TIM-3 is constitutively expressed on subsets of macrophages and dendritic cells. Its expression on other cells of the innate immune system and its role in fetomaternal tolerance has not yet been explored. Here we investigate the role of TIM-3 expressing innate immune cells in the regulation of tolerance at the fetomaternal interface (FMI) using an allogeneic mouse model of pregnancy. Blockade of TIM-3 results in accumulation of inflammatory granulocytes and macrophages at the utero-placental interface and up regulation of pro-inflammatory cytokines. Furthermore, TIM-3 blockade inhibits the phagocytic potential of uterine macrophages resulting in a build up of apoptotic bodies at the utero-placental interface that elicits a local immune response. In response to inflammatory cytokines, Ly-6ChiGneg M-MDSCs (monocytic myeloid derived suppressor cells) expressing iNOS and arginase 1 are induced. However, these suppressive cells fail to down-regulate the inflammatory cascade induced by inflammatory granulocytes (Ly-6Cint Ghi) and apoptotic cells; the increased production of IFNγ and TNFα by inflammatory granulocytes leads to abrogation of tolerance at the fetomaternal interface and fetal rejection. These data highlight the interplay between cells of the innate immune system at the FMI and their influence on successful pregnancy in mice.

The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti-inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFNγ with induction of indoleamine 2,3-dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3Kα pathways interplay regulates IFNγ-induced IDO production in MSC. Chemical phosphoinositide 3-kinase (PI3K) pan-inhibition, PI3Kα-specific inhibition or shRNA knockdown diminished IFNγ-induced IDO production. This effect involved PI3Kα-mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3Kα mutant failed to induce basal IDO production, but shifted MSC into an MSC2-like phenotype by strongly enhancing IDO production in response to IFNγ as compared to controls. STAT1 overexpression strongly enhanced MSC-mediated T-cell suppression. The same effect could be induced using short-term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype.

Clinical trials investigating mesenchymal stromal cell (MSC) therapy for bronchopulmonary dysplasia have been initiated; however, the optimal delivery route and functional effects of MSC therapy in newborns remain incompletely established. We studied the morphologic and functional effects of intranasal versus i.p. MSC administration in a rodent model of neonatal lung injury. Cultured human cord tissue MSCs (0.1, 0.5, or 1 × 10(6) cell per pup) were given intranasally or i.p. to newborn severe combined immunodeficiency-beige mice exposed to 90% O2 from birth; sham controls received an equal volume of phosphate-buffered saline. Lung mechanics, engraftment, lung growth, and alveolarization were evaluated 8 weeks after transplantation. High-dose i.p. MSC administration to newborn mice exposed to 90% O2 resulted in the restoration of normal lung compliance, elastance, and pressure-volume loops (tissue recoil). Histologically, high-dose i.p. MSC administration was associated with alveolar septal widening, suggestive of interstitial matrix modification. Intranasal MSC or lower-dose i.p. administration had no significant effects on lung function or alveolar remodeling. Pulmonary engraftment was rare in all the groups. These findings suggest that high-dose systemic administration of human cultured MSCs can restore normal compliance in neonatally injured lungs, possibly by paracrine modulation of the interstitial matrix. Intranasal delivery had no obvious pulmonary effects.

We encapsulated cyclosporine A (CsA) in poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (PEG-PLGA) nanoparticles (NPs) by nanoprecipitation of CsA and PEG-PLGA. The resulting CsA/PEG-PLGA-NPs were <100 nm in diameter with a narrow particle size distribution. The NP size could be controlled by tuning the polymer concentration, solvent, or water/solvent ratio during formulation. The PEGylated NPs maintained non-aggregated in salt solution. Solid NPs lyoprotected with bovine serum albumin were prepared for the convenience of storage and transportation. The release kinetics of CsA (55.6% released on Day 1) showed potential for maintaining therapeutic CsA concentrations in vivo. In T-cell assays, both free CsA and CsA/PEG-PLGA-NPs suppressed T-cell proliferation and production of inflammatory cytokines dose dependently. In a mixed lymphocyte reaction assay, the IC50 values for free CsA and CsA/PEG-PLGA-NPs were found to be 30 and 35 ng/mL, respectively. This nanoparticulate CsA delivery technology constitutes a strong basis for future targeted delivery of immunosuppressive drugs with improved efficiency and potentially reduced toxicity.

Type 1 diabetes (T1D) remains a major health problem worldwide, with a steadily rising incidence yet no cure. Phosphoinositide 3-kinase-γ (PI3Kγ), a member of a family of lipid kinases expressed primarily in leukocytes, has been the subject of substantial research for its role in inflammatory diseases. However, the role of PI3Kγ inhibition in suppressing autoimmune T1D remains to be explored. We tested the role of the PI3Kγ inhibitor AS605240 in preventing and reversing diabetes in NOD mice and assessed the mechanisms by which this inhibition abrogates T1D. Our data indicate that the PI3Kγ pathway is highly activated in T1D. In NOD mice, we found upregulated expression of phosphorylated Akt (PAkt) in splenocytes. Notably, T regulatory cells (Tregs) showed significantly lower expression of PAkt compared with effector T cells. Inhibition of the PI3Kγ pathway by AS605240 efficiently suppressed effector T cells and induced Treg expansion through the cAMP response element-binding pathway. AS605240 effectively prevented and reversed autoimmune diabetes in NOD mice and suppressed T-cell activation and the production of inflammatory cytokines by autoreactive T cells in vitro and in vivo. These studies demonstrate the key role of the PI3Kγ pathway in determining the balance of Tregs and autoreactive cells regulating autoimmune diabetes.

Regulatory T-cells (Tregs) play a pivotal role in the maintenance of immune tolerance and hold great promise as cell therapy for a variety of immune-mediated diseases. However, the cellular mechanisms that regulate Treg maintenance and homeostasis have yet to be fully explored. While Tregs express Granzyme-B (GrB) to suppress effector T-cells via direct-killing, the mechanisms by which they protect themselves from GrB-mediated self-inflicted damage are unknown. We show, for the first time, that both iTregs and nTregs increase their intracellular expression of GrB and its endogenous inhibitor, Serine Protease Inhibitor-6 (Spi6) upon activation. Sub-cellular fractionation and measurement of GrB activity in the cytoplasm of Tregs show that activated Spi6−/− Tregs had significantly higher cytoplasmic GrB activity. We observed an increase in GrB-mediated apoptosis in Spi6−/− nTregs and impaired suppression of alloreactive T-cells in vitro. Spi6−/− Tregs were rescued from apoptosis by the addition of a GrB inhibitor (Z-AAD-CMK) in vitro. Furthermore, adoptive transfer experiments showed that Spi6−/− nTregs were less effective than WT nTregs in suppressing Graft-versus-host-disease (GVHD) due to their impaired survival, as shown in our in vivo bioluminescence imaging. Finally, Spi6-deficient recipients rejected MHC class II-mismatch heart allografts at a much faster rate and showed a higher rate of apoptosis among Tregs, as compared to WT recipients. Our data demonstrate, for the first time, a novel role for Spi6 in Treg homeostasis by protecting activated Tregs from GrB-mediated injury. These data could have significant clinical implications for Treg-based therapy in immune-mediated diseases.

Whereas DC have increasingly been recognized for their role in activating the inflammatory cascades during IRIs, the mechanisms by which oxidative stress enhances DC activation remain to be explored. We examined the role of oxidative stress on two important features of DC: T cell activation and trafficking. Bone marrow-derived OS-DC were compared with untreated DC. DC exposed to oxidative stress augmented allogeneic T cell proliferation and showed increased migration in a chemotaxis chamber. These results were confirmed by using hypoxanthine and xanthine oxidase as another inducer of oxidative stress. We used OT-II and OT-I mice to assess the effect of oxidative stress on DC activation of OVA-specific CD4(+) and CD8(+) T cells, respectively. Oxidative stress increased DC capacity to promote OVA-specific CD4(+) T cell activity, demonstrated by an increase in their proliferation and production of IFN-γ, IL-6, and IL-2 proinflammatory cytokines. Whereas oxidative stress increased the DC ability to stimulate IFN-γ production by OVA-specific CD8(+) T cells, cellular proliferation and cytotoxicity were not affected. Compared with untreated DC, oxidative stress significantly reduced the capacity of DC to generate T(regs), which were restored by using anti-IL-6. With regard to DC trafficking, whereas oxidative stress increased DC expression of p-Akt and p-NF-κB, targeting PI3Kγ and NF-κB pathways abrogated the observed increase in DC migration. Our data propose novel insights on the activation of DC by oxidative stress and provide rationales for targeted therapies, which can potentially attenuate IRI.

Type 1 diabetes (T1D) is an immune-mediated disease resulting in destruction of insulinproducing pancreatic beta cells. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties, garnering increasing attention as cellular therapy for T1D and other immunologic diseases. However, MSCs generally lack homing molecules, hindering their colonization at inflammatory sites following intravenous (IV) administration. Here, we analyzed whether enforced E-selectin ligand expression on murine MSCs could impact their effect in reversing hyperglycemia in nonobese diabetic (NOD) mice. Although murine MSCs natively do not express the E-selectin-binding determinant sialyl Lewis x (sLe x ), we found that fucosyltransferasemediated a(1,3)-exofucosylation of murine MSCs resulted in sLe x display uniquely on cell surface CD44 thereby creating hematopoietic cell E-/L-selectin ligand (HCELL), the E-selectin-binding glycoform of CD44. Following IV infusion into diabetic NOD mice, allogeneic HCELL 1 MSCs showed threefold greater peri-islet infiltrates compared to buffer-treated (i.e., HCELL 2 ) MSCs, with distribution in proximity to E-selectin-expressing microvessels. Exofucosylation had no effect on MSC immunosuppressive capacity in in vitro assays; however, although engraftment was temporary for both HCELL 1 and HCELL 2 MSCs, administration of HCELL 1 MSCs resulted in durable reversal of hyperglycemia, whereas only transient reversal was observed following administration of HCELL 2 MSCs. Notably, exofucosylation of MSCs generated from CD44 2/2 mice induced prominent membrane expression of sLe x , but IV administration of these MSCs into hyperglycemic NOD mice showed no enhanced pancreatotropism or reversal of hyperglycemia. These findings provide evidence that glycan engineering to enforce HCELL expression boosts trafficking of infused MSCs to pancreatic islets of NOD mice and substantially improves their efficacy in reversing autoimmune diabetes. STEM CELLS 2013;33:1523-1531