Exopolysaccharide-Treated Dendritic Cells Effectively Ameliorate Acute Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is a primary and often lethal complication of allogenic hematopoietic stem cell transplantation (HSCT). Prophylactic regimens for GVHD are given as standard pretransplantation therapy; however, up to 50% of these patients develop acute GVHD (aGVHD) and require additional immunosuppressive intervention. Using a mouse GVHD model, we previously showed that injecting mice with exopolysaccharide (EPS) from Bacillus subtilis prior to GVHD induction significantly increased 80-day survival after transplantation of complete allogeneic major histocompatibility complex-mismatched cells. To ask whether EPS might also inhibit GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of A2neg human peripheral blood mononuclear cells (PBMCs). Because we could not inject human donors with EPS, we transferred EPS-pretreated dendritic cells (DCs) to inhibit aGVHD. We derived these DCs from CD34+ human cord blood cells, treated them with EPS, and then injected them together with PBMCs into the NSG-HLA-A2 mice. We found that all mice that received untreated DCs were dead by day 35, whereas 25% of mice receiving EPS-treated DCs (EPS-DCs) survived. This DC cell therapy could be readily translatable to humans, because we can generate large numbers of human EPS-DCs and use them as an "off the shelf" treatment for patients undergoing HSCT.

Antigenic responses are hallmarks of fibrotic interstitial lung diseases independent of underlying etiologies.

Interstitial lung diseases (ILD) are heterogeneous conditions that may lead to progressive fibrosis and death of affected individuals. Despite diversity in clinical manifestations, enlargement of lung-associated lymph nodes (LLN) in fibrotic ILD patients predicts worse survival. Herein, we revealed a common adaptive immune landscape in LLNs of all ILD patients, characterized by highly activated germinal centers and antigen-activated T cells including regulatory T cells (Tregs). In support of these findings, we identified serum reactivity to 17 candidate auto-antigens in ILD patients through a proteome-wide screening using phage immunoprecipitation sequencing. Autoantibody responses to actin binding LIM protein 1 (ABLIM1), a protein highly expressed in aberrant basaloid cells of fibrotic lungs, were correlated with LLN frequencies of T follicular helper cells and Tregs in ILD patients. Together, we demonstrate that end-stage ILD patients have converging immune mechanisms, in part driven by antigen-specific immune responses, which may contribute to disease progression.

Gut microbiota modulates bleomycin-induced acute lung injury response in mice.

BACKGROUND: Airway instillation of bleomycin (BLM) in mice is a widely used, yet challenging, model for acute lung injury (ALI) with high variability in treatment scheme and animal outcomes among investigators. Whether the gut microbiota plays any role in the outcome of BLM-induced lung injury is currently unknown. METHODS: Intratracheal instillation of BLM into C57BL/6 mice was performed. Fecal microbiomes were analyzed by 16s rRNA amplicon and metagenomic sequencing. Germ-free mice conventionalization and fecal microbiota transfer between SPF mice were performed to determine dominant commensal species that are associated with more severe BLM response. Further, lungs and gut draining lymph nodes of the mice were analyzed by flow cytometry to define immunophenotypes associated with the BLM-sensitive microbiome. RESULTS: Mice from two SPF barrier facilities at the University of Chicago exhibited significantly different mortality and weight loss during BLM-induced lung injury. Conventionalizing germ-free mice with SPF microbiota from two different housing facilities recapitulated the respective donors' response to BLM. Fecal microbiota transfer from the facility where the mice had worse mortality into the mice in the facility with more survival rendered recipient mice more susceptible to BLM-induced weight loss in a dominant negative manner. BLM-sensitive phenotype was associated with the presence of Helicobacter and Desulfovibrio in the gut, decreased Th17-neutrophil axis during steady state, and augmented lung neutrophil accumulation during the acute phase of the injury response. CONCLUSION: The composition of gut microbiota has significant impact on BLM-induced wasting and death suggesting a role of the lung-gut axis in lung injury.

IRF4 expression by lung dendritic cells drives acute but not Trm cell-dependent memory Th2 responses.

Expression of the transcription factor interferon regulatory factor 4 (IRF4) is required for the development of lung conventional DCs type 2 (cDC2s) that elicit Th2 responses, yet how IRF4 functions in lung cDC2s throughout the acute and memory allergic response is not clear. Here, we used a mouse model that loses IRF4 expression after lung cDC2 development to demonstrate that mice with IRF4-deficient DCs display impaired memory responses to allergen. This defect in the memory response was a direct result of ineffective Th2 induction and impaired recruitment of activated effector T cells to the lung after sensitization. IRF4-deficient DCs demonstrated defects in their migration to the draining lymph node and in T cell priming. Finally, T cells primed by IRF4-competent DCs mediated potent memory responses independently of IRF4-expressing DCs, demonstrating that IRF4-expressing DCs are not necessary during the memory response. Thus, IRF4 controlled a program in mature DCs governing Th2 priming and effector responses, but IRF4-expressing DCs were dispensable during tissue-resident memory T cell-dependent memory responses.

A Gata3 enhancer necessary for ILC2 development and function.

The type 2 helper effector program is driven by the master transcription factor GATA3 and can be expressed by subsets of both innate lymphoid cells (ILCs) and adaptive CD4+ T helper (Th) cells. While ILC2s and Th2 cells acquire their type 2 differentiation program under very different contexts, the distinct regulatory mechanisms governing this common program are only partially understood. Here we show that the differentiation of ILC2s, and their concomitant high level of GATA3 expression, are controlled by a Gata3 enhancer, Gata3 +674/762, that plays only a minimal role in Th2 cell differentiation. Mice lacking this enhancer exhibited defects in several but not all type 2 inflammatory responses, depending on the respective degree of ILC2 and Th2 cell involvement. Our study provides molecular insights into the different gene regulatory pathways leading to the acquisition of the GATA3-driven type 2 helper effector program in innate and adaptive lymphocytes.

IL-33-mediated Eosinophilia Protects against Acute Lung Injury.

Pneumonia-induced lung injury and acute respiratory distress syndrome can develop because of an inappropriate inflammatory response to acute infections, leading to a compromised alveolar barrier. Recent work suggests that hospitalized patients with allergies/asthma are less likely to die of pulmonary infections and that there is a correlation between survival from acute respiratory distress syndrome and higher eosinophil counts; thus, we hypothesized that eosinophils associated with a type 2 immune response may protect against pneumonia-induced acute lung injury. To test this hypothesis, mice were treated with the type 2-initiating cytokine IL-33 intratracheally 3 days before induction of pneumonia with airway administration of a lethal dose of Staphylococcus aureus. Interestingly, IL-33 pretreatment promoted survival by inhibiting acute lung injury: amount of BAL fluid proinflammatory cytokines and pulmonary edema were both reduced, with an associated increase in oxygen saturation. Pulmonary neutrophilia was also reduced, whereas eosinophilia was strongly increased. This eosinophilia was key to protection; eosinophil reduction eliminated both IL-33-mediated protection against mortality and inhibition of neutrophilia and pulmonary edema. Together, these data reveal a novel role for eosinophils in protection against lung injury and suggest that modulation of pulmonary type 2 immunity may represent a novel therapeutic strategy.

Sirolimus augments hematopoietic stem and progenitor cell regeneration following hematopoietic insults.

The role of mammalian target of rapamycin and its suppressor sirolimus in the regulation of hematopoietic stem and progenitor cells (HSPCs) is controversial. We show here that sirolimus enhanced regeneration of HSPCs in mice exposed to sublethal total body irradiation (TBI) and other regenerative stressors. Sorted Lin- CD150+ bone marrow cells from sirolimus-treated TBI mice had increased expression of c-Kit and other hematopoietic genes. HSPCs from sirolimus-treated TBI mice were functionally competent when tested by competitive engraftment in vivo. Postradiation regeneration of HSPCs in mice treated with sirolimus was accompanied by decreased γ-H2AX levels detected by flow cytometry and increased expression of DNA repair genes by quantitative polymerase chain reaction. Reduction of cell death and DNA damage post-radiation by sirolimus was associated with enhanced clearance of cellular reactive oxygen species (ROS) in HSPCs. Increased HSPC recovery with sirolimus was also observed in mice injected with hematoxic agents, busulfan and 5-fluorouracil. In contrast, sirolimus showed no effect on HSPCs in normal mice at steady state, but stimulated HSPC expansion in mice carrying the Wv mutation at the c-Kit locus. In human to mouse xenotransplantation, sirolimus enhanced engraftment of irradiated human CD34+ cells. In summary, our results are consistent with sirolimus' acceleration of HSPC recovery in response to hematopoietic stress, associated with reduced DNA damage and ROS. Sirolimus might have clinical application for the treatment and prevention of hematopoietic injury.

Distinct Graft-Specific TCR Avidity Profiles during Acute Rejection and Tolerance.

Mechanisms implicated in robust transplantation tolerance at the cellular level can be broadly categorized into those that inhibit alloreactive T cells intrinsically (clonal deletion and dysfunction) or extrinsically through regulation. Here, we investigated whether additional population-level mechanisms control T cells by examining whether therapeutically induced peripheral transplantation tolerance could influence T cell populations' avidity for alloantigens. Whereas T cells with high avidity preferentially accumulated during acute rejection of allografts, the alloreactive T cells in tolerant recipients retained a low-avidity profile, comparable to naive mice despite evidence of activation. These contrasting avidity profiles upon productive versus tolerogenic stimulation were durable and persisted upon alloantigen re-encounter in the absence of any immunosuppression. Thus, peripheral transplantation tolerance involves control of alloreactive T cells at the population level, in addition to the individual cell level. Controlling expansion or eliminating high-affinity, donor-specific T cells long term may be desirable to achieve robust transplantation tolerance in the clinic.

PD-1 deficiency augments bone marrow failure in a minor-histocompatibility antigen mismatch lymphocyte infusion model.

Although PD-1 blockade has revolutionized cancer immunotherapy, immune-related adverse events (irAEs) present life-threatening complications. Recent reports of aplastic anemia (AA) as irAEs implicate PD-1/PD-L1 as important in preventing immune-mediated destruction of the hematopoietic niche. Infusion of PD-1-deficient (PD-1 knockout [KO]) lymph node (LN) cells into minor-antigen mismatched mice resulted in early mortality, as well as more severe bone marrow (BM) hypoplasia, anemia, and BM microarchitecture disruption in PD-1 KO LN-infused mice relative to mice that received B6 LN cell infusion. Mice that received PD-1 KO LN cells had more CD8+ T-cell infiltration of the BM and greater expansion of H60-specific CD8+ T cells than did their B6 LN-infused counterparts. In the spleen, CD8+ T cells were skewed to an effector memory phenotype, suggesting accelerated differentiation of PD-1 KO T cells. Our data suggest that PD-1 dysregulation has a role in murine BM failure and vigilance in irAE monitoring may be desirable to treat early AA and related cytopenias.

Rapamycin is highly effective in murine models of immune-mediated bone marrow failure.

Acquired aplastic anemia, the prototypical bone marrow failure disease, is characterized by pancytopenia and marrow hypoplasia. Most aplastic anemia patients respond to immunosuppressive therapy, usually with anti-thymocyte globulin and cyclosporine, but some relapse on cyclosporine withdrawal or require long-term administration of cyclosporine to maintain blood counts. In this study, we tested efficacy of rapamycin as a new or alternative treatment in mouse models of immune-mediated bone marrow failure. Rapamycin ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of cyclosporine. Rapamycin effectively reduced Th1 inflammatory cytokines interferon-γ and tumor necrosis factor-α, increased the Th2 cytokine interleukin-10, stimulated expansion of functional regulatory T cells, eliminated effector CD8+ T cells (notably T cells specific to target cells bearing minor histocompatibility antigen H60), and preserved hematopoietic stem and progenitor cells. Rapamycin, but not cyclosporine, reduced the proportion of memory and effector T cells and maintained a pool of naïve T cells. Cyclosporine increased cytoplasmic nuclear factor of activated T-cells-1 following T-cell receptor stimulation, whereas rapamycin suppressed phosphorylation of two key signaling molecules in the mammalian target of rapamycin pathway, S6 kinase and protein kinase B. In summary, rapamycin was an effective therapy in mouse models of immune-mediated bone marrow failure, acting through different mechanisms to cyclosporine. Its specific expansion of regulatory T cells and elimination of clonogenic CD8+ effectors support its potential clinical utility in the treatment of aplastic anemia.