Morphological landscapes from high content imaging reveal cytokine priming strategies that enhance mesenchymal stromal cell immunosuppression.

Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify "priming" conditions that produce MSCs with consistent immunomodulatory function; however, challenges remain with predicting and understanding how priming impacts MSC behavior. The purpose of this study was to develop a high throughput, image-based approach to assess MSC morphology in response to combinatorial priming treatments and establish morphological profiling as an effective approach to screen the effect of manufacturing changes (i.e., priming) on MSC immunomodulation. We characterized the morphological response of multiple MSC lines/passages to an array of Interferon-gamma (IFN-γ) and tumor necrosis factor-⍺ (TNF-⍺) priming conditions, as well as the effects of priming on MSC modulation of activated T cells and MSC secretome. Although considerable functional heterogeneity, in terms of T-cell suppression, was observed between different MSC lines and at different passages, this heterogeneity was significantly reduced with combined IFN-γ/TNF-⍺ priming. The magnitude of this change correlated strongly with multiple morphological features and was also reflected by MSC secretion of immunomodulatory factors, for example, PGE2, ICAM-1, and CXCL16. Overall, this study further demonstrates the ability of priming to enhance MSC function, as well as the ability of morphology to better understand MSC heterogeneity and predict changes in function due to manufacturing.

Morphological profiling using machine learning reveals emergent subpopulations of interferon-γ-stimulated mesenchymal stromal cells that predict immunosuppression.

BACKGROUND: Although a preponderance of pre-clinical data demonstrates the immunosuppressive potential of mesenchymal stromal cells (MSCs), significant heterogeneity and lack of critical quality attributes (CQAs) based on immunosuppressive capacity likely have contributed to inconsistent clinical outcomes. This heterogeneity exists not only between MSC lots derived from different donors, tissues and manufacturing conditions, but also within a given MSC lot in the form of functional subpopulations. We therefore explored the potential of functionally relevant morphological profiling (FRMP) to identify morphological subpopulations predictive of the immunosuppressive capacity of MSCs derived from multiple donors, manufacturers and passages. METHODS: We profiled the single-cell morphological response of MSCs from different donors and passages to the functionally relevant inflammatory cytokine interferon (IFN)-γ. We used the machine learning approach visual stochastic neighbor embedding (viSNE) to identify distinct morphological subpopulations that could predict suppression of activated CD4+ and CD8+ T cells in a multiplexed quantitative assay. RESULTS: Multiple IFN-γ-stimulated subpopulations significantly correlated with the ability of MSCs to inhibit CD4+ and CD8+ T-cell activation and served as effective CQAs to predict the immunosuppressive capacity of additional manufactured MSC lots. We further characterized the emergence of morphological heterogeneity following IFN-γ stimulation, which provides a strategy for identifying functional subpopulations for future single-cell characterization and enrichment techniques. DISCUSSION: This work provides a generalizable analytical platform for assessing functional heterogeneity based on single-cell morphological responses that could be used to identify novel CQAs and inform cell manufacturing decisions.

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.

Killer B lymphocytes and their fas ligand positive exosomes as inducers of immune tolerance.

Induction of immune tolerance is a key process by which the immune system is educated to modulate reactions against benign stimuli such as self-antigens and commensal microbes. Understanding and harnessing the natural mechanisms of immune tolerance may become an increasingly useful strategy for treating many types of allergic and autoimmune diseases, as well as for improving the acceptance of solid organ transplants. Our laboratory and others have been interested in the natural ability of some B lymphocytes to express the death-inducing molecule Fas ligand (FasL), and their ability to kill T helper (TH) lymphocytes. We have recently shown that experimental transformation of human B cells by a non-replicative variant of Epstein-Barr virus (EBV) consistently resulted in high expression of functional FasL protein. The production and release of FasL(+) exosomes that co-expressed major histocompatibility complex (MHC) class II molecules and had the capacity to kill antigen-specific TH cells was also observed. Several lines of evidence indicate that FasL+ B cells and FasL(+)MHCII(+) exosomes have important roles in natural immune tolerance and have a great deal of therapeutic potential. Taken together, these findings suggest that EBV-immortalized human B lymphoblastoid cell lines could be used as cellular factories for FasL(+) exosomes, which would be employed to therapeutically establish and/or regain immune tolerance toward specific antigens. The goals of this review are to summarize current knowledge of the roles of FasL(+) B cells and exosomes in immune regulation, and to suggest methods of manipulating killer B cells and FasL(+) exosomes for clinical purposes.

Mesenchymal stem cells in the treatment of inflammatory and autoimmune diseases in experimental animal models.

Multipotent mesenchymal stromal cells [also known as mesenchymal stem cells (MSCs)] are currently being studied as a cell-based treatment for inflammatory disorders. Experimental animal models of human immune-mediated diseases have been instrumental in establishing their immunosuppressive properties. In this review, we summarize recent studies examining the effectiveness of MSCs as immunotherapy in several widely-studied animal models, including type 1 diabetes, experimental autoimmune arthritis, experimental autoimmune encephalomyelitis, inflammatory bowel disease, graft-vs-host disease, and systemic lupus erythematosus. In addition, we discuss mechanisms identified by which MSCs mediate immune suppression in specific disease models, and potential sources of functional variability of MSCs between studies.

Characterization and activity of Fas ligand producing CD5⁺ B cells.

B lymphocytes make several contributions to immune regulation including production of antibodies with regulatory properties, release of immune suppressive cytokines, and expression of death-inducing ligands. A role for Fas ligand (FasL)-expressing "killer" B cells in regulating T helper cell survival and chronic inflammation has been demonstrated in animal models of schistosome worm infection, asthma, and autoimmune arthritis. Interestingly, a population of CD5(+) B cells found in the spleen and lungs of naïve mice constitutively expresses FasL and has potent killer function against T helper cells that is antigen-specific and FasL-dependent. Killer B cells therefore represent a novel target for immune modulation in many disease settings. Our laboratory has recently published methods of characterizing FasL(+) B cells and inducing their proliferation in vitro. This chapter will describe detailed methods of identifying and expanding killer B cells from mice, detecting FasL expression in B cells, and performing functional killing assays against antigen-specific TH cells.

Human B Cell-Derived Lymphoblastoid Cell Lines Constitutively Produce Fas Ligand and Secrete MHCII(+)FasL(+) Killer Exosomes.

Immune suppression mediated by exosomes is an emerging concept with potentially immense utility for immunotherapy in a variety of inflammatory contexts, including allogeneic transplantation. Exosomes containing the apoptosis-inducing molecule Fas ligand (FasL) have demonstrated efficacy in inhibiting antigen-specific immune responses upon adoptive transfer in animal models. We report here that a very high frequency of human B cell-derived lymphoblastoid cell lines (LCL) constitutively produce MHCII(+)FasL(+) exosomes that can induce apoptosis in CD4(+) T cells. All LCL tested for this study (>20 independent cell lines) showed robust expression of FasL, but had no detectable FasL on the cell surface. Given this intracellular sequestration, we hypothesized that FasL in LCL was retained in the secretory lysosome and secreted via exosomes. Indeed, we found both MHCII and FasL proteins present in LCL-derived exosomes, and using a bead-based exosome capture assay demonstrated the presence of MHCII(+)FasL(+) exosomes among those secreted by LCL. Using two independent experimental approaches, we demonstrated that LCL-derived exosomes were capable of inducing antigen-specific apoptosis in autologous CD4(+) T cells. These results suggest that LCL-derived exosomes may present a realistic source of immunosuppressive exosomes that could reduce or eliminate T cell-mediated responses against donor-derived antigens in transplant recipients.

Interleukin-5 supports the expansion of fas ligand-expressing killer B cells that induce antigen-specific apoptosis of CD4(+) T cells and secrete interleukin-10.

Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL(+) killer B cells, showing they are enriched in the IgM(high)CD5(+)CD1d(high) B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL(+) B cells, but most FasL(+) B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL(+) B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL(+) cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4(+) T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL(+) B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders.

Multiple mechanisms of immune suppression by B lymphocytes.

Suppression of the immune system after the resolution of infection or inflammation is an important process that limits immune-mediated pathogenesis and autoimmunity. Several mechanisms of immune suppression have received a great deal of attention in the past three decades. These include mechanisms related to suppressive cytokines, interleukin (IL)-10 and transforming growth factor (TGF)-ß, produced by regulatory cells, and mechanisms related to apoptosis mediated by death ligands, Fas ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), expressed by killer or cytotoxic cells. Despite many lines of evidence supporting an important role for B lymphocytes as both regulatory and killer cells in many inflammatory settings, relatively little attention has been given to understanding the biology of these cells, their relative importance or their usefulness as therapeutic targets. This review is intended to give an overview of the major mechanisms of immunosuppression used by B lymphocytes during both normal and inflammatory contexts. The more recent discoveries of expression of granzyme B, programmed death 1 ligand 2 (PD-L2) and regulatory antibody production by B cells as well as the interactions of regulatory and killer B cells with regulatory T cells, natural killer T (NKT) cells and other cell populations are discussed. In addition, new evidence on the basis of independent characterizations of regulatory and killer CD5(+) B cells point toward the concept of a multipotent suppressor B cell with seemingly high therapeutic potential.

Single-nucleotide polymorphisms in the IL2RA gene are associated with age at diagnosis in late-onset Finnish type 1 diabetes subjects.

The onset of type 1 diabetes can occur at any age, with as many as half of all cases diagnosed after age 15. Despite this wide distribution in age at diagnosis, most genetic studies focus on cases diagnosed in childhood or during early adulthood. To better understand the genetics of late-onset type 1 diabetes, we collected a Finnish case/control cohort with all cases diagnosed between ages 15 and 40. We genotyped 591 probands and 1,538 control subjects at regions well established as susceptibility loci in early onset type 1 diabetes. These loci were then tested for disease association and age-at-diagnosis effects. Using logistic regression, we found that single-nucleotide polymorphisms (SNPs) at the INS, PTPN22, and IFIH1 loci were associated with late-onset disease (OR (95%CI) = 0.57(0.47-0.69), p = 2.77 x 10(-9); OR (95%CI) = 1.50 (1.27-1.78), p = 3.98 x 10(-6); and OR (95%CI) = 0.81(0.71-0.93), p = 0.0028, respectively). In contrast, a disease association was not detected for two SNPs at the IL2RA locus (rs11594656 and rs41295061). Despite this, we did find an independent age-at-diagnosis effect for each IL2RA SNP using a multivariate Cox proportional hazards model (p = 0.003, 0.002, respectively). Taken together, polymorphisms at the IL2RA locus were a major determinant of age at diagnosis in our cohort with an effect at par with the HLA-DQ2/DQ8 genotype as measured by hazard ratios. These findings suggest that the IL2RA locus controls both the susceptibility to disease and its time of occurrence. Thus, we believe the IL2/IL2R axis represents a potential therapeutic target for delaying the onset of disease.