The blood endothelial cell chamber - An innovative system to study immune responses in drug development.

The risk for adverse immune-mediated reactions, associated with the administration of certain immunotherapeutic agents, should be mitigated early. Infusion reactions to monoclonal antibodies and other biopharmaceuticals, known as cytokine release syndrome, can arise from the release of cytokines via the drug target cell, as well as the recruitment of immune effector cells. While several in vitro cytokine release assays have been proposed up to date, many of them lack important blood components, required for this response to occur. The blood endothelial cell chamber model is an in vitro assay, composed of freshly drawn human whole blood and cultured human primary endothelial cells. Herein, its potential to study the compatibility of immunotherapeutics with the human immune system was studied by evaluating three commercially available monoclonal antibodies and bacterial endotoxin lipopolysaccharide. We demonstrate that the anti-CD28 antibody TGN1412 displayed an adaptive cytokine release profile and a distinct IL-2 response, accompanied with increased CD3+ cell recruitment. Alemtuzumab exhibited a clear cytokine response with a mixed adaptive/innate source (IFNγ, TNFα and IL-6). Its immunosuppressive nature is observed in depleted CD3+ cells. Cetuximab, associated with low infusion reactions, showed a very low or absent stimulatory effect on proinflammatory cytokines. In contrast, bacterial endotoxin demonstrated a clear innate cytokine response, defined by TNFα, IL-6 and IL-1ß release, accompanied with a strong recruitment of CD14+CD16+ cells. Therefore, the blood endothelial cell chamber model is presented as a valuable in vitro tool to investigate therapeutic monoclonal antibodies with respect to cytokine release and vascular immune cell recruitment.

Mesenchymal stromal cells support endothelial cell interactions in an intramuscular islet transplantation model.

BACKGROUND: Mesenchymal stromal cells (MSC) have been under investigation for a number of therapies and have lately been in focus as immunosuppressive actors in the field of transplantation. Herein we have extended our previously published in vitro model of MSC-islets in an experimental setting of islet transplantation to the abdominal muscle. Human islets coated with luciferase-GFP transduced human MSC were transplanted to the abdomen muscle tissue of NOD-scid ILR2γ(null) mice and cellular interactions were investigated by confocal microscopy. RESULTS: The MSC reduced fibrotic encapsulation and facilitated endothelial cell interactions. In particular, we show a decreased fraction of αSMA expressing fibrotic tissue surrounding the graft in presence of MSC-islets compared to islets solely distributed into the muscle tissue. Also, in the presence of MSC, human islet endothelial cells migrated from the center of the graft out into the surrounding tissue forming chimeric blood vessels with recipient endothelial cells. Further, in the graft periphery, MSC were seen interacting with infiltrating macrophages. CONCLUSIONS: Here, in our experimental in vivo model of composite human islets and luciferase-GFP-transduced human MSC, we enable the visualization of close interactions between the MSC and the surrounding tissue. In this model of transplantation the MSC contribute to reduced fibrosis and increased islet endothelial cell migration. Furthermore, the MSC interact with the recipient vasculature and infiltrating macrophages.

The cerebrospinal fluid cytokine signature of multiple sclerosis: a homogenous response that does not conform to the Th1/Th2/Th17 convention.

In this cross-sectional study, we wanted to identify key cytokines characteristic of different stages of multiple sclerosis (MS). To this end, cerebrospinal fluid from patients with MS was investigated with a multiplexed fluorescent bead-based immunoassay. In total 43 cytokines were assessed and related to clinical and imaging data. Increased levels of CCL22, CXCL10 and sCD40L characterized relapsing-remitting MS patients with the presence of gadolinium-enhancing lesions; decreased CCL2 and increased CXCL1 and CCL5 were typical of relapsing-remitting MS patients irrespectively of the presence of gadolinium-enhancing lesions. These homogenous patterns of cytokine activation do not conform to conventional Th1/Th2/Th17 responses.

Intranasal delivery of central nervous system-retargeted human mesenchymal stromal cells prolongs treatment efficacy of experimental autoimmune encephalomyelitis.

Treatment with mesenchymal stromal cells (MSCs) is currently of interest for a number of diseases including multiple sclerosis. MSCs are known to target inflamed tissues, but in a therapeutic setting their systemic administration will lead to few cells reaching the brain. We hypothesized that MSCs may target the brain upon intranasal administration and persist in central nervous system (CNS) tissue if expressing a CNS-targeting receptor. To demonstrate proof of concept, MSCs were genetically engineered to express a myelin oligodendrocyte glycoprotein-specific receptor. Engineered MSCs retained their immunosuppressive capacity, infiltrated into the brain upon intranasal cell administration, and were able to significantly reduce disease symptoms of experimental autoimmune encephalomyelitis (EAE). Mice treated with CNS-targeting MSCs were resistant to further EAE induction whereas non-targeted MSCs did not give such persistent effects. Histological analysis revealed increased brain restoration in engineered MSC-treated mice. In conclusion, MSCs can be genetically engineered to target the brain and prolong therapeutic efficacy in an EAE model.

T-cell responses after haematopoietic stem cell transplantation for aggressive relapsing-remitting multiple sclerosis.

Autologous haematopoietic stem cell transplantation (HSCT) for relapsing-remitting multiple sclerosis is a potentially curative treatment, which can give rise to long-term disease remission. However, the mode of action is not yet fully understood. The aim of the study was to evaluate similarities and differences of the CD4(+) T-cell populations between HSCT-treated patients (n = 12) and healthy controls (n = 9). Phenotyping of memory T cells, regulatory T (Treg) cells and T helper type 1 (Th1) and type 17 (Th17) cells was performed. Further, T-cell reactivity to a tentative antigen, myelin oligodendrocyte glycoprotein, was investigated in these patient populations. Patients treated with natalizumab (n = 15) were included as a comparative group. White blood cells were analysed with flow cytometry and T-cell culture supernatants were analysed with magnetic bead panel immunoassays. HSCT-treated patients had similar levels of Treg cells and of Th1 and Th17 cells as healthy subjects, whereas natalizumab-treated patients had lower frequencies of Treg cells, and higher frequencies of Th1 and Th17 cells. Cells from HSCT-treated patients cultured with overlapping peptides from myelin oligodendrocyte glycoprotein produced more transforming growth factor-ß1 than natalizumab-treated patients, which suggests a suppressive response. Conversely, T cells from natalizumab-treated patients cultured with those peptides produced more interleukin-17 (IL-17), IL-1 and IL-10, indicating a Th17 response. In conclusion, we demonstrate circumstantial evidence for the removal of autoreactive T-cell clones as well as development of tolerance after HSCT. These results parallel the long-term disease remission seen after HSCT.

CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model. METHODS: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery. RESULTS: The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs. CONCLUSION: CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

AdCD40L immunogene therapy for bladder carcinoma--the first phase I/IIa trial.

PURPOSE: Immunotherapy with Bacillus Calmette-Guerin (BCG) instillation is recommended for high-risk, non-muscle invasive bladder cancer. Bacillus Calmette-Guerin is not effective in advanced tumors, and better alternatives are warranted. Immunostimulating gene therapy with adenoviral vectors expressing CD40 ligand (AdCD40L) has shown efficacy in tumor models. CD40 ligand stimulates systemic immunity and may be effective in local and invasive human disease. EXPERIMENTAL DESIGN: Patients with invasive bladder cancer scheduled for cystectomy or patients with T(a) tumors were enrolled in a phase I/IIa trial. Patients were treated with three cycles of intrabladder Clorpactin WCS-90 prewash, followed by AdCD40L instillation 1 week apart. Safety, gene transfer, immune effects, and antitumor responses were monitored. RESULTS: All eight recruited patients were treated as scheduled, and therapy was well tolerated. The main adverse effect was transient local pain during prewash. Postoperatively, urinary tract infections and one case of late septicemia with elevated potassium were reported. No adverse events were ascribed to vector therapy. Gene transfer was detected in biopsies, and bladders were heavily infiltrated with T cells. The effector marker IFN-gamma increased in biopsies, whereas levels of circulating T regulatory cells were reduced. Histologic evaluation indicated that AdCD40L therapy reduced the load of malignant cells. CONCLUSIONS: To our knowledge, this is the first report on immunogene therapy in bladder cancer and the first using AdCD40L in vivo. Local AdCD40L gene therapy was safe, boosted immune activation, and should be further evaluated as a single or an adjuvant therapy for urothelial malignancies.

T regulatory cells lacking CD25 are increased in MS during relapse.

Dysregulation of inflammatory responses is considered to be a key element in autoreactive immune responses. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4(+)CD25(+)FoxP3(+) Tregs in autoimmunity has been extensively investigated. Recently, it was shown that Tregs in systemic lupus erythematosus lacked CD25 but were biologically functional. These data warrants for further investigation of CD25(- ) Tregs in human autoimmunity. We analyzed relapsing-remitting multiple sclerosis (MS) patients by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3, and the IL7R (CD127). Further, the level of Tregs was compared in remitting and relapsing patients and correlated with disease duration. Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls (p < 0.05), indicating that Tregs attempt to restrain immune activity during relapse. The proportion of Tregs tended to be decreased with disease duration, while CD25(+)CD4(+) and CD25(+)CD8(+) effector T-cell proportions were elevated and positively correlated with overall disease duration (p < 0.05). In conclusion, while MS patients in remission have normal levels of Tregs of different phenotype, relapsing patients show an increased proportion of systemic CD25(-)FoxP3(+) Tregs. With time, the proportion of Tregs decrease while effector T cells expand.

Local AdCD40L gene therapy is effective for disseminated murine experimental cancer by breaking T-cell tolerance and inducing tumor cell growth inhibition.

CD40 ligand (CD40L) is one of the most potent stimulators of Th1-type immunity through its maturation of dendritic cells that, in turn, stimulate effector cells such as T cells and NK cells. Lately, CD40-mediated cell growth inhibition and apoptosis have been in focus for the development of novel cancer treatment regiments, including recombinant soluble CD40L or CD40-stimulating antibodies. In this study, intravesical CD40L gene transfer through adenoviral vectors (AdCD40L) was used to treat an aggressive model of disseminated bladder cancer (MB49/C57BL/6). Three weekly AdCD40L vector instillations increased overall survival of tumor-bearing mice (mean 18.5 d, control mice 13 d). Furthermore, bladder tumors were eradicated (2 of 10) simultaneously as lung metastases (6 of 10) were cleared. FoxP3 levels were similar in the tumors of AdCD40L-treated mice and control mice but the tumor-infiltrating effector T cells in AdCD40L-treated mice were cytotoxic (CD107a+) in contrast to those in control-treated tumors. Furthermore, AdCD40L gene therapy could induce cell growth inhibition and cell death in the MB49 tumor cells in vitro and in vivo. However, this effect was not potent enough to cure growing tumors in immunodeficient mice. In conclusion, AdCD40L gene therapy is potent for disseminated cancer both by activation of T cells and controlling tumor cell growth and viability.

The T-cell pool is anergized in patients with multiple sclerosis in remission.

Relapsing-remitting multiple sclerosis (RRMS) is a complex autoimmune disease of the central nervous system with oscillating phases of relapse and remission. RRMS has been considered to be driven by T helper type 1 (Th1) lymphocytes but new data indicate the involvement of Th17 responses. In the present study, blood samples from patients (n=48) and healthy individuals (n=44) were evaluated for their immunological status. T cells from patients with RRMS expressed high levels of the activation marker CD28 (P<0.05) and secreted both interferon-gamma (CD8: P<0.05) and interleukin-17 upon polyclonal mitogen or myelin oligodendrocyte glycoprotein antigen stimulation. However, T cells from patients with RRMS in remission, in contrast to relapse, had poor proliferative capacity (P<0.05) suggesting that they are controlled and kept in anergy. This anergy could be broken with CD28 stimulation that restored the T-cell replication. Furthermore, the patients with RRMS had normal levels of CD4(+) Foxp3(+) T regulatory cells but the frequency of Foxp3(+) cells lacking CD127 (interleukin-7 receptor) was lower in patients with MS (mean 12%) compared to healthy controls (mean 29%). Still, regulatory cells (CD25(+) sorted cells) from patients with RRMS displayed no difference in suppressive capacity. In conclusion, patients in relapse/remission demonstrate in vitro T-cell responses that are both Th1 and Th17 that, while in remission, appear to be controlled by tolerogenic mechanisms yet to be investigated.

AdCD40L gene therapy counteracts T regulatory cells and cures aggressive tumors in an orthotopic bladder cancer model.

PURPOSE: The aim of this study was to develop an immunostimulating gene therapy for the treatment of orthotopic bladder carcinoma by transferring the gene for CD40L into the tumor site. CD40L stimulation of dendritic cells induces interleukin-12 expression that drives Th1 type of immune responses with activation of cytotoxic T cells. EXPERIMENTAL DESIGN: The gene for murine CD40L was transferred into bladders of tumor-bearing mice using an adenoviral vector construct. To facilitate viral uptake, the bladders were pretreated with Clorpactin. Survival of mice as well as transgene expression and immunologic effect, such as resistance to tumor challenge and presence of T regulatory cells, were monitored. RESULTS: On viral vector instillation, CD40L expression could be detected by reverse transcription-PCR. As a sign of transgene function, interleukin-12 (IL-12) expression was significantly increased. AdCD40L gene therapy cured 60% of mice with preestablished tumors. The cured mice were completely resistant to subcutaneous challenge with MB49 tumor cells, whereas the growth of a syngeneic irrelevant tumor was unaltered. Furthermore, the mRNA expression level of the T regulatory cell transcription factor Foxp3 was evaluated both in tumor biopsies and lymph nodes. There were no differences within the tumors of the different treatment groups. However, Foxp3 mRNA levels were down-regulated in the lymph nodes of AdCD40L-treated mice. Correspondingly, T cells from AdCD40L-treated mice were not able to inhibit proliferation of naive T cells as opposed to T cells from control-treated, tumor-bearing mice. CONCLUSIONS: AdCD40L gene therapy evokes Th1 cytokine responses and counteracts T regulatory cell development and/or function.