Humanized anti-IL-26 monoclonal antibody as a novel targeted therapy for chronic graft-versus-host disease

IL-26 is a Th17 cytokine, with its gene being absent in rodents. To characterize the in vivo immunological effects of IL-26 in chronic systemic inflammation, we used human IL26 transgenic (hIL-26Tg) mice and human umbilical cord blood mononuclear cells (hCBMC) in mouse allogeneic-graft-versus-host disease (GVHD) and chronic xenogeneic-GVHD model, respectively. Transfer of bone marrow and spleen T cells from hIL-26Tg mice into B10.BR mice resulted in GVHD progression, with clinical signs of tissue damage in multiple organs. IL-26 markedly increased neutrophil levels both in the GVHD-target tissues and peripheral blood. Expression levels of Th17 cytokines in hIL-26Tg mice-derived donor CD4 T cells were significantly increased, whereas IL-26 did not affect cytotoxic function of donor CD8 T cells. In addition, granulocyte-colony stimulating factor, IL-1β, and IL-6 levels were particularly enhanced in hIL-26Tg mice. We also developed a humanized neutralizing anti-IL-26 monoclonal antibody (mAb) for therapeutic use, and its administration after onset of chronic xenogeneic-GVHD mitigated weight loss and prolonged survival, with preservation of graft-versus-leukemia effect. Taken together, our data elucidate the in vivo immunological effects of IL-26 in chronic GVHD models and suggest that a humanized anti-IL-26 mAb may be a potential therapeutic agent for the treatment of chronic GVHD. IL-26 is a Th17 cytokine, with its gene being absent in rodents. To characterize the in vivo immunological effects of IL-26 in chronic systemic inflammation, we used human IL26 transgenic (hIL-26Tg) mice and human umbilical cord blood mononuclear cells (hCBMC) in mouse allogeneic-graft-versus-host disease (GVHD) and chronic xenogeneic-GVHD model, respectively. Transfer of bone marrow and spleen T cells from hIL-26Tg mice into B10.BR mice resulted in GVHD progression, with clinical signs of tissue damage in multiple organs. IL-26 markedly increased neutrophil levels both in the GVHD-target tissues and peripheral blood. Expression levels of Th17 cytokines in hIL-26Tg mice-derived donor CD4 T cells were significantly increased, whereas IL-26 did not affect cytotoxic function of donor CD8 T cells. In addition, granulocyte-colony stimulating factor, IL-1β, and IL-6 levels were particularly enhanced in hIL-26Tg mice. We also developed a humanized neutralizing anti-IL-26 monoclonal antibody (mAb) for therapeutic use, and its administration after onset of chronic xenogeneic-GVHD mitigated weight loss and prolonged survival, with preservation of graft-versus-leukemia effect. Taken together, our data elucidate the in vivo immunological effects of IL-26 in chronic GVHD models and suggest that a humanized anti-IL-26 mAb may be a potential therapeutic agent for the treatment of chronic GVHD. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a well-established procedure for hematological malignancies or bone marrow (BM) failure syndromes,1Beura LK Rosato PC Masopust D Implications of resident memory T cells for transplantation.Am J Transplant. 2017; 17: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 2Copelan EA Hematopoietic stem-cell transplantation.N Engl J Med. 2006; 354: 1813-1826Crossref PubMed Scopus (1690) Google Scholar, 3Anasetti C Logan BR Lee SJ et al.Peripheral-blood stem cells versus bone marrow from unrelated donors.N Engl J Med. 2012; 367: 1487-1496Crossref PubMed Scopus (633) Google Scholar with acute and chronic graft-versus-host disease (GVHD) due to an immunological attack on target recipient organs by donor allogeneic T cells being significant complications. While suppression of donor allogeneic T cells is highly effective for preventing GVHD, complications such as loss of graft-versus-leukemia (GVL) effect or increased opportunistic infections can occur.4Chang YJ Zhao XY Huang XJ Strategies for enhancing and preserving anti-leukemia effects without aggravating graft-versus-host disease.Front Immunol. 2018; 9: 3041Crossref PubMed Scopus (39) Google Scholar In-depth understanding of the molecular mechanisms involved in human GVHD will improve allo-HSCT clinical outcome. IL-26 belonging to the IL-10 cytokine family,5Kotenko SV The family of IL-10-related cytokines and their receptors: related, but to what extent?.Cytokine Growth Factor Rev. 2002; 13: 223-240Crossref PubMed Scopus (191) Google Scholar,6Donnelly RP Sheikh F Dickensheets H Savan R Young HA Walter MR Interleukin-26: an IL-10-related cytokine produced by Th17 cells.Cytokine Growth Factor Rev. 2010; 21: 393-401Crossref PubMed Scopus (100) Google Scholar is conserved in several vertebrate species but not found in mice and rats.7Schoenborn JR Dorschner MO Sekimata M et al.Comprehensive epigenetic profiling identifies multiple distal regulatory elements directing transcription of the gene encoding interferon-gamma.Nat Immunol. 2007; 8: 732-742Crossref PubMed Scopus (242) Google Scholar IL-26 is known as a Th17 cytokine, while NK cells, macrophages, bronchial epithelial cells, and synoviocytes have capacity to produce IL-26.8Corvaisier M Delneste Y Jeanvoine H et al.IL-26 is overexpressed in rheumatoid arthritis and induces proinflammatory cytokine production and Th17 cell generation.PLoS Biol. 2012; 10: e1001395Crossref PubMed Scopus (119) Google Scholar, 9Che KF Tengvall S Levanen B et al.Interleukin-26 in antibacterial host defense of human lungs. Effects on neutrophil mobilization.Am J Respir Crit Care Med. 2014; 190: 1022-1031Crossref PubMed Scopus (49) Google Scholar, 10Che KF Kaarteenaho R Lappi-Blanco E et al.Interleukin-26 production in human primary bronchial epithelial cells in response to viral stimulation: modulation by Th17 cytokines.Mol Med. 2017; 23: 247-257Crossref PubMed Scopus (18) Google Scholar, 11Itoh T Hatano R Horimoto Y et al.IL-26 mediates epidermal growth factor receptor-tyrosine kinase inhibitor resistance through endoplasmic reticulum stress signaling pathway in triple-negative breast cancer cells.Cell Death Dis. 2021; 12: 520Crossref PubMed Scopus (6) Google Scholar IL-20RA/IL-10RB heterodimer is an IL-26 receptor, and IL-26 binding to IL-20RA/IL-10RB results in functional activation via STAT3 phosphorylation.12Hor S Pirzer H Dumoutier L et al.The T-cell lymphokine interleukin-26 targets epithelial cells through the interleukin-20 receptor 1 and interleukin-10 receptor 2 chains.J Biol Chem. 2004; 279: 33343-33351Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar However, IL-26 can activate human monocytes and NK cells,8Corvaisier M Delneste Y Jeanvoine H et al.IL-26 is overexpressed in rheumatoid arthritis and induces proinflammatory cytokine production and Th17 cell generation.PLoS Biol. 2012; 10: e1001395Crossref PubMed Scopus (119) Google Scholar,13Miot C Beaumont E Duluc D et al.IL-26 is overexpressed in chronically HCV-infected patients and enhances TRAIL-mediated cytotoxicity and interferon production by human NK cells.Gut. 2015; 64: 1466-1475Crossref PubMed Scopus (39) Google Scholar despite the lack of IL-20RA expression on these cell types.14Wolk K Kunz S Asadullah K Sabat R Cutting edge: immune cells as sources and targets of the IL-10 family members?.J Immunol. 2002; 168: 5397-5402Crossref PubMed Scopus (508) Google Scholar IL-26 also acts on vascular endothelial cells to stimulate angiogenesis and activates EGFR-tyrosine kinase inhibitor-associated bypass pathway to promote drug resistance in triple-negative breast cancer cells despite a lack of IL-20RA expression.11Itoh T Hatano R Horimoto Y et al.IL-26 mediates epidermal growth factor receptor-tyrosine kinase inhibitor resistance through endoplasmic reticulum stress signaling pathway in triple-negative breast cancer cells.Cell Death Dis. 2021; 12: 520Crossref PubMed Scopus (6) Google Scholar,15Itoh T Hatano R Komiya E et al.Biological effects of IL-26 on T cell-mediated skin inflammation, Including Psoriasis.J Invest Dermatol. 2019; 139: 878-889Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar These findings strongly suggest the existence of an unidentified IL-26 receptor besides IL-20RA/IL-10RB. Furthermore, IL-26 is a cationic and amphipathic cytokine with characteristics of an antimicrobial peptide,16Meller S Di Domizio J Voo KS et al.T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26.Nat Immunol. 2015; 16: 970-979Crossref PubMed Scopus (0) Google Scholar,17Dang AT Teles RM Weiss DI et al.IL-26 contributes to host defense against intracellular bacteria.J Clin Invest. 2019; 129: 1926-1939Crossref PubMed Scopus (28) Google Scholar capable of binding to DNA/RNA released from bacteria or dying cells or neutrophil extracellular traps (NETs), triggering inflammatory cytokine production from plasmacytoid dendritic cells, monocytes, and neutrophils in a TLR9 or STING- and inflammasome-dependent manner,16Meller S Di Domizio J Voo KS et al.T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26.Nat Immunol. 2015; 16: 970-979Crossref PubMed Scopus (0) Google Scholar,18Poli C Augusto JF Dauve J et al.IL-26 confers proinflammatory properties to extracellular DNA.J Immunol. 2017; 198: 3650-3661Crossref PubMed Scopus (55) Google Scholar indicating the diverse mechanism of action of IL-26. Without accessible animal models due partly to the absence of the IL26 gene in mice, IL-26 role in inflammatory disorders is not clearly characterized. Utilizing human IL26 bacterial artificial chromosome (BAC) transgenic (hIL-26Tg) mice,19Collins PL Chang S Henderson M et al.Distal regions of the human IFNG locus direct cell type-specific expression.J Immunol. 2010; 185: 1492-1501Crossref PubMed Scopus (29) Google Scholar,20Collins PL Henderson MA Aune TM Lineage-specific adjacent IFNG and IL26 genes share a common distal enhancer element.Genes Immun. 2012; 13: 481-488Crossref PubMed Scopus (24) Google Scholar we recently investigated the in vivo effects of IL-26 in acute local inflammation models and showed that vascularization and immune cell infiltration were enhanced in the skin of imiquimod-applied hIL-26Tg mice.15Itoh T Hatano R Komiya E et al.Biological effects of IL-26 on T cell-mediated skin inflammation, Including Psoriasis.J Invest Dermatol. 2019; 139: 878-889Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar,21Corridoni D Antanaviciute A Gupta T et al.Single-cell atlas of colonic CD8(+) T cells in ulcerative colitis.Nat Med. 2020; 26: 1480-1490Crossref PubMed Scopus (64) Google Scholar These findings strongly suggest that IL-26 may represent a novel therapeutic target for inflammatory disorders, although IL-26 role in chronic inflammation still needs clarification. In the murine models utilizing hIL-26Tg mice, human IL-26 is not expressed constitutively but is induced only when the cells capable of expressing IL-26 are activated, which resembles the human condition. However, a potential drawback of the hIL-26 BAC Tg mouse model is that the expression level of human IL-26 in murine CD4 T cells is quite low as compared with the activated human CD4 T cells,20Collins PL Henderson MA Aune TM Lineage-specific adjacent IFNG and IL26 genes share a common distal enhancer element.Genes Immun. 2012; 13: 481-488Crossref PubMed Scopus (24) Google Scholar resulting in suboptimal biological effects of human IL-26 in the inflammation models involving hIL-26Tg mice. For these reasons, inflammation models utilizing human T cells are essential to investigate the inherent functions of human IL-26. Due to the reasons above, in the present study, we examined mouse allogeneic (allo)-GVHD model utilizing hIL-26Tg mice and chronic xenogeneic (xeno)-GVHD model utilizing human umbilical cord blood mononuclear cells (hCBMC) to evaluate the in vivo immunological effects of IL-26 in the pathology of chronic systemic inflammation, and created a humanized neutralizing anti-IL-26 monoclonal antibody (mAb) based on our recently developed murine mAb22Hatano R Itoh T Otsuka H et al.Characterization of novel anti-IL-26 neutralizing monoclonal antibodies for the treatment of inflammatory diseases including psoriasis.MAbs. 2019; 11: 1428-1442Crossref PubMed Scopus (9) Google Scholar as a potential treatment for chronic GVHD. Serum samples were obtained from nine mild acute skin GVHD patients (6 males and 3 females, 2 grade I and 7 grade II, age 47.56 ± 9.62) and six moderate/severe chronic lung GVHD patients (4 males and 2 females, 2 severe, and 4 moderate, age 48.33 ± 15.04). Serum was collected from patients following GVHD diagnosis at Sapporo Medical University. Serum was collected from 12 healthy volunteers (8 males and 4 females, age 46.83 ± 11.47) at Juntendo University. All serum samples were stored at −80°C. Mononuclear cells isolated from human cord blood with Ficoll density gradation method were purchased from RIKEN BioResource Center. Female B10.BR (H-2k) and NOD/Shi-scidIL2rγnull (NOG) (H-2d) mice were purchased from Sankyo Labo-Service and In-Vivo Science, respectively. C57BL/6 (H-2b) mice carrying a 190-kb BAC transgene with human IFNG and IL26 gene (hIL-26Tg) and a BAC Tg-deleting conserved noncoding sequence (CNS) positioned at 77 kb upstream of the IFNG transcription start site (ΔCNS-77Tg) were developed in Dr. Aune’s laboratory.19Collins PL Chang S Henderson M et al.Distal regions of the human IFNG locus direct cell type-specific expression.J Immunol. 2010; 185: 1492-1501Crossref PubMed Scopus (29) Google Scholar,20Collins PL Henderson MA Aune TM Lineage-specific adjacent IFNG and IL26 genes share a common distal enhancer element.Genes Immun. 2012; 13: 481-488Crossref PubMed Scopus (24) Google Scholar Human IL-26 expression was completely abrogated in ΔCNS-77Tg mice.20Collins PL Henderson MA Aune TM Lineage-specific adjacent IFNG and IL26 genes share a common distal enhancer element.Genes Immun. 2012; 13: 481-488Crossref PubMed Scopus (24) Google Scholar Although both hIL-26Tg mice and ΔCNS-77Tg mice-derived CD4 T cells were capable of producing human IFN-γ, it has been reported that human IFN-γ does not transduce intracellular signals through murine IFNGR nor exhibit biological activity.23Gibbs VC Williams SR Gray PW et al.The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer.Mol Cell Biol. 1991; 11: 5860-5866PubMed Google Scholar All mice used in this study were housed in a specific pathogen-free facility in micro-isolator cages, and used at 10–12 weeks of age. All other information is detailed in Supplementary methods. We first used hIL-26Tg mice for a complete MHC-mismatched allo-GVHD model, having previously demonstrated that human IL-26 can act on both murine and human cells.11Itoh T Hatano R Horimoto Y et al.IL-26 mediates epidermal growth factor receptor-tyrosine kinase inhibitor resistance through endoplasmic reticulum stress signaling pathway in triple-negative breast cancer cells.Cell Death Dis. 2021; 12: 520Crossref PubMed Scopus (6) Google Scholar,15Itoh T Hatano R Komiya E et al.Biological effects of IL-26 on T cell-mediated skin inflammation, Including Psoriasis.J Invest Dermatol. 2019; 139: 878-889Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar,24Ohnuma K Hatano R Aune TM et al.Regulation of pulmonary graft-versus-host disease by IL-26+CD26+CD4 T lymphocytes.J Immunol. 2015; 194: 3697-3712Crossref PubMed Scopus (33) Google Scholar ΔCNS-77Tg mice lacking human IL-26 transcription were used as controls. The basic characteristics of spleen T cells in hIL-26Tg mice were generally similar as those in control mice (Figure S1). Transplantation of T cell-depleted bone marrow (TCD-BM) alone hardly affected survival and weight of B10.BR recipients (Figure 1A,B). In contrast, acute weight loss occurred until one-week post-transplant of donor BM plus splenic T cells, followed by a slight body weight recovery and then a gradual decrease due to allo-GVHD. B10.BR mice receiving hIL-26Tg mice-derived BM and splenic T cells (hIL-26Tg-B10.BR mice) exhibited significantly decreased overall survival (p = .0215) and body weight, compared to recipients transplanted with ΔCNS-77Tg mice-derived BM and splenic T cells (ΔCNS-77Tg-B10.BR mice) (Figure 1A,B). Blood leukocytes of recipient mice were mostly from donor-derived H-2Db+H-2Dk− cells (Figure 1C). Although there was no difference in donor CD4 and CD8 T cell expansion between allo-GVHD groups, donor granulocyte levels were apparently increased in hIL-26Tg-B10.BR mice compared to ΔCNS-77Tg-B10.BR mice (Figures 1C, Figure S2). GVHD manifestations were hardly observed in B10.BR mice receiving TCD-BM alone (Figure 1D,E). In contrast, histiocytes in pulmonary alveoli, portal inflammatory cell infiltration around portal vein, and severe colitis with ulceration were observed in hIL-26Tg-B10.BR mice, with significantly higher pathological scores of these organs compared to those of control mice, whereas skin GVHD manifestations were moderate in this model (Figures 1D,E, Figure S3). Moreover, collagen deposition was clearly observed in the portal area of the liver of hIL-26Tg-B10.BR mice (Figure 1F). Considering the survival period of recipient mice after transplantation, the manifestation of colon and liver GVHD and lung inflammation, and the fibroproliferation in the liver, this allo-GVHD model appears to display the characteristics of both acute and chronic GVHD. Taken together, our data indicate that IL-26 further exacerbates systemic GVHD progression and severity. Our next studies examined the mechanisms involved in IL-26-mediated allo-GVHD. CD45+ leukocytes were localized in the lung of normal B10.BR or B10.BR recipients receiving TCD-BM alone while being at low levels in the liver. Both allo-GVHD groups demonstrated increased levels of donor leukocytes in the lung and particularly in the liver (Figure 2A). There was a higher percentage of CD4 T cells than CD8 T cells in donor leukocytes in B10.BR mice receiving TCD-BM alone, with the ratio reversed in the allo-GVHD mice (Figure 2B). The percentage of donor CD4 and CD8 T cells in hIL-26Tg-B10.BR and ΔCNS-77Tg-B10.BR mice were similar, whereas neutrophil levels were markedly increased in lung, liver, spleen, and colon of hIL-26Tg-B10.BR mice, as well as a trend toward an enhanced level of donor monocytes and macrophages (Figure 2B, Figures S4 and S5). Meanwhile, B cell percentage was prominently decreased in the allo-GVHD mice as compared with recipients receiving TCD-BM alone (Figure 2B, Figure S6). We next evaluated the effect of anti-mouse Ly6G mAb to determine whether the increased neutrophil levels in the GVHD-target organs from hIL-26Tg-B10.BR mice were associated with GVHD exacerbation. Although anti-Ly6G mAb administration did not result in complete neutrophil depletion, levels of neutrophil infiltration in the lung and liver of hIL-26Tg-B10.BR mice were decreased, concurrent with a trend toward reduced allo-GVHD-associated lethality (p = .1546) and weight loss (Figure S7). These results suggest that the increased neutrophil levels in the GVHD-target organs are at least partially responsible for GVHD exacerbation. In contrast with mice receiving TCD-BM alone, most of the donor CD4 and CD8 T cells in the allo-GVHD mice expressed PD-1, suggesting a TCR-stimulated phenotype (Figure 2C,E). Human IL26 mRNA expression was observed only in donor CD4 T cells transplanted from hIL-26Tg but not ΔCNS-77Tg mice (Figure 2D). Expression levels of all the genes examined in donor CD4 and CD8 T cells purified from allo-GVHD mice were higher than from mice receiving TCD-BM alone. Mouse Il17a and Il21 levels in donor CD4 T cells in the liver of hIL-26Tg-B10.BR mice were significantly higher than those from ΔCNS-77Tg-B10.BR mice, with similar levels of effector molecules in donor CD8 T cells from hIL-26Tg-B10.BR mice and control recipients (Figure 2D,F). Higher expression level of mouse IL-17A in donor CD4 T cells in the liver of hIL-26Tg-B10.BR mice was also confirmed by flow cytometry (Figure S8). Similar results were observed for lung and spleen donor T cells (data not shown). Plasma levels of human IL-26 and mouse IL-17A were similar to those shown in Figure 2D,G. Inflammatory factors highly associated with human IL-26 expression were identified by multiplex assays. Among 23 cytokines and chemokines evaluated, plasma levels of mouse RANTES, IL-1α, IL-1β, IL-6, and granulocyte-colony stimulating factor (G-CSF) in hIL-26Tg-B10.BR mice were significantly higher than those in ΔCNS-77Tg-B10.BR mice (Figure 2H). Furthermore, quantitative RT-PCR analyses confirmed the enhanced mRNA expression of these genes, particularly Il1b, Il6, and Csf3, in the GVHD-target organs of hIL-26Tg-B10.BR mice (Figure 2I). Our data hence indicate that IL-26 markedly increases neutrophil levels in GVHD-target tissues and peripheral blood, and augments Th17 response associated with enhanced levels of G-CSF, IL-1β, and IL-6. Since IL-26 and DNA have synergistic effect on IL-1β and IL-6 production,18Poli C Augusto JF Dauve J et al.IL-26 confers proinflammatory properties to extracellular DNA.J Immunol. 2017; 198: 3650-3661Crossref PubMed Scopus (55) Google Scholar we next analyzed Il1b and Il6 expression in mouse myeloid cells. Human IL-26 plus mouse genomic DNA stimulated both Il1b and Il6 mRNA expression and IL-1β and IL-6 production from primary mouse splenic CD11b+ cells (Figure 3A,B), but not Il12b and Il23a expression (data not shown). Similar results were observed for the mouse macrophage cell line RAW264.7 (Figure S9). Other cytokines besides TGF-β and IL-6 are involved in the functional maturation and maintenance of Th17 cells.25Bettelli E Carrier Y Gao W et al.Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.Nature. 2006; 441: 235-238Crossref PubMed Scopus (5552) Google Scholar, 26Chung Y Chang SH Martinez GJ et al.Critical regulation of early Th17 cell differentiation by interleukin-1 signaling.Immunity. 2009; 30: 576-587Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar, 27McGeachy MJ Chen Y Tato CM et al.The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo.Nat Immunol. 2009; 10: 314-324Crossref PubMed Scopus (784) Google Scholar, 28Korn T Bettelli E Gao W et al.IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells.Nature. 2007; 448: 484-487Crossref PubMed Scopus (1506) Google Scholar Since we demonstrated that IL-26 exposure was associated with enhanced IL-1β and IL-6 expression, we next examined IL-1β and IL-6 effect on Th17 polarization and activation. Exogenous mouse IL-1β and IL-6 augmented mouse Il17a and human IL26 expression, respectively, in hIL-26Tg mice-derived splenic CD4+ T cells costimulated via CD3 and CD28 in the presence of mouse TGF-β1, IL-1β, and IL-6 (Figure 3C). In contrast, stimulation with TGF-β1 and IL-6 but not IL-1β was essential to induce CD4+ T cell Rorc expression, the master transcription factor driving Th17 cell differentiation (Figure 3C), while Il21 expression was regulated by IL-6 alone (Figure 3C). TGF-β1 and IL-6 stimulation suppressed Ifng expression while slightly enhanced Il4 expression (Figure 3C). Mouse cytokine and Rorc levels were similar in ΔCNS-77Tg mice-derived and hIL-26Tg mice-derived splenic CD4+ T cells, while human IL26 expression was never observed in ΔCNS-77Tg mice-derived CD4+ T cells in all stimulatory conditions (data not shown). These results indicate that both IL-1β and IL-6 are essential for the marked increase in mouse IL-17A and human IL-26 expression in CD4+ T cells of hIL-26Tg mice. We also examined the association between IL-26-mediated positive-feedback and G-CSF expression, a critical factor for granulopoiesis.29Lieschke GJ Grail D Hodgson G et al.Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.Blood. 1994; 84: 1737-1746Crossref PubMed Google Scholar Stimulation with mouse IL-1β dramatically induced G-CSF production from RAW264.7 cells, with additional enhancement by mouse IL-17A, genomic DNA, and human IL-26 (Figure 3D). Meanwhile, mouse IL-1β and IL-17A synergistically enhanced G-CSF production from NIH3T3 cells, with minimal effect by mouse IL-6, genomic DNA, and human IL-26 (Figure 3E). To develop a novel IL-26-targeted therapy, we have succeeded in developing humanized mAb h69-10 with strong binding affinity and neutralizing activity against IL-26 as compared with the original murine mAb m69-10 (Figure S10). Acute GVHD is caused by T cells within the original stem cell transplant, whereas chronic GVHD is theoretically caused by allo- (auto-) reactive T cells that have matured through the host thymus. We previously established a chronic xeno-GVHD model,24Ohnuma K Hatano R Aune TM et al.Regulation of pulmonary graft-versus-host disease by IL-26+CD26+CD4 T lymphocytes.J Immunol. 2015; 194: 3697-3712Crossref PubMed Scopus (33) Google Scholar which has two inherent advantages that allow us to investigate the therapeutic effect of anti-IL-26 mAb. One is human IL-26 expression level in human CD4 T cells is much higher than hIL-26Tg mice-derived CD4 T cells, and the other is disease progression is moderate as compared with the hIL-26Tg-B10.BR allo-GVHD model. m69-10 or h69-10 treatment following the appearance of GVHD clinical symptoms on day +28 markedly increased overall survival of hCBMC-NOG mice (Figure 4A), with stable body weight for up to 7 weeks post-transplantation (Figure 4B). In this model, recipient-derived hematopoietic stem cells were not destroyed completely by sublethal irradiation, resulting in the development of chimeric mice. Human leukocytes were mainly composed of T cells, whereas mouse leukocytes were mostly granulocytes and monocytes/macrophages. Although anti-IL-26 mAb administration did not affect donor CD4 and CD8 T cell expansion, there was a trend toward a decrease in the percentage of mouse granulocytes in hCBMC-NOG mice receiving m69-10 or h69-10 (Figure 4C). Although consolidation in lung, regenerative change in bile duct epithelium, cholestasis in hepatocytes, and acanthosis, follicular dropout, sclerosis of reticular dermis, and fat loss in skin were often observed in hCBMC-NOG mice receiving control IgG, progression of these systemic GVHD symptoms were prominently suppressed in m69-10 or h69-10-administered hCBMC-NOG mice, with hardly any GVHD manifestation observed in colon tissues of all mice (Figure 4D,E, Figure S11). Furthermore, significant decrease in resistance and elastance, and increase in compliance were observed in pulmonary functions of hCBMC-NOG mice receiving anti-IL-26 mAb (Figure 4F). Serum alanine transaminase activity was also elevated in mice treated with control IgG compared to anti-IL-26 mAb (Figure 4G). These data indicate that both h69-10 and m69-10 treatment significantly impeded chronic GVHD development. Administration of h69-10 or m69-10 markedly reduced the total number of donor leukocytes in both lung and liver of hCBMC-NOG mice, while the percentage of CD4 and CD8 T cells in donor leukocytes of mice receiving anti-IL-26 mAb was nearly identical to that of mice receiving control IgG, indicating that anti-IL-26 mAb treatment decreased the absolute number of donor CD4 and CD8 T cells in the GVHD-target organs (Figure 5A–C). Anti-IL-26 mAb administration also significantly reduced the total number of recipient leukocytes in lung and the percentage of neutrophils in recipient leukocytes in the spleen, lung, or liver of hCBMC-NOG mice, indicating that anti-IL-26 mAb treatment reduced neutrophils systemically, but particularly in lung of hCBMC-NOG mice (Figure 5A–C, Figure S12). Most of donor CD4 and CD8 T cells in hCBMC-NOG recipients expressed PD-1, suggesting a TCR-stimulated phenotype (Figure 5D). In addition, anti-IL-26 mAb treatment markedly decreased human IL26, IL17A, and IL21 but not IFNG and IL4 expression levels in human CD4 T cells (Figure 5E, Figure S13), plasma levels of human IL-26 and IL-17A (Figure 5F), and mRNA expression levels of mouse Ccl3, Ccl5, Cxcl1, Cxcl2, Il1b, Il6, and Csf3 in lung or liver of hCBMC-NOG recipients (Figure 5G). While the lung of hCBMC-NOG mice receiving control IgG manifested significant peribronchiolar and perivascular collagen deposition and enhanced α-smooth muscle actin-positive myofibroblast level as compared with normal NOG mice (Figure, 5H,I, Figure S14), levels of collagen deposition, myofibroblasts as well as IL-26+ cells were prominently decreased in the lung of anti-IL-26 mAb-treated hCBMC-NOG mice (Figure 5H,I). Taken together, our data indicate that anti-IL-26 mAb treatment significantly suppresses T cell and neutrophil infiltration into GVHD-target organs, Th17 response, and fibroproliferation. Since GVHD and GVL are highly linked immune reactions,30Wu CJ Ritz J Revealing tumor immunity after hematopoietic stem cell transplantation.Clin Cancer Res. 2009; 15: 4515-4517Crossref PubMed Scopus (8) Google Scholar our next studies evaluated the potential influence of anti-IL-26 mAb on GVL effect. To stably develop tumor-disseminated recipient mice, firefly luciferase-transfected A20 (A20-luc) cells were inoculated on day +28 after hCBMC transplantation, followed by anti-IL-26 mAb treatment (Figure 6A). NOG mice inoculated with A20-luc alone displayed prominent tumor proliferation (Figure 6B,C), succumbing to high tumor burden within 3 weeks post-tumor inoculation. hCBMC-NOG recipients receiving control IgG exhibited clinical symptoms of GVHD such as weight loss and alopecia, although tumor progression was clearly suppressed, possibly by human T cells. In contrast, anti-IL-26 mAb treatment suppressed both tumor progression and GVHD manifestations