Morphological features of IFN-γ-stimulated mesenchymal stromal cells predict overall immunosuppressive capacity.

Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSC-based therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ-enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.

Restoring immune tolerance in neuromyelitis optica: Part I.

Neuromyelitis optica (NMO) and spectrum disorder (NMO/SD) represent a vexing process and its clinical variants appear to have at their pathogenic core the loss of immune tolerance to the aquaporin-4 water channel protein. This process results in a characteristic pattern of astrocyte dysfunction, loss, and demyelination that predominantly affects the spinal cord and optic nerves. Although several empirical therapies are currently used in the treatment of NMO/SD, none has been proven effective in prospective, adequately powered, randomized trials. Furthermore, most of the current therapies subject patients to long-term immunologic suppression that can cause serious infections and development of cancers. The following is the first of a 2-part description of several key immune mechanisms in NMO/SD that might be amenable to therapeutic restoration of immune tolerance. It is intended to provide a roadmap for how potential immune tolerance restorative techniques might be applied to patients with NMO/SD. This initial installment provides a background rationale underlying attempts at immune tolerization. It provides specific examples of innovative approaches that have emerged recently as a consequence of technical advances. In several autoimmune diseases, these strategies have been reduced to practice. Therefore, in theory, the identification of aquaporin-4 as the dominant autoantigen makes NMO/SD an ideal candidate for the development of tolerizing therapies or cures for this increasingly recognized disease.

Combination Therapy Using IL-2/IL-2 Monoclonal Antibody Complexes, Rapamycin, and Islet Autoantigen Peptides Increases Regulatory T Cell Frequency and Protects against Spontaneous and Induced Type 1 Diabetes in Nonobese Diabetic Mice.

Regulatory T cells (Treg) play a crucial role in the maintenance of self-tolerance. In this study, we sought to expand Ag-specific Tregs in vivo and investigate whether the expanded Tregs can prevent or delay the development of type 1 diabetes (T1D) in the NOD mouse model. NOD mice were treated with a combination of IL-2/anti-IL-2 Ab complex, islet Ag peptide, and rapamycin. After the combined treatment, CD4(+)CD25(+)Foxp3(+) Tregs were significantly expanded in vivo, they expressed classical Treg markers, exerted enhanced suppressive functions in vitro, and protected against spontaneous development of T1D in NOD mice. Moreover, treated mice were almost completely protected from the adoptively transferred, aggressive form of T1D caused by in vitro-activated cytotoxic islet Ag-specific CD8 T cells. Protection from T1D was transferrable by Tregs and could be attributed to reduced islet infiltration of immune cells as well as the skewing of the immune response toward a Th2 cytokine profile. This new method of peripheral immune regulation could potentially contribute to development of novel immunotherapeutic strategies to prevent the development of T1D or to promote tolerance to islet transplants without using immunosuppressive drugs for long terms.

Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro.

INTRODUCTION: Mesenchymal stem cells (MSCs) have immunosuppressive activity. They do not induce allospecific T cell responses, making them promising tools for reducing the severity of graft versus host disease (GVHD) as well as treating various immune diseases. Currently, there is a need in the MSC field to develop a robust in vitro bioassay which can characterize the immunosuppressive function of MSCs. METHODS: Murine clonal CD4 and CD8 T cells were stimulated with cognate peptide antigen and antigen presenting cells (APCs) in the absence or presence of human MSCs, different aspects of T cell activation were monitored and analyzed using flow cytometry, real time RT-PCR and cytokine measurement. RESULTS: Human MSCs (hMSCs) can alter multiple aspects of murine T cell activation induced by stimulation with specific antigen, including: reduced proliferation, inhibited or stimulated cell surface marker expression (CD25, CD69, CD44 and CD62L), inhibited mRNA expression of transcription factors (T-bet and GATA-3) and decreased cytokine expression (interferon-gamma, interleukin-10). Disappearance of activation-induced cluster formation and decreased apoptosis of CD8 T cells were also observed. Moreover, the effects are specific to MSCs; incubating the T cells with non-MSC control cell lines had no effect on T cell proliferation and activation. CONCLUSIONS: Clonal murine T cells can be used to measure, characterize, and quantify the in vitro immunosuppressive activity of human MSCs, representing a promising approach to improve bioassays for immunosuppression.

The apoptotic pathway contributing to the deletion of naive CD8 T cells during the induction of peripheral tolerance to a cross-presented self-antigen.

The maintenance of T cell tolerance in the periphery proceeds through several mechanisms, including anergy, immuno-regulation, and deletion via apoptosis. We examined the mechanism underlying the induction of CD8 T cell peripheral tolerance to a self-Ag expressed on pancreatic islet beta-cells. Following adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. Additional experiments revealed that the induction of clone 4 T cell apoptosis during peripheral tolerance occurred via an intrinsic death pathway that could be inhibited by overexpression of Bcl-2 or targeted deletion of the proapoptotic molecule, Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis whereas Bim-/- clone 4 cells were not autoaggressive. Examination of the upstream molecular mechanisms contributing to clone 4 T cell apoptosis revealed that it proceeded in a p53, E2F1, and E2F2-independent manner. Taken together, these data reveal that initiation of clone 4 T cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl-2-sensitive and at least partially Bim-dependent mechanism.

N-terminal trimer extension of nominal CD8 T cell epitopes is sufficient to promote cross-presentation to cognate CD8 T cells in vivo.

Cross-priming is the process in which Ag-presenting dendritic cells (DCs) acquire, process, and present Ags scavenged from other cells, and use these cells to activate naive CD8 T cells. Cross-priming of cognate CD8 cells can result in either tolerance or immunity, depending upon the activation status of the Ag-presenting DC. Previous studies have shown that nominal peptide is inefficiently cross-presented and that proteins and large polypeptides that require proteasomal processing are the main source of naturally cross-presented Ags. In this study we show that N-terminal extension of nominal peptide by as few as three residues is sufficient to produce a substrate for TAP-dependent cross-presentation that is highly efficient in cross-priming murine CD8 T cells in vivo. On a molar basis, cross-priming with 3-mer-extended peptide is 20-fold more efficient than priming with intact protein. This method of peptide extension should prove of great value in facilitating in vivo studies of CD8 immunity and tolerance that rely on cross-presentation.

Tissue-resident memory CD8+ T cells can be deleted by soluble, but not cross-presented antigen.

Under noninflammatory conditions, both naive and central memory CD8 T cells can be eliminated in the periphery with either soluble peptide or cross-presented Ag. Here, we assess the tolerance susceptibility of tissue-resident memory CD8 T cells in mice to these two forms of tolerogen. Soluble peptide specifically eliminated the majority of memory CD8 cells present in both lymphoid and extralymphoid tissues including lung and liver, but was unable to reduce the number present in the CNS. In contrast, systemic cross-presentation of Ag by dendritic cells resulted in successful elimination of memory cells only from the spleen, with no significant reduction in the numbers of tissue-resident memory cells in the lung. The fact that tissue-resident memory cells were unable to access cross-presented Ag suggests that either the memory cells in the lung do not freely circulate out of the tissue, or that they circulate through a region in the spleen devoid of cross-presented Ag. Thus, although tissue-resident memory cells are highly susceptible to tolerance induction, both the form of tolerogen and location of the T cells can determine their accessibility to tolerogen and the degree to which they are successfully deleted from specific tissues.

Regulation of expression of Bcl-2 protein family member Bim by T cell receptor triggering.

Bim, a proapoptotic BH3-only member of the Bcl-2 protein family, is required for central and peripheral deletion of T lymphocytes. Mechanisms regulating Bim activity in T cells remain poorly understood. We show that expression of Bim is up-regulated in human T cells after polyclonal or specific T cell receptor triggering. Induction of Bim was affected by the agonistic potency of MHC:peptide ligands. Peptides that failed to induce Bim expression, failed to induce apoptosis in specific T cells, whereas partially agonistic ligands, which trigger death receptor-independent activation-induced cell death (AICD), induced Bim, but were inefficient in up-regulating Bcl-X(L). Activation of protein kinase C and calcineurin appeared to be necessary and sufficient for Bim up-regulation after T cell receptor ligation. Immunosuppressive drugs known to prevent T cell deletion in vivo, such as cyclosporin A or FK506, blocked Bim up-regulation and rescued T cells from death receptor-independent AICD, whereas rapamycin, which allows the development of stable immunological tolerance, did not exhibit these activities. These results define a new mode of Bim regulation, strongly implicate Bim as a mediator of AICD, and suggest that Bim up-regulation can be targeted to influence the outcome of specific immune responses.

Soluble factors released by virus specific activated cytotoxic T-lymphocytes induce apoptotic death of astroglioma cell lines.

Astrocytomas and astrogliomas represent the most common types of primary tumors in human central nervous system and are associated with high mortality due to the absence of efficient therapy. Here we demonstrate that, upon antigen-specific activation, cytotoxic T-lymphocytes (CTLs) secrete products that inhibit proliferation and induce apoptosis in a significant proportion of astroglioma cell lines. This effect is tumor specific in that normal cultured astrocytes do not develop apoptotic changes upon exposure to supernatant of activated CTLs. Experiments with purified lymphokines and lymphokine specific blocking antibodies indicate that synergistic activities of tumor necrosis factor (TNF)-alpha and interferon (INF)-gamma are required for the apoptosis inducing effect on some astroglioma cell lines. However, this effect appears to be dependent on additional factors produced by activated CTLs. Our results suggest that local application of factors released by activated CTLs or induction of CTL migration and activation in the tumor site may have a therapeutic effect in patients with astrogliomas.

Tetramer binding and secretion of interferon-gamma in response to antigenic stimulation are compatible with a range of affinities of MHC:TCR interaction and distinct programs of cytotoxic T-lymphocyte activation.

Tetramer staining and detection of IFN-gamma secretion in response to specific stimulation are widely used to quantify and isolate specific T-cells. However, it remains unclear how these assays reflect different functional outcomes of T-cell triggering with MHC:peptide ligands. An immunogenic EBV-derived A11-restricted CTL peptide epitope and its partially agonistic analogue trigger different programs of activation induced cell death (AICD) in specific CTLs. In this study we analysed a panel of CTL clones, bulk CTL cultures and PBMCs isolated from HLA A11-positive EBV-infected individuals for their ability to bind tetrameric complexes assembled with either of the two peptides and correlated tetramer binding with the activity of the peptides in functional assays. This analysis demonstrates that specific tetramer staining and secretion of IFN-gamma are compatible with at least two activation programs in CTLs. One of these programs corresponds to full-scale CTL activation and death of a proportion of activated T-cells in a Fas-dependent manner. In contrast, the alternative program is characterized by selective induction of IFN-gamma and TNF-alpha, absence of proliferative response and Fas-independent cell death. These findings may have important implications for the evaluation of data obtained with MHC:peptide tetramers and IFN-gamma secretion assays, especially in experimental systems with extensive antigenic variability.