T cells become licensed in the lung to enter the central nervous system.

The blood­brain 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.

Risk factors associated with exposure to bovine respiratory disease pathogens during the peri-weaning period in dairy bull calves.

BACKGROUND: Bovine respiratory disease (BRD) remains among the leading causes of death of cattle internationally. The objective of this study was to identify risk factors associated with exposure to BRD pathogens during the peri-weaning period (day (d)-14 to d 14 relative to weaning at 0) in dairy bull calves using serological responses to these pathogens as surrogate markers of exposure. Clinically normal Holstein-Friesian and Jersey breed bull calves (n = 72) were group housed in 4 pens using a factorial design with calves of different breeds and planes of nutrition in each pen. Intrinsic, management and clinical data were collected during the pre-weaning (d - 56 to d - 14) period. Calves were gradually weaned over 14 days (d - 14 to d 0). Serological analysis for antibodies against key BRD pathogens (BRSV, BPI3V, BHV-1, BHV-4, BCoV, BVDV and H. somni) was undertaken at d - 14 and d 14. Linear regression models (for BVDV, BPI3V, BHV-1, BHV-4, BCoV and H. somni) and a single mixed effect random variable model (for BRSV) were used to identify risk factors for changes in antibody levels to these pathogens. RESULTS: BRSV was the only pathogen which demonstrated clustering by pen. Jersey calves experienced significantly lower changes in BVDV S/P than Holstein-Friesian calves. Animals with a high maximum respiratory score (≥8) recorded significant increases in H. somni S/P during the peri-weaning period when compared to those with respiratory scores of ≤3. Haptoglobin levels of between 1.32 and 1.60 mg/ml at d - 14 were significantly associated with decreases in BHV-1 S/N during the peri-weaning period. Higher BVDV S/P ratios at d - 14 were significantly correlated with increased changes in serological responses to BHV-4 over the peri-weaning period. CONCLUSIONS: Haptoglobin may have potential as a predictor of exposure to BHV-1. BRSV would appear to play a more significant role at the 'group' rather than 'individual animal' level. The significant associations between the pre-weaning levels of antibodies to certain BRD pathogens and changes in the levels of antibodies to the various pathogens during the peri-weaning period may reflect a cohort of possibly genetically linked 'better responders' among the study population.

Donor bone marrow-derived dendritic cells prolong corneal allograft survival and promote an intragraft immunoregulatory milieu.

Investigations into cell therapies for application in organ transplantation have grown. Here, we describe the ex vivo generation of donor bone marrow-derived dendritic cells (BMDCs) and glucocorticoid-treated BMDCs with potent immunomodulatory properties for application in allogeneic transplantation. BMDCs were treated with dexamethasone (Dexa) to induce an immature, maturation-resistant phenotype. BMDC and Dexa BMDC phenotype, antigen presenting cell function, and immunomodulatory properties were fully characterized. Both populations display significant immunomodulatory properties, including, but not limited to, a significant increase in mRNA expression of programmed death-ligand 1 and indoleamine 2,3-dioxygenase. BMDCs and Dexa BMDCs display a profound impaired capacity to stimulate allogeneic lymphocytes. Moreover, in a fully MHC I/II mismatched rat corneal transplantation model, injection of donor-derived, untreated BMDC or Dexa BMDCs (1 × 10(6) cells, day -7) significantly prolonged corneal allograft survival without the need for additional immunosuppression. Although neovascularization was not reduced and evidence of donor-specific alloantibody response was detected, a significant reduction in allograft cellular infiltration combined with a significant increase in the ratio of intragraft FoxP3-expressing regulatory cells was observed. Our comprehensive analysis demonstrates the novel cellular therapeutic approach and significant effect of donor-derived, untreated BMDCs and Dexa BMDCs in preventing corneal allograft rejection.

Gene therapy approaches to prevent corneal graft rejection: where do we stand?

Cornea transplantation (penetrating keratoplasty) is the most frequently performed transplant procedure in humans. Despite advances in microsurgery and immunosuppressive treatment protocols, a significant number of corneal grafts still undergo immune-mediated allograft rejection. Topical treatment with corticosteroids is currently the gold standard and while this treatment is effective in many corneal transplant patients, it is much less effective in 'high-risk' patients with previous episodes of neovascularisation or graft rejection. Therefore, alternative approaches such as genetic modification of donor corneas are needed to prevent corneal transplant rejection. Cornea transplantation holds the unique advantage in that gene therapy can be used to modify allografts ex vivo prior to transplantation. Many preclinical studies using local (and systemic) gene transfer have been performed to date and many different gene transfer vehicles (gene therapy vectors) and therapeutic strategies (immunomodulatory or graft-protective) have been investigated to prevent corneal allograft rejection. The most recent gene therapy applications to prevent corneal allograft rejection will be reviewed in this article. Moreover, it will be discussed why the development of clinical trials for the genetic modification of corneal grafts prior to transplantation is lagging behind of those for the treatment of inherited retinal diseases.

Immunogenicity of allogeneic mesenchymal stem cells.

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-1ß, 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.

Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy.

Two photon microscopy (TPM) recently emerged as optical tool for the visualization of immune processes hundreds of micrometers deep in living tissue and organs. Here we summarize recent work on exploiting this technology to study brain antigen specific T cells. These cells are the cause of Experimental Autoimmune Encephalomyelitis (EAE) an autoimmune disease model of Multiple Sclerosis. TPM studies elucidated the dynamics of the autoaggressive effector T cells in peripheral immune milieus during preclinical EAE, where the cells become reprogrammed to enter their target organ. These studies revealed an unexpectedly lively locomotion behavior of the cells interrupted only by short-lasting contacts with the local immune stroma. Live T cell behavior was furthermore studied within the acutely inflamed CNS. Two distinct migratory patterns of the T cells were found: the majority of cells (60-70%) moved fast and seemingly unhindered through the compact CNS parenchyma. The motility of the other cell fraction was highly confined. The cells swung around a fixed cell pole forming long-lasting contacts to putative local antigen presenting cells.

Macrophages behavior on different NIPAm-based thermoresponsive substrates.

Thermoresponsive materials and surfaces are widely used in cell culture applications. There is a lot of research work employing thermoresponsive materials with various structure and compositions. However, little is known about the immunological response to the thermoresponsive materials. Macrophage-like transformed murine cell line RAW264.7 was selected as it is a widely used standard model for immune activation analysis. This study proposes to compare the effects of thermoresponsive films with various compositions on macrophage cells. Thermoresponsive materials are a useful utility as a non-enzymatic harvesting system for tissue culture. As RAW264.7 cells are difficult to remove from the substrate by enzymatic methods we also explored the possibility to use thermoresponsive materials for the macrophage cultivation. Spin coating and solvent casting was used to produce films of N-isopropylacrylamide-based polymers from the nanometer to micrometer range. Successful cell adhesion and proliferation was highly dependent on the thickness and composition of the coating. RAW264.7 cells were successfully detached from the coatings upon temperature reduction. Furthermore, results indicate that the RAW264.7 cells remained inactivated as cell secreted cytokine remained at a low level and the surface receptor profile of RAW264.7 was not altered when cells were detached in this manner.

Adenoviral transduction of mesenchymal stem cells: in vitro responses and in vivo immune responses after cell transplantation.

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.

Gene therapy in transplantation: Toward clinical trials.

The genetic modification of organs or cells is an attractive approach to protect allogeneic transplants from acute rejection and other complications. The transplant setting offers a unique opportunity to utilize ex vivo gene therapy for the modification of allogeneic organs and tissues prior to implantation. However, significant challenges exist in the application of this concept to human organ transplantation, including the large number of potential molecular targets, the diversity and safety profile of available vector delivery systems and the merging of gene-based therapies with existing immunosuppressive regimens. Accordingly, many different therapeutic concepts and vector systems have been investigated in preclinical studies with the aim of prolonging allograft survival. However, the translation of promising gene therapy strategies to transplant clinical trials has lagged behind the progress made in other medical fields. This review describes the recent preclinical applications of gene transfer to transplantation, and critically evaluates the degree to which gene therapy has been tested clinically in organ transplant recipients.

Instant effect of soluble antigen on effector T cells in peripheral immune organs during immunotherapy of autoimmune encephalomyelitis.

i.v. infusion of native autoantigen or its altered peptide variants is an important therapeutic option for the treatment of autoimmune diseases, because it selectively targets the disease-inducing T cells. To learn more about the mechanisms and kinetics of this approach, we visualized the crucial initial effects of i.v. infusion of peptides or intact protein on GFP-tagged autoaggressive CD4(+) effector T cells using live-video and two-photon in situ imaging of spleens in living animals. We found that the time interval between i.v. injection of intact protein to first changes in T cell behavior was extremely short; within 10 min after protein application, the motility of the T cells changed drastically. They slowed down and became tethered to local sessile stromal cells. A part of the cells aggregated to form clusters. Within the following 20 min, IFN-gamma mRNA was massively (>100-fold) up-regulated; surface IL-2 receptor and OX-40 (CD 134) increased 1.5 h later. These processes depleted autoimmune T cells in the blood circulation, trapping the cells in the peripheral lymphoid organs and thus preventing them from invading the CNS. This specific blockage almost completely abrogated CNS inflammation and clinical disease. These findings highlight the speed and efficiency of antigen recognition in vivo and add to our understanding of T cell-mediated autoimmunity.

Transition from enhanced T cell infiltration to inflammation in the myelin-degenerative central nervous system.

Myelin degeneration in the central nervous system (CNS) is often associated with elevated numbers of T cells in brain and spinal cord (SC). In some degenerative diseases, this T cell immigration has no clinical relevance, in others, it may precede severe inflammation and tissue damage. We studied T cells in the myelin-degenerative SC of transgenic (tg) Lewis rats overexpressing the proteolipid protein (PLP). These lymphocytes are T(H)1/T(C)1 cells and represent different T cell clones unique to individual animals. The SC-infiltrating CD8(+) T cell pool is more restricted than its CD4(+) counterpart, possibly due to constrictions in the peripheral CD8(+) T cell repertoire. Some SC-infiltrating T cells are highly motile and cover large distances within their target tissue, others are tethered to MHC class II(+) microglia cells. The activation of the tethered cells may trigger the formation of inflammatory foci and could pave the way for inflammation in degenerative CNS disease.