A Simple Cuff Technique for Murine Left Lung Transplantation.

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an efficient and highly reliable "cuff technique" of transplantation. This article describes a sophisticated and comprehensive method for orthotopic lung transplantation in a vascularized orthotopic lung model, representing the most physiologic and clinically relevant model of mouse lung transplantation to date. The transplantation process consists of two distinct stages: donor harvest and subsequent implantation into the recipient. The method has been successfully mastered, and with several months of sufficient training, a skilled practitioner can perform the procedure in approximately 90 min from skin-to-skin. Surprisingly, once individuals overcome the initial learning curve, the survival rate during the perioperative period approaches nearly 100%. The mouse model allows for the use of multiple commercially available transgenic and mutant strains of mice, enabling the study of tolerance and rejection. Additionally, the unique features of this model make it a valuable tool for investigating tumor biology and immunology.

Monitoring regulatory T cells as a prognostic marker in lung transplantation.

Lung transplantation is the major surgical procedure, which restores normal lung functioning and provides years of life for patients suffering from major lung diseases. Lung transplant recipients are at high risk of primary graft dysfunction, and chronic lung allograft dysfunction (CLAD) in the form of bronchiolitis obliterative syndrome (BOS). Regulatory T cell (Treg) suppresses effector cells and clinical studies have demonstrated that Treg levels are altered in transplanted lung during BOS progression as compared to normal lung. Here, we discuss levels of Tregs/FOXP3 gene expression as a crucial prognostic biomarker of lung functions during CLAD progression in clinical lung transplant recipients. The review will also discuss Treg mediated immune tolerance, tissue repair, and therapeutic strategies for achieving in-vivo Treg expansion, which will be a potential therapeutic option to reduce inflammation-mediated graft injuries, taper the toxic side effects of ongoing immunosuppressants, and improve lung transplant survival rates.

Loss of stromal cell Thy-1 plays a critical role in lipopolysaccharide induced chronic lung allograft dysfunction.

BACKGROUND: Long-term survival of lung transplants lags behind other solid organs due to early onset of a fibrotic form of chronic rejection known as chronic lung allograft dysfunction (CLAD). Preventing CLAD is difficult as multiple immunologic and physiologic insults contribute to its development. Targeting fibroblast activation, which is the final common pathway leading to CLAD, offers the opportunity to ameliorate fibrosis irrespective of the initiating insult. Thy-1 is a surface glycoprotein that controls fibroblast differentiation and activation. METHODS: To study the role of Thy-1 in CLAD, we utilized the minor antigen mismatched C57BL/6 (B6wild-type) or B6Thy-1-/-→C57BL/10 (B10) model of murine orthotopic lung transplantation with postoperative bacterial infection modeled by intratracheal lipopolysaccharide (LPS) administration. The effects of LPS on Thy-1 expression, proliferation, and gene expression were assessed in fibroblasts in vitro and the therapeutic potential of Thy-1 replacement was assessed in vivo. RESULTS: More severe CLAD was evident in B6Thy-1-/- →B10 grafts compared to B6wild-type →B10 grafts. LPS further accentuated fibrosis in B6wild-type →B10 grafts with some, but limited, effects on B6Thy-1-/- →B10 grafts. LPS contributed to Thy-1 loss from Thy-1(+) fibroblasts in vitro due to a decrease in mRNA expression. In addition, LPS promoted proliferation and upregulation of multiple inflammatory pathways in Thy-1(-) fibroblasts by gene expression analysis. Most importantly, replacement of Thy-1 through exogenous administration ameliorated the fibrotic phenotype post-LPS mediated modeling of infection. CONCLUSIONS: Our findings suggest that the loss of Thy-1 on fibroblasts is a previously unrecognized cause of CLAD and its replacement may offer therapeutic applications for amelioration of this disease post-transplantation in the setting of infectious stress responses.

Ischemia reperfusion injury facilitates lung allograft acceptance through IL-33-mediated activation of donor-derived IL-5 producing group 2 innate lymphoid cells.

Pathways regulating lung alloimmune responses differ from most other solid organs and remain poorly explored. Based on our recent work identifying the unique role of eosinophils in downregulating lung alloimmunity, we sought to define pathways contributing to eosinophil migration and homeostasis. Using a murine lung transplant model, we have uncovered that immunosuppression increases eosinophil infiltration into the allograft in an IL-5-dependent manner. IL-5 production depends on immunosuppression-mediated preservation of donor-derived group 2 innate lymphoid cells (ILC2). We further describe that ischemia reperfusion injury upregulates the expression of IL-33, which functions as the dominant and nonredundant mediator of IL-5 production by graft-resident ILC2. Our work thus identifies unique cellular mechanisms that contribute to lung allograft acceptance. Notably, ischemia reperfusion injury, widely considered to be solely deleterious to allograft survival, can also downregulate alloimmune responses by initiating unique pathways that promote IL-33/IL-5/eosinophil-mediated tolerance.

Solving the Conundrum of Eosinophils in Alloimmunity.

Eosinophils are bone-marrow-derived granulocytes known for their ability to facilitate clearance of parasitic infections and their association with asthma and other inflammatory diseases. The purpose of this review is to discuss the currently available human observational and animal experimental data linking eosinophils to the immunologic response in solid organ transplantation. First, we present observational human studies that demonstrate a link between transplantation and eosinophils yet were unable to define the exact role of this cell population. Next, we describe published experimental models and demonstrate a defined mechanistic role of eosinophils in downregulating the alloimmune response to murine lung transplants. The overall summary of this data suggests that further studies are needed to define the role of eosinophils in multiple solid organ allografts and points to the possibility of manipulating this cell population to improve graft survival.

Retargeting IL-2 Signaling to NKG2D-Expressing Tumor-Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy.

Ex vivo expansion followed by reinfusion of tumor-infiltrating leukocytes (TILs) has been used successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine IL-2 to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 after reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic because of its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. In this study, we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high-affinity IL-2R for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident CTLs, such as CD8+ T cells, NK cells, and γδT cells, whereas wild-type IL-2 provides a growth advantage for CD4+Foxp3+ T cells as well as myeloid cells. OMCPmutIL-2-expanded CTLs express higher levels of tumor-homing receptors, such as LFA-1, CD49a, and CXCR3, which correlate with TIL localization to the tumor bed after i.v. injection. Consistent with this, OMCPmutIL-2-expanded TILs provided superior tumor control compared with those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D-expressing CTLs rather than indiscriminate expansion of all TILs.

Deciphering the role of eosinophils in solid organ transplantation.

Eosinophils are rare granulocytes that belong to the innate arm of the immune system. This cell population is traditionally defined as a destructive and cytotoxic mediator in asthma and helminth infection. Limited data in transplantation have suggested that eosinophils play a similar role in potentiating deleterious organ inflammation and immunologic rejection. Contrary to this long-held notion, recent data have uncovered the possibility that eosinophils play an alternative role in immune homeostasis, defense against a wide range of pathogens, as well as downregulation of deleterious inflammation. Specifically, translational data from small animal models of lung transplantation have demonstrated a critical role for eosinophils in the downregulation of alloimmunity. These findings shed new light on the unique immunologic features of the lung allograft and demonstrate that environmental polarization may alter the phenotype and function of leukocyte populations previously thought to be static in nature. In this review, we provide an update on eosinophils in the homeostasis of the lung as well as other solid organs.

Vendor-specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4+ Foxp3+ regulatory T cell levels.

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4+ Foxp3+ regulatory T cells (Tregs ) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad-spectrum antibiotics decreases Tregs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3-driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of Tregs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through Treg -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period.

Eosinophils downregulate lung alloimmunity by decreasing TCR signal transduction.

Despite the accepted notion that granulocytes play a universally destructive role in organ and tissue grafts, it has been recently described that eosinophils can facilitate immunosuppression-mediated acceptance of murine lung allografts. The mechanism of eosinophil-mediated tolerance, or their role in regulating alloimmune responses in the absence of immunosuppression, remains unknown. Using lung transplants in a fully MHC-mismatched BALB/c (H2d) to C57BL/6 (H2b) strain combination, we demonstrate that eosinophils downregulate T cell-mediated immune responses and play a tolerogenic role even in the absence of immunosuppression. We further show that such downregulation depends on PD-L1/PD-1-mediated synapse formation between eosinophils and T cells. We also demonstrate that eosinophils suppress T lymphocyte responses through the inhibition of T cell receptor/CD3 (TCR/CD3) subunit association and signal transduction in an inducible NOS-dependent manner. Increasing local eosinophil concentration, through administration of intratracheal eotaxin and IL-5, can ameliorate alloimmune responses in the lung allograft. Thus, our data indicate that eosinophil mobilization may be utilized as a novel means of lung allograft-specific immunosuppression.

Modulation of NKG2D, NKp46, and Ly49C/I facilitates natural killer cell-mediated control of lung cancer.

Natural killer (NK) cells play a critical role in controlling malignancies. Susceptibility or resistance to lung cancer, for example, specifically depends on NK cell function. Nevertheless, intrinsic factors that control NK cell-mediated clearance of lung cancer are unknown. Here we report that NK cells exposed to exogenous major histocompatibility class I (MHCI) provide a significant immunologic barrier to the growth and progression of malignancy. Clearance of lung cancer is facilitated by up-regulation of NKG2D, NKp46, and other activating receptors upon exposure to environmental MHCI. Surface expression of the inhibitory receptor Ly49C/I, on the other hand, is down-regulated upon exposure to tumor-bearing tissue. We thus demonstrate that NK cells exhibit dynamic plasticity in surface expression of both activating and inhibitory receptors based on the environmental context. Our data suggest that altering the activation state of NK cells may contribute to immunologic control of lung and possibly other cancers.