The blood–brain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.

Mesenchymal stem cells (MSCs) inhibit proliferation of allogeneic T cells and express low levels of major histocompatibility complex class I (MHCI), MHCII and vascular adhesion molecule-1 (VCAM-1). We investigated whether their immunosuppressive properties and low immunophenotype protect allogeneic rat MSCs against cytotoxic lysis in vitro and result in a reduced immune response in vivo. Rat MSCs were partially protected against alloantigen-specific cytotoxic T cells in vitro. However, after treatment with IFN-γ and IL-1β, MSCs upregulated MHCI, MHCII and VCAM-1, and cytotoxic lysis was significantly increased. In vivo, allogeneic T cells but not allogeneic MSCs induced upregulation of the activation markers CD25 and CD71 as well as downregulation of CD62L on CD4+ T cells from recipient rats. However, intravenous injection of allo-MSCs in rats led to the formation of alloantibodies with the capacity to facilitate complement-mediated lysis, although IgM levels were markedly decreased compared with animals that received T cells. The allo-MSC induced immune response was sufficient to lead to significantly reduced survival of subsequently injected allo-MSCs. Interestingly, no increased immunogenicity of IFN-γ stimulated allo-MSCs was observed in vivo. Both the loss of protection against cytotoxic lysis under inflammatory conditions and the induction of complement-activating antibodies will likely impact the utility of allogeneic MSCs for therapeutic applications.

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) that is initiated when self-reactive T cells enter the brain and become locally activated after encountering their specific nervous antigens. When and where the disease-relevant antigen encounters occur is unclear. Here we combined fluorescently labeled nuclear factor of activated T cells (NFAT) with histone protein H2B to create a broadly applicable molecular sensor for intravital imaging of T cell activation. In experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, we report that effector T cells entering the CNS become activated after short contacts with leptomeningeal phagocytes. During established disease, the activation process is extended to the depth of the CNS parenchyma, where the cells form contacts with microglia and recruited phagocytes. We show that it is the activation processes during the preclinical phase rather than during established disease that are essential for the intensity and duration of the disease bout.

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y These authors contributed equally to this work.Mesenchymal stem cells (MSCs) are being investigated extensively due to their ability to dampen immune responses. Here, we tested the ability of MSCs from three distinct sources to prolong rat corneal allograft survival. A fully allogeneic rat cornea transplant model (DA to LEW) was used. Recipient rats received 1 Â 10 6 MSCs (syn [LEW], allo [DA] or third-party [Wistar Furth]) intravenously 7 days before transplantation and again on the day of transplantation (day 0). A high percentage of untreated and syn-MSC treated allografts were rejected (80% and 100%, respectively). Preactivation of syn-MSCs with interferon gamma also failed to prolong allograft survival. Conversely, corneal allograft survival was significantly prolonged in allo-MSC treated (90%) and third-party MSC treated (80%) allograft recipients. Flow cytometric analysis revealed less infiltrating natural killer T cells in corneas of both allo-and third-party MSC treated animals, coupled with a higher proportion of splenic CD4þFoxp3þ regulatory T cells, compared to controls. In the case of allo-and third-party MSCs, results from a delayed-type hypersensitivity assay clearly showed that hypo-responsiveness was specific for corneal donor-associated alloantigens. Thus, allo-and third-party MSC treatment prolongs corneal allograft survival by suppressing peripheral immune responses and promoting an intragraft immunoregulatory milieu.

In a model of peritoneal metastasis in immune-competent mice, we show that nuclear factor (NF)-κB inhibition in CT26 colon cancer cells prevents metastasis. NF-κB inhibition, by stable overexpression of IκB-α super-repressor, induced differential polarization of co-cultured macrophages to an M1-like anti-tumour phenotype in vitro. NF-κB-deficient cancer cell-conditioned media (CT26/IκB-α SR) induced interleukin (IL)-12 and nitric oxide (NO) synthase (inducible NO synthase (iNOS)) expression in macrophages. Control cell (CT26/EV) conditioned media induced high levels of IL-10 and arginase in macrophages. In vivo, this effect translated to reduction in metastasis in mice injected with CT26/ IκB-α SR cells and was positively associated with increased CD8(+)CD44(+)CD62L(-) and CD4(+)CD44(+)CD62L(-) effector T cells. Furthermore, inhibition of NF-κB activity induced high levels of NO in infiltrating immune cells and decreases in matrix metalloproteinase-9 expression, simultaneous with increases in tissue inhibitor of metalloproteinases 1 and 2 within tumours. CT26/IκB-α SR tumours displayed increased pro-inflammatory gene expression, low levels of angiogenesis and extensive intratumoral apoptosis, consistent with the presence of an anti-tumour macrophage phenotype. Macrophage depletion reduced tumour size in CT26/EV-injected animals and increased tumour size in CT26/IκB-α SR cells compared with untreated tumours. Our data demonstrate, for the first time, that an important implication of targeting tumour cell NF-κB is skewing of macrophage polarization to an anti-tumour phenotype. This knowledge offers novel therapeutic opportunities for anticancer treatment.

To investigate the role of lentivirus-mediated overexpression of programmed death-ligand 1 (PD-L1) on rat corneal allograft survival. A fully allogeneic rat cornea transplant model was used for in vivo studies. Lentiviral (LV) vectors are efficient tools for ex vivo genetic modification of cultured corneas. LV vector encoding for PD-L1 (LV.PD-L1) and LV vector encoding for eGFP (LV.eGFP, as control) were constructed and tested. PD-L1 or eGFP expression was increased on corneal cells upon LV.PD-L1 and LV.eGFP transduction, respectively. Both allogeneic controls and allogeneic LV.eGFP transduced corneas were uniformly rejected (MST: 13.8 ± 1.7 days and 12.3 ± 1.9 days, respectively). In contrast, allogeneic LV.PD-L1 transduced corneas showed a high percentage (83%) of graft survival (MST > 30 days, n = 5, 15 days, n = 1). Graft opacity of PD-L1 transduced corneas was present but was significantly reduced compared to control or eGFP expressing corneas. Flow cytometric analysis revealed that percentages of CD3 + CD8 + CD161 + and CD3 + CD8 + CD161 -lymphocytes were decreased in animals receiving LV.PD-L1 transduced corneas compared to animals grafted with LV.eGFP transduced corneas. Moreover, reduced expression of proinflammatory cytokines (IFN-c and IL-6) in PD-L1 transduced corneas compared to allogeneic controls was also observed. Local PD-L1 gene transfer in cultured corneas is a promising approach for the prolongation of corneal allograft survival and attenuation of graft rejection.

Adult mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential which makes them attractive targets for regenerative medicine applications. However, to date, therapeutic success of MSC-therapy is limited and the genetic modification of MSCs using viral vectors is one option to improve their therapeutic potential. Ex-vivo genetic modification of MSCs using recombinant adenovirus (Ad) could be promising to reduce undesired immune responses as Ad will be removed before cell/tissue transplantation. In this regard, we investigated whether Ad-modification of MSCs alters their immunological properties in vitro and in vivo. We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86. Moreover, Ad-transduction caused no significant changes in terms of pro-inflammatory cytokine expression, chemokine and chemokine receptor and Toll-like receptor expression. In addition, Ad-modification of MSCs had no affect on their ability to suppress T cell proliferation in vitro. In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues. Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.