Prevention of chronic graft-versus-host disease (cGVHD) remains a major challenge in allogeneic hematopoietic cell transplantation (HCT), due to limited understanding of cGVHD pathogenesis and lack of appropriate animal models. Here, we report that, in classical acute GVHD models with C57BL/6 donors and MHC-mismatched BALB/c recipients and with C3H.SW donors and MHC-matched C57BL/6 recipients, GVHD recipients surviving for more than 60 days after HCT developed cGVHD characterized by cutaneous fibrosis, tissue damage in the salivary gland and the presence of serum autoantibodies. Donor CD8+ T cells were more potent than CD4+ T cells for inducing cGVHD. The recipient thymus and de novo-generated, donor-derived CD4+ T cells were required for induction of cGVHD by donor CD8+ T cells but not by donor CD4+ T cells. Donor CD8+ T cells preferentially damaged recipient medullary thymic epithelial cells and impaired negative selection, resulting in production of autoreactive CD4+ T cells that perpetuated damage to the thymus and augmented the development of cGVHD. Short-term anti-CD4 monoclonal antibody treatment early after HCT enabled recovery from thymic damage and prevented cGVHD. These results demonstrate that donor CD8+ T cells cause cGVHD solely through thymic-dependent mechanisms, while CD4+ T cells can cause cGVHD through either thymic-dependent or independent mechanisms.

Programmed death ligand-1 (PD-L1) interacts with programmed death-1 (PD-1) and the immunostimulatory molecule CD80 and functions as a checkpoint to regulate immune responses. The interaction of PD-L1 with CD80 alone has been shown to exacerbate the severity of graft-versus-host disease (GVHD), whereas costimulation of CD80 and PD-1 ameliorates GVHD. Here we have demonstrated that temporary depletion of donor CD4+ T cells early after hematopoietic cell transplantation effectively prevents GVHD while preserving strong graft-versus-leukemia (GVL) effects in allogeneic and xenogeneic murine GVHD models. Depletion of donor CD4+ T cells increased serum IFN-γ but reduced IL-2 concentrations, leading to upregulation of PD-L1 expression by recipient tissues and donor CD8+ T cells. In GVHD target tissues, the interactions of PD-L1 with PD-1 on donor CD8+ T cells cause anergy, exhaustion, and apoptosis, thereby preventing GVHD. In lymphoid tissues, the interactions of PD-L1 with CD80 augment CD8+ T cell expansion without increasing anergy, exhaustion, or apoptosis, resulting in strong GVL effects. These results indicate that the outcome of PD-L1-mediated signaling in CD8+ T cells depends on the presence or absence of CD4+ T cells, the nature of the interacting receptor expressed by CD8+ T cells, and the tissue environment in which the signaling occurs.

• Antibodies produced by donor B cells are required for thymic and lymphoid damage in mice with chronic GVHD.• Antibody-producing donor B cells associate with infiltration of Th17 cells in the skin and perpetuation of cutaneous chronic GVHD in mice.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome mediated by pathogenic CD4+ T and B cells, but the function of extrafollicular and germinal center CD4+ T and B interactions in cGVHD pathogenesis remains largely unknown. Here we show that extrafollicular CD4+ T and B interactions are sufficient for inducing cGVHD, while germinal center formation is dispensable. The pathogenesis of cGVHD is associated with the expansion of extrafollicular CD44hiCD62loPSGL-1loCD4+ (PSGL-1loCD4+) T cells. These cells express high levels of ICOS, and the blockade of ICOS/ICOSL interaction prevents their expansion and ameliorates cGVHD. Expansion of PSGL-1loCD4+ T cells is also prevented by BCL6 or Stat3 deficiency in donor CD4+ T cells, with the induction of cGVHD ameliorated by BCL6 deficiency and completely suppressed by Stat3 deficiency in donor CD4+ T cells. These results support that Stat3- and BCL6-dependent extrafollicular CD4+ T and B interactions play critical functions in the pathogenesis of cGVHD.

The circadian system regulates numerous physiological processes including immune responses. Here, we show that mice deficient of the circadian clock genes and [ double knockout (DKO)] develop an autoimmune phenotype including high serum IgG concentrations, serum antinuclear antibodies, and precipitation of IgG, IgM, and complement 3 in glomeruli and massive infiltration of leukocytes into the lungs and kidneys. Flow cytometry of lymphoid organs revealed decreased pre-B cell numbers and a higher percentage of mature recirculating B cells in the bone marrow, as well as increased numbers of B2 B cells in the peritoneal cavity of DKO mice. The B cell receptor (BCR) proximal signaling pathway plays a critical role in autoimmunity regulation. Activation of DKO splenic B cells elicited markedly enhanced tyrosine phosphorylation of cellular proteins compared with cells from control mice, suggesting that overactivation of the BCR-signaling pathway may contribute to the autoimmunity phenotype in the DKO mice. In addition, the expression of, the deficiency of which contributes to the pathogenesis of systemic lupus erythematosus, was significantly down-regulated in DKO B cells. Our results suggest that B cell development, the BCR-signaling pathway, and expression are regulated by circadian clock CRY proteins and that their dysregulation through loss of CRY contributes to autoimmunity.

The pathogenesis of human H5N1 influenza remains poorly understood and controversial. 'Cytokine storm' has been hypothesized to be the main cause of the severity of this disease. However, the significance of this hypothesis has been called into question by a recent report, which demonstrates that inhibition of the cytokine response did not protect against lethal H5N1 influenza infection in mice. Here we showed discrepant findings in two adult H5N1 autopsies and a fetus obtained at autopsy which also raise doubt about this hypothesis. Antigens of 10 cytokines/chemokines which were found to be significantly elevated in previous H5N1-infected patients and in vitro experiments, and mRNA of eight of these, were absent from the lungs of a pregnant woman and her fetus. In contrast, antigens of seven cytokines/chemokines and mRNA of six of these were found to be increased in the lungs of a male autopsy. The cells expressing these cytokines and chemokines were identified as type II pneumocytes, bronchial epithelial cells, macrophages and vascular endothelial cells. Levels of cytokines and chemokines in the serum of the male case were also significantly higher than those of infectious (infection other than by H5N1) and non-infectious controls. In comparison with results from our previous study, it appeared that the male case had increased cytokine/chemokine expression but reduced viral load, while the pregnant female had diminished cytokine/chemokine expression but a significantly increased viral load in the lungs. These disparate findings in these two cases suggest that 'cytokine storm' alone could not be a sufficient explanation for the severe lung injury of this newly emerging disease.

Interactions of B7H1 (PD-L1) with its two ligands, PD-1 and CD80, on T cells play a pivotal role in controlling T cell activation, proliferation, anergy, and apoptosis. However, the interactions between the two pathways remain unknown. Using an alloimmune response model of graft-versus-host disease (GVHD), we report here that: 1) Comparison of proliferation and apoptosis of wild-type (WT) and PD-1−/− CD4+ conventional T (Tcon) cells in WT and B7H1−/− recipients has revealed that B7H1/CD80 interaction per se augments T cell proliferation, and this interaction augments T cell apoptosis mediated by B7H1/PD-1 interaction. This observation was recapitulated in an in vitro mixed lymphocyte reaction assay. 2) Specific blockade of the B7H1/CD80 axis by anti-B7H1 mAb reduces WT-alloreactive Tcon cell proliferation, IL-2 production, expression of PD-1, and apoptosis, resulting in worsening GVHD. In contrast, specific blockade of B7H1/CD80 interaction reduces donor PD-1−/− Tcon cell proliferation without impact on apoptosis, resulting in ameliorating GVHD. 3) B7H1 fused to an immunoglobulin Fc domain (B7H1-Ig), when produced in vivo by hydrodynamic injection of B7H1-Ig plasmid, ameliorates GVHD by augmenting proliferation and apoptosis of WT- alloreactive Tcon cells. Conversely, B7H1-Ig treatment has no impact on apoptosis but augments PD-1−/− T cell proliferation and worsens GVHD. These results indicate that B7H1/CD80 interaction augments Tcon cell proliferation, IL-2 production, and expression of PD-1, which leads to increased apoptosis mediated by the B7H1/PD1 pathway. Additionally, by engaging both PD-1 and CD80, B7H1-Ig can be a powerful therapeutic reagent for down-regulating the T cell immune response.

It has been proposed that immune injury is the central mechanism of pathogenesis of the infectious disease, severe acute respiratory syndrome (SARS). To gain a better understanding of immune injury in the spleen, we investigated the number and distribution of various immune cell types in the spleens of SARS patients. We performed autopsies on six confirmed SARS cases, with six normal subjects as controls; spleen samples from these autopsies were examined with hematoxylin and eosin (H&E) sections, in situ hybridization for SARS virus genomic sequences, and immunohistochemistry with seven monoclonal antibodies to five cell types. The number and distribution of these cells were measured and analyzed using an image analysis system. SARS genomic sequences were detected in all SARS spleens. The SARS spleens all had severe damage to the white pulp and showed an alteration of the normal distribution of various cell types. Immunocytes in the red pulp were decreased by 68.0-90.7% except for CD68+ macrophages and human leukocyte antigen (HLA)-DR positive antigen-presenting cells (APC), which were decreased to a lesser degree. On average, CD68+ macrophages were increased in size by 2.21-fold. We hypothesize that the collapse of the splenic immune system plays a key role in the clinical outcome of these patients.