TL1A priming induces a multi-cytokine Th9 cell phenotype that promotes robust allergic inflammation in murine models of asthma.

Multi-cytokine-producing Th9 cells secrete IL-9 and type 2 cytokines and mediate mouse and human allergic inflammation. However, the cytokines that promote a multi-cytokine secreting phenotype have not been defined. Tumor necrosis factor superfamily member TL1A signals through its receptor DR3 to increase IL-9. Here we demonstrate that TL1A increases expression of IL-9 and IL-13 co-expressing cells in murine Th9 cell cultures, inducing a multi-cytokine phenotype. Mechanistically, this is linked to histone modifications allowing for increased accessibility at the Il9 and Il13 loci. We further show that TL1A alters the transcription factor network underlying expression of IL-9 and IL-13 in Th9 cells and increases binding of transcription factors to Il9 and Il13 loci. TL1A-priming enhances the pathogenicity of Th9 cells in murine models of allergic airway disease through the increased expression of IL-9 and IL-13. Lastly, in both chronic and memory-recall models of allergic airway disease, blockade of TL1A signaling decreases the multi-cytokine Th9 cell population and attenuates the allergic phenotype. Taken together, these data demonstrate that TL1A promotes the development of multi-cytokine Th9 cells that drive allergic airway diseases and that targeting pathogenic T helper cell-promoting cytokines could be an effective approach for modifying disease.

LILRB3 Modulates Acute Myeloid Leukemia Progression and Acts as an Effective Target for CAR T-cell Therapy.

Identifying novel cell surface receptors that regulate leukemia cell differentiation and can be targeted to inhibit cellular proliferation is crucial to improve current treatment modalities in acute myeloid leukemia (AML), especially for relapsed or chemotherapy-refractory leukemia. Leukocyte immunoglobulin-like receptor type B (LILRB) is an immunomodulatory receptor originally found to be expressed in myeloid cells. In this study, we found that LILRB receptors can be induced under inflammatory stimuli and chemotherapy treatment conditions. Blockade of LILRB3 inhibited leukemia cell proliferation and leukemia progression. In addition, treatment with LILRB3 blocking antibodies upregulated myeloid lineage differentiation transcription factors, including PU.1, C/EBP family, and IRF, whereas phosphorylation of proliferation regulators, for example, AKT, cyclin D1, and retinoblastoma protein, was decreased. Conversely, transcriptomic analysis showed LILRB3 activation by agonist antibodies may enhance leukemia survival through upregulation of cholesterol metabolism, which has been shown to promote leukemia cell survival. Moreover, LILRB3-targeted CAR T cells exhibited potent antitumor effects both in vitro and in vivo. Taken together, our results suggest that LILRB3 is a potentially potent target for multiple treatment modalities in AML. SIGNIFICANCE: LILRB3 regulates differentiation and proliferation in acute myeloid leukemia and can be targeted with monoclonal antibodies and CAR T cells to suppress leukemia growth.

The Dichotomy of Interleukin-9 Function in the Tumor Microenvironment.

Interleukin 9 (IL-9) is a cytokine with potent proinflammatory properties that plays a central role in pathologies such as allergic asthma, immunity to parasitic infection, and autoimmunity. More recently, IL-9 has garnered considerable attention in tumor immunity. Historically, IL-9 has been associated with a protumor function in hematological malignancies and an antitumor function in solid malignancies. However, recent discoveries of the dynamic role of IL-9 in cancer progression suggest that IL-9 can act as both a pro- or antitumor factor in various hematological and solid malignancies. This review summarizes IL-9-dependent control of tumor growth, regulation, and therapeutic applicability of IL-9 blockade and IL-9-producing cells in cancer.

The World according to IL-9.

Among the cytokines regulating immune cells, IL-9 has gained considerable attention for its ability to act on multiple cell types as a regulator of beneficial and pathologic immune responses. Yet, it is still not clearly defined how IL-9 impacts immune responses. IL-9 demonstrates a remarkable degree of tissue-specific functionality and has cellular sources that vary by tissue site and the context of the inflammatory milieu. Here, we provide perspective to summarize the biological activities of IL-9 and highlight cell type-specific roles in the immune pathogenesis of diseases. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial and where it has the potential to complicate clinical outcomes.

Interleukin-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration in allergic airway inflammation.

Allergic asthma is a chronic lung disease characterized by airway hyperresponsiveness and cellular infiltration that is exacerbated by immunoglobulin E-dependent mast cell (MC) activation. Interleukin-9 (IL-9) promotes MC expansion during allergic inflammation but precisely how IL-9 expands tissue MCs and promotes MC function is unclear. In this report, using multiple models of allergic airway inflammation, we show that both mature MCs (mMCs) and MC progenitors (MCp) express IL-9R and respond to IL-9 during allergic inflammation. IL-9 acts on MCp in the bone marrow and lungs to enhance proliferative capacity. Furthermore, IL-9 in the lung stimulates the mobilization of CCR2+ mMC from the bone marrow and recruitment to the allergic lung. Mixed bone marrow chimeras demonstrate that these are intrinsic effects in the MCp and mMC populations. IL-9-producing T cells are both necessary and sufficient to increase MC numbers in the lung in the context of allergic inflammation. Importantly, T cell IL-9-mediated MC expansion is required for the development of antigen-induced and MC-dependent airway hyperreactivity. Collectively, these data demonstrate that T cell IL-9 induces lung MC expansion and migration by direct effects on the proliferation of MCp and the migration of mMC to mediate airway hyperreactivity.

Rapamycin Treatment Alleviates Chronic GVHD-Induced Lupus Nephritis in Mice by Recovering IL-2 Production and Regulatory T Cells While Inhibiting Effector T Cells Activation.

In this study, we test the therapeutic effects of rapamycin in a murine model of SLE-like experimental lupus nephritis induced by chronic graft-versus-host disease (cGVHD). Our results suggest that rapamycin treatment reduced autoantibody production, inhibited T lymphocyte and subsequent B cell activation, and reduced inflammatory cytokine and chemokine production, thereby protecting renal function and alleviating histological lupus nephritis by reducing the occurrence of albuminuria. To explore the potential mechanism of rapamycin's reduction of kidney damage in mice with lupus nephritis, a series of functional assays were conducted. As expected, rapamycin remarkably inhibited the lymphocytes' proliferation within the morbid mice. Interestingly, significantly increased proportions of peripheral CD4+FOXP3+ and CD4+CD25high T cells were observed in rapamycin-treated group animals, suggesting an up-regulation of regulatory T cells (Tregs) in the periphery by rapamycin treatment. Furthermore, consistent with the results regarding changes in mRNA abundance in kidney by real-time PCR analysis, intracellular cytokine staining demonstrated that rapamycin treatment remarkably diminished the secretion of Th1 and Th2 cytokines, including IFN-γ, IL-4 and IL-10, in splenocytes of the morbid mice. However, the production of IL-2 from splenocytes in rapamycin-treated mice was significantly higher than in the cells from control group animals. These findings suggest that rapamycin treatment might alleviate systemic lupus erythematosus (SLE)-like experimental lupus nephritis through the recovery of IL-2 production, which promotes the expansion of regulatory T cells while inhibiting effector T cell activation. Our studies demonstrated that, unlike other commonly used immunosuppressants, rapamycin does not appear to interfere with tolerance induction but permits the expansion and suppressive function of Tregs in vivo.

Long-Range Cationic Disordering Induces two Distinct Degradation Pathways in Co-Free Ni-Rich Layered Cathodes.

Ni-rich layered oxides are one of the most attractive cathode materials in high-energy-density lithium-ion batteries, their degradation mechanisms are still not completely elucidated. Herein, we report a strong dependence of degradation pathways on the long-range cationic disordering of Co-free Ni-rich Li1-m (Ni0.94 Al0.06 )1+m O2 (NA). Interestingly, a disordered layered phase with lattice mismatch can be easily formed in the near-surface region of NA particles with very low cation disorder (NA-LCD, m≤0.06) over electrochemical cycling, while the layered structure is basically maintained in the core of particles forming a "core-shell" structure. Such surface reconstruction triggers a rapid capacity decay during the first 100 cycles between 2.7 and 4.3 V at 1 C or 3 C. On the contrary, the local lattice distortions are gradually accumulated throughout the whole NA particles with higher degrees of cation disorder (NA-HCD, 0.06≤m≤0.15) that lead to a slow capacity decay upon cycling.

Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9.

Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4+ T cell-derived IL-9 promotes the expansion of both CD11c+ and CD11c- interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1+ but not Arg1- lung macrophages to Il9r-/- mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy.

Patients with Lung Cancer of Different Racial Backgrounds Harbor Distinct Immune Cell Profiles.

Tumors accumulated with infiltrated immune cells (hot tumors) have a higher response rate to immune checkpoint blockade, when compared with those with minimal T-cell infiltration (cold tumors). We report here that patients with lung cancer with different racial backgrounds harbored distinct immune cell profiles in the tumor microenvironment. Compared with African Americans (AA), Caucasian Americans (CA) exhibited increased immune cell infiltration and vasculature, and increased survival. Changes of survival and immune profile were most pronounced among active smokers and nonsmokers, compared with former smokers and total patients. Neighborhood analysis showed that immune cells accumulated around cancer cells in CAs but not AAs. Our findings reveal intrinsic biological differences between AA and CA patients with lung cancer, suggesting that treatment plans should be tailored for patients with different racial backgrounds. Significance: We report biological racial differences among patients with lung cancer where Caucasians present a hot tumor microenvironment compared with cold tumor in AAs. Treatment plans should be customized to maximize therapeutic outcomes.

Virus-Mimic mRNA Vaccine for Cancer Treatment.

An effective therapeutic cancer vaccine should be empowered with the capacity to overcome the immunosuppressive tumor microenvironment. Here, the authors describe a mRNA virus-mimicking vaccine platform that is comprised of a phospholipid bilayer encapsulated with a protein-nucleotide core consisting of antigen-encoding mRNA molecules, unmethylated CpG oligonucleotides and positively charged proteins. In cell culture, VLVP potently stimulated bone marrow-derived dendritic cells (BMDCs) to express inflammatory cytokines that facilitated dendritic cell (DC) maturation and promoted antigen processing and presentation. In tumor-bearing mice, VLVP treatment stimulated proliferation of antigen-specific CD8+T cells in the lymphatic organs and T cell infiltration into the tumor bed, resulting in potent anti-tumor immunity. Cytometry by time of flight (CyTOF) analysis revealed that VLVP treatment stimulated a 5-fold increase in tumor-associated CD8+DCs and a 4-fold increase in tumorinfiltrated CD8+T cells, with concurrent decreases in tumor-associated bone marrow-derived suppressor cells and arginase 1- expressing suppressive DCs. Finally, CpG oligonucleotide is an essential adjuvant for vaccine activity. Inclusion of CpG not only maximized vaccine activity but also prevented PD-1 expression in T cells, serving the dual roles as a potent adjuvant and a checkpoint blockade agent.

Myeloid-derived suppressor cells as cellular immunotherapy in transplantation and autoimmune diseases.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which have been characterized for their immunosuppressive capacity through multiple mechanisms. These cells have been extensively studied in the field of tumor immunity. Emerging evidence has highlighted its essential role in maintaining immune tolerance in transplantation and autoimmunity. Because of their robust immune inhibitory activities, there has been growing interest in MDSC-based cellular therapy. Various pre-clinical studies have demonstrated that the adoptive transfer of MDCS represented a promising therapeutic strategy for immune-related disorders. In this review, we summarize relevant studies of MDSC-based cell therapy in transplantation and autoimmune diseases and discuss the challenges and future directions for clinical application of MDSC-based cell therapy.

ST2/MyD88 Deficiency Protects Mice against Acute Graft-versus-Host Disease and Spares Regulatory T Cells.

Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (HCT). Plasma levels of soluble membrane-bound ST2 (ST2) are elevated in human and murine aGVHD and correlated to type 1 T cells response. ST2 signals through the adapter protein MyD88. The role of MyD88 in T cells during aGVHD has yet to be elucidated. We found that knocking out MyD88 in the donor T cells protected against aGVHD independent of IL-1R and TLR4 signaling in two murine HCT models. This protection was entirely driven by MyD88-/- CD4 T cells. Transplanting donor MyD88-/- conventional T cells (Tcons) with wild-type (WT) or MyD88-/- regulatory T cells (Tregs) lowered aGVHD severity and mortality. Transcriptome analysis of sorted MyD88-/- CD4 T cells from the intestine 10 d post-HCT showed lower levels of Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, Batf, and Jak2 Transplanting donor ST2-/- Tcons with WT or ST2-/- Tregs showed a similar phenotype with what we observed when using donor MyD88-/- Tcons. Decreased ST2 was confirmed at the protein level with less secretion of soluble ST2 and more expression of ST2 compared with WT T cells. Our data suggest that Treg suppression from lack of MyD88 signaling in donor Tcons during alloreactivity uses the ST2 but not the IL-1R or TLR4 pathways, and ST2 represents a potential aGVHD therapeutic target sparing Tregs.

The mechanistic study behind suppression of GVHD while retaining GVL activities by myeloid-derived suppressor cells.

Graft-versus-host disease (GVHD) is a major barrier to the widespread use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for treating hematologic malignancies. Myeloid-derived suppressor cells (MDSCs) have been recognized as crucial immunosuppressive cells in various pathologic settings. Here, we investigated whether the unique functional properties of MDSCs could be harnessed to control allo-HSCT-associated GVHD. Using multiple murine GVHD/GVL models including both MHC-mismatched and miHA-mismatched, we demonstrated that treatment with CD115+ MDSCs efficiently suppressed GVHD but did not significantly impair graft-versus-leukemia (GVL) activity, leading to 80 and 67% protection in treated mice in GVHD and GVL models, respectively. The mechanism for this dissociation of GVHD from GVL, specifically the emergence of donor-derived NKG2D+ CD8 T cells with a memory phenotype in MDSC-treated recipient mice, was identified. NKG2D expression on donor T cells was required for eradication of allogeneic lymphoma cells. Furthermore, long-term surviving MDSC recipients that exhibited cytolytic activities against allogeneic leukemia cells had a significantly increased percentage of T regulatory cells and, more importantly, NKG2D+ CD8 T cells. These findings indicate that MDSCs can be used as a novel cell-based therapy to suppress GVHD while maintaining GVL activities through selective induction of NKG2D+ CD8 memory T cells.

Rorc restrains the potency of ST2+ regulatory T cells in ameliorating intestinal graft-versus-host disease.

Soluble stimulation-2 (ST2) is increased during graft-versus-host disease (GVHD), while Tregs that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3- T cells and Tregs that were collected and sorted from different Foxp3 reporter mice indicated that in mice that developed GVHD, ST2+ Tregs were thymus derived and predominantly localized to the intestine. ST2-/- Treg transplantation was associated with reduced total intestinal Treg frequency and activation. ST2-/- versus WT intestinal Treg transcriptomes showed decreased Treg functional markers and, reciprocally, increased Rorc expression. Rorc-/- T cells transplantation enhanced the frequency and function of intestinal ST2+ Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2-producing type 1 and increased IL-4/IL-10-producing type 2 T cells. Cotransfer of ST2+ Tregs sorted from Rorc-/- mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs, and decreased type 1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33-stimulated Tregs (TregIL-33) expressed higher amphiregulin and displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show that inverse expression of ST2 and RORγt in intestinal Tregs determines GVHD and that TregIL-33 has potential as a cellular therapy avenue for preventing GVHD.

Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity.

Tumor-associated myeloid cells maintain immunosuppressive microenvironments within tumors. Identification of myeloid-specific receptors to modulate tumor-associated macrophage and myeloid-derived suppressor cell (MDSC) functions remains challenging. The leukocyte immunoglobulin-like receptor B (LILRB) family members are negative regulators of myeloid cell activation. We investigated how LILRB targeting could modulate tumor-associated myeloid cell function. LILRB2 antagonism inhibited receptor-mediated activation of SHP1/2 and enhanced proinflammatory responses. LILRB2 antagonism also inhibited AKT and STAT6 activation in the presence of M-CSF and IL-4. Transcriptome analysis revealed that LILRB2 antagonism altered genes involved in cell cytoskeleton remodeling, lipid/cholesterol metabolism, and endosomal sorting pathways, as well as changed differentiation gene networks associated with inflammatory myeloid cells as opposed to their alternatively activated phenotype. LILRB2 blockade effectively suppressed granulocytic MDSC and Treg infiltration and significantly promoted in vivo antitumor effects of T cell immune checkpoint inhibitors. Furthermore, LILRB2 blockade polarized tumor-infiltrating myeloid cells from non-small cell lung carcinoma tumor tissues toward an inflammatory phenotype. Our studies suggest that LILRB2 can potentially act as a myeloid immune checkpoint by reprogramming tumor-associated myeloid cells and provoking antitumor immunity.

From proteomics to discovery of first-in-class ST2 inhibitors active in vivo.

Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.

Leukocyte immunoglobulin-like receptors in human diseases: an overview of their distribution, function, and potential application for immunotherapies.

Myeloid-derived suppressor cells (MDSCs), a population of immature myeloid cells expanded and accumulated in tumor-bearing mice and in patients with cancer, have been shown to mediate immune suppression and to promote tumor progression, thereby, posing a major hurdle to the success of immune-activating cancer therapies. MDSCs, like their healthy counterparts, such as monocytes/macrophages and granulocytes, express an array of costimulatory and coinhibitory molecules as well as myeloid activators and inhibitory receptors, such as leukocyte immunoglobulin-like receptors (LILR) A and B. This review summarizes current findings on the LILR family members in various diseases, their potential roles in the pathogenesis, and possible strategies to revert or enhance the suppressive function of MDSCs for the benefit of patients by targeting LILRs.

Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease.

Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA-transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA- transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT.

ST2 blockade reduces sST2-producing T cells while maintaining protective mST2-expressing T cells during graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a devastating complication after allogeneic hematopoietic cell transplantation (HCT). We previously identified high plasma soluble suppression of tumorigenicity 2 (sST2) as a biomarker of the development of GVHD and death. sST2 sequesters interleukin-33 (IL-33), limiting its availability to T cells expressing membrane-bound ST2 (mST2) [T helper 2 (TH2) cells and ST2(+)FoxP3(+) regulatory T cells]. We report that blockade of sST2 in the peritransplant period with a neutralizing monoclonal antibody (anti-ST2 mAb) reduced GVHD severity and mortality. We identified intestinal stromal cells and T cells as major sources of sST2 during GVHD. ST2 blockade decreased systemic interferon-γ, IL-17, and IL-23 but increased IL-10 and IL-33 plasma levels. ST2 blockade also reduced sST2 production by IL-17-producing T cells while maintaining protective mST2-expressing T cells, increasing the frequency of intestinal myeloid-derived suppressor cells, and decreasing the frequency of intestinal CD103 dendritic cells. Finally, ST2 blockade preserved graft-versus-leukemia activity in a model of green fluorescent protein (GFP)-positive MLL-AF9 acute myeloid leukemia. Our findings suggest that ST2 is a therapeutic target for severe GVHD and that the ST2/IL-33 pathway could be investigated in other T cell-mediated immune disorders with loss of tolerance.

[Influence of excessive complement activation on pathological process of acute graft versus host disease in mice].

This study was aimed to explore the influence of excessive complement activation on the pathological process of acute graft-versus-host disease (aGVHD) in mice. A murine model with aGVHD was established by injecting cell mixture containing splenocytes and bone marrow cells at 2:1 ratio from donor C57BL/6(H-2K(b)) mice into recipient BALB/c (H-2K(d)) mice within 4-6 hours after 8 Gy (60)Co γ-ray total body irradiation. The mice received syngeneic bone marrow transplantation were used as control group. After transplantation, the mice were monitored daily for body weight and mortality. At day 14, all mice were sacrificed and each liver was freshly dissociated for histological analysis. The hepatic mRNA abundance for complement components C3a and C5a as well as receptors for these two anaphylatoxin were tested by real-time quantitative PCR method. And the levels of C3a and C5a production in liver were detected by ELISA. The deposition of complement C3 in liver was determined by immunofluorescence staining using frozen section. The results indicated that as compared with syngeneic bone-marrow transplantation control group, experimental animals underwent aGVHD characterized by weight loss, depilation, diarrhea and lassitude. Interestingly, the hepatic mRNA expression for complement anaphylatoxin family member C3a and C5a as well as their receptors C3aR and C5aR1 in mice with aGVHD were significantly up-regulated in comparison with control group (p < 0.05). Consistently, the content of C3a and C5a in liver increased markedly in mice with aGVHD (p < 0.01). For animals ongoing aGVHD, complement component C3 depositions were observed in hepatic portal areas, around which massive inflammatory cell infiltration was also observed. It is concluded that in aGVHD animals, excessive complement activation occurs, and the activated complement components participate in pathological process of the aGVHD.