Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+ T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Genetic modifications designed for xenotransplantation attenuate sialoadhesin-dependent binding of human erythrocytes to porcine macrophages.

The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.

Extensive germline genome engineering in pigs.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Keratinocyte-Derived Chemokines Orchestrate T-Cell Positioning in the Epidermis during Vitiligo and May Serve as Biomarkers of Disease.

Vitiligo is an autoimmune disease of the skin that results in the destruction of melanocytes and the clinical appearance of white spots. Disease pathogenesis depends on IFN-γ and IFN-γ-induced chemokines to promote T-cell recruitment to the epidermis where melanocytes reside. The skin is a complex organ, with a variety of resident cell types. We sought to better define the microenvironment and distinct cellular contributions during autoimmunity in vitiligo, and we found that the epidermis is a chemokine-high niche in both a mouse model and human vitiligo. Analysis of chemokine expression in mouse skin showed that CXCL9 and CXCL10 expression strongly correlate with disease activity, whereas CXCL10 alone correlates with severity, supporting them as potential biomarkers for following disease progression. Further studies in both our mouse model and human patients showed that keratinocytes were the major chemokine producers throughout the course of disease, and functional studies using a conditional signal transducer and activator of transcription (STAT)-1 knockout mouse showed that IFN-γ signaling in keratinocytes was critical for disease progression and proper autoreactive T-cell homing to the epidermis. In contrast, epidermal immune cell populations including endogenous T cells, Langerhans cells, and γδ T cells were not required. These results have important clinical implications, because topical therapies that target IFN-γ signaling in keratinocytes could be safe and effective new treatments, and skin expression of these chemokines could be used to monitor disease activity and treatment responses.

Immunomodulatory activity of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) were discovered as a rare population of non-hematopoietic stem cells that reside in the bone marrow and interact closely with hematopoietic stem cells to support their growth and differentiation. MSCs are multipotent cells that have the ability to differentiate into cells of the mesenchymal lineage including adipocytes, osteocytes and chondrocytes and they have been reported to home to areas of tissue injury and participate in tissue repair. More recently, MSCs have also been described to possess anti-inflammatory and immunomodulatory properties that can affect multiple arms of the immune system. MSCs have been shown to inhibit T and B cell proliferation, downregulate the lytic activity of cytotoxic T lymphocytes and NK cells, inhibit the maturation and antigen-presenting function of dendritic cells and modulate macrophage function through both contact-dependent and contact-independent mechanisms. The administration of MSCs in models of autoimmune disease such as collagen-induced arthritis, EAE and autoimmune diabetes has provided additional evidence for an immunoregulatory role of MSCs supporting their use in controlling autoimmunity. The administration of allogeneic MSCs as immunosuppressive agents represents a viable approach as they appear to be largely non-immunogenic and clinical trials with allogeneic MSCs are currently underway in graftversus- host disease, Crohn's disease and type I diabetes indications. The immunomodulatory properties, mechanism of action and potential clinical utility of MSCs are reviewed herein.

IgM monomers accelerate disease manifestations in autoimmune-prone Fas-deficient mice.

The monomeric form of IgM, also known as low molecular weight IgM, is found in increased concentrations in patients chronically infected with a variety of viral and bacterial pathogens or suffering from various autoimmune diseases. Whether monomeric IgM contributes to the disease process, however, is not known. To address this question, transgenic mice were created that secreted elevated levels of IgM monomers. In normal mice (C57BL/6), the presence of IgM monomers did not alter the composition of the immune system significantly: lymphocyte subsets and serum antibody levels were normal, with the exception of increased levels of IgM due to the presence of the monomers. Immune responses also appeared to be normal. Transgenic mice did develop antinuclear antibodies (ANA) earlier than non-transgenic littermates, but did not develop further indications of autoimmune disease. When the transgene was expressed in the autoimmune-prone strain of mice, B6.MRL-Tnfrsf6(lpr) (B6/lpr), these mice developed autoimmune manifestations more rapidly than non-transgenic littermates, including hypergammaglobulinemia, splenomegaly, and ANA production. Transgenic mice also displayed earlier evidence of immune complex deposition in the kidneys. From these results, we conclude that monomeric IgM does not induce autoimmune disease, but its presence can accelerate the onset of autoimmune manifestations in otherwise autoimmune prone animals.

Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells.

Secreted IgM and complement are important mediators in the optimal initiation of primary T-dependent humoral immune responses. Secreted IgM serves as a natural adjuvant by enhancing the immunogenicity of protein antigens, perhaps as a result of IgM's ability to facilitate antigen deposition onto follicular dendritic cells (FDCs) and promote rapid germinal center (GC) formation. To understand how IgM enhances adaptive immune responses, we investigated the mechanism by which IgM-containing immune complexes (IgM-IC) are transported to FDCs as a first step in GC formation. We demonstrate that IgM-IC localize first to the splenic marginal zone (MZ) where the IgM-IC bind MZ B cells in a complement and complement receptor (CR1/2) dependent process. MZ B cells then transport the IgM-IC into the follicle for deposition onto FDCs. Mice with reduced numbers of MZ B cells trap IgM-IC on FDC less efficiently, whereas mice with reduced numbers of follicular B cells trap IgM-IC normally. The functional elimination of MZ B cells abrogates the ability of FDCs to trap IgM-IC. Transfer of B cells with associated IgM-IC into naive mice results in deposition of IgM-IC onto FDC by MZ B cells. The results demonstrate an IgM and complement-dependent role for MZ B cells in the fate of antigen early in the initial phases of T-dependent immune responses. The data also establish an important role for CR1/2 on MZ B cells in the efficient binding and transport of IgM-IC to FDCs, which we suggest is an important first step in initiating adaptive immune responses.

Synergistic roles of IgM and complement in antigen trapping and follicular localization.

We have investigated the roles of IgM and complement (C) in the enhancement of primary immune responses and the localization of protein antigen (Ag) in the spleen. Pentameric but not monomeric IgM enhances antibody (Ab) responses in both wild-type and secretory micro-deficient (micro(s) (-/-)) mice, indicating that a single IgM clone is sufficient as long as it activates C. Ag localizes on follicular dendritic cells (FDC) within 16 h after injection of immune complexes (IC) containing pentameric but not monomeric IgM. Surprisingly, pentameric IgM-containing IC were trapped in spleens of C3-depleted and Cr2-deficient mice. However, the IC were found only in the marginal zone (MZ), associated predominantly with MZ macrophages. IC were also detected in the MZ in normal mice within 1 h after injection, but associated with other cells in addition to MZ macrophages. The results demonstrate an important role for pentameric IgM in the initiation of Ag trapping, a step independent of C3 activation and of the interaction of IC with CR1 and CR2. The data also provide direct evidence that C3 activation is required for the next phase of localization, in which Ag moves from the MZ to FDC, with consequent enhancement of specific immune responses.