinocytes.
Fig. 6
Fig. 6
TCR repertoires in acute and chronic GVHD humanised mice. (a,b) TRA and TRB repertoire of (a) skin T cells and spleen CD4+ T cells from cGVHD humanised mice (n = 3) and (b) spleen, lung and liver CD4+ and CD8+ T cells from aGVHD humanised mice (n = 3). When same clones accounted for >1%, they are shown using the same colour. (c) Venn diagrams show overlap of 25 most frequent clones among spleen, lung, and liver.
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