Comprehensive Analysis of Chemokines in Host Organs and Their Corresponding Receptors on Donor T-Cells in Xenogeneic Gvhd Model
Authors: Kawasaki Yasufumi , Sato Kazuya , Nakano Hirofumi , Mashima Kiyomi , Minakata Daisuke , Yamasaki Ryoko , Morita Kaoru , Ashizawa Masahiro , Yamamoto Chihiro , Hatano Kaoru , Fujiwara Shin-ichiro , Oh Iekuni , Ohmine Ken , Muroi Kazuo , Kanda Yoshinobu 
Published: October 2019

Abstract
Background
After hematopoietic stem cell transplantation, donor T-cells home to secondary lymphoid organs and recognize alloantigens within MHC molecules presented by host APCs. Following activation, donor T-cells acquire effector functions and then migrate into host organs along the chemokine gradients. Animal models targeting chemokine signals for prevention or treatment of GVHD have shown promising results; however, there have been significant inconsistencies among studies probably due to differences in species and conditioning regimens. The aim of this study is to evaluate the role of chemokines and their receptors, CCR5 (receptor of CCL3-5) and CXCR3 (receptor of CXCL9-10), in human T-cell homing and the development of GVHD using xenogeneic GVHD mouse model.
Methods
NOG mice received 250cGy of total body irradiation (TBI) if not otherwise specified, and were subsequently injected intravenously with human pan T-cells. All mice developed severe GVHD and died within 2 weeks, while the mice that received TBI only survived without any symptoms of GVHD. Peripheral blood was collected from mice at a certain interval for chemokine measurement. To assess the expression of chemokine receptors and genes associated with T-cell homing, cells were harvested from GVHD target organs of mice at day 9. For CCR5 blockage, mice were treated with 31 mg/kg maraviroc once daily by oral gavage after transplantation.
Results
Extensive infiltration of human T-cells and tissue destruction were observed in lungs and liver, but less severely in colon of GVHD mice. Consistent with this, quantitative real-time PCR analysis for five chemokine-related genes detected up-regulation of murine CXCL9 and CXCL10 in lungs, CCL4 in lungs and liver, but no up-regulation in colon. Similarly, the multiplex analysis of nine chemokines in plasma showed a marked increase in murine CCL4, CXCL9, and CXCL10 in GVHD mice. These observations suggest that the increased expression of CCL4, CXCL9, and CXCL10 on individual organs and following their systemic release play a critical role in the homing of allogeneic T-cells.
Quantitative real-time PCR analysis of 84 genes associated with chemokines and chemokine receptors in human T-cells obtained from GVHD target organs revealed down-regulation of 36 genes, most of which are critical for T-cell homing into lymph nodes, such as CCL21 (-6.73-fold) and its receptor, CCR7 (-51.6-fold), and up-regulation of 16 genes such as CCL3 (225.5-fold), CCL4 (25.2-fold), CCR1 (11.4-fold), CCR5 (3.94-fold), and CXCL10 (2.88-fold).
Focusing on chemokine receptors on human T-cells, flow cytometric analysis showed significantly higher expression of CCR5 on CD4+ and CD8+ T-cells, and CXCR3 on CD4+ T-cells in GVHD mice, whereas CXCR3 on CD8+ T-cells was strongly expressed even in resting state. Tissue damages were less apparent in GVHD mice that received human T-cells only compared with irradiated GVHD mice. Consistent with this, not only a total number but also the proliferation rate of human T-cells was decreased in non-irradiated GVHD mice. Also, non-irradiated GVHD mice showed significantly decreased plasma CCL4 and CXCL10 levels in plasma, and lower expression of CCR5 on CD4+ and CD8+ T-cells, and CXCR3 on CD4+ T-cells. The same was observed, to a significantly greater extent, in MHC class I/II deficient mice, suggesting that recognition of host MHC molecules by T-cells are critical for both host and donor chemokine signals. Taken together, TBI promotes host chemokine secretion and chemokine receptor expression on donor T-cells, leading to faster recruitment of donor T-cells into host organs and their proliferation.
Contrary to the previous reports, CCR5 inhibitor treatment failed to attenuate GVHD and to improve the survival of mice. Although none of chemokine ligands but CCL4 was up-regulated on the liver, the number of infiltrated T-cells and tissue destruction were almost equivalent compared to the control. These observations indicate that compensatory chemokine pathways involving alternative receptors for CCL3-5, such as CCR1 and CCR2 on effector T-cells may overcome CCR5 blockage.
Conclusion
This study firstly provides a comprehensive picture of human T-cell homing through CCR5 and CXCR3 signaling in xenogeneic GVHD models. Our data supports the development of novel preventive and therapeutic strategies targeting chemokine signaling for GVHD.

Disclosures
Fujiwara: Shire: Consultancy; Pfizer: Consultancy; Chugai: Consultancy; Kirin: Consultancy; Kyowa-Hakko: Consultancy; Astellas: Consultancy. Ohmine:Kyowa Hakko Kirin: Speakers Bureau; Takara Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Celgene Corporation: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Alexion Pharmaceuticals: Speakers Bureau; Ono Pharmaceutical: Consultancy. Muroi:Japanese Red Cross Society: Speakers Bureau; Dickinson and Company: Speakers Bureau; Becton: Speakers Bureau; JCR: Speakers Bureau. Kanda:Taisho-Toyama: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Tanabe-Mitsubishi: Research Funding; CSL Behring: Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; MSD: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Nippon-Shinyaku: Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.