Expression of αvβ3 and αvβ8 integrins during oligodendrocyte precursor differentiation in the presence and absence of axons

We have shown previously that switching of the αv-associated β1 and β5 integrin subunits during differentiation of myelin-forming oligodendrocytes may regulate important aspects of cell behaviour such as migration (Milner et al., 1996: J Neurosci 16:7240-7252). In this study we have examined the developmental regulation of other αv-associated β subunits in oligodendroglial cell cultures and also the control of their expression by neurons, using xenocultures to distinguish glial and neuronal integrins. We have found that oligodendroglia express αvβ8 in addition to the previously-described αvβ1, αvβ3, and αvβ5. β8 and β3 together comprise the 80kD band seen in αv immunoprecipitations that represents the most abundant αv-associated β subunit and show reciprocal patterns of expression during development. αvβ8 is expressed at high levels on oligodendrocyte precursors and differentiated oligodendrocytes but diminishes during the intermediate stages of differentiation. αvβ3, in contrast, shows an opposite pattern of expression, with the highest levels seen at the intermediate stages of differentiation and little expression on either oligodendrocyte precursors and differentiated oligodendrocytes. The expression of αvβ3 is not altered by coculture with neurons, unlike that of αvβ8, in which the decrease seen at the intermediate stages of differentiation is less marked in the presence of neurones. Our results confirm that switching of αv-associated β subunits is an important feature of oligodendrocyte differentiation and suggest that αvβ8 and αvβ3 have distinct functions during myelination

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/ macrophages

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers. © 1991.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Induction and Potential Reversal of a T Cell Exhaustion-Like State: In Vitro Potency Assay for Functional Screening of Immune Checkpoint Drug Candidates

Tumor infiltrating lymphocytes (TIL) entering the tumor microenvironment (TME) encounter many suppressive factors resulting in a spectrum of possible differentiation paths, many with overlapping characteristics. This differentiation spectrum includes T cell anergy, unresponsiveness, quiescence, tolerance, dysfunction/suppression, and exhaustion, each with many subtypes. Immune checkpoint blockade (ICB) cancer drug therapies have focused on reinvigorating exhausted T cells (T-EX) and dysfunctional T cells (herein referred to as TEX). Many factors have been attributed to as causative to this exhausted state including sustained surface expression of multiple co-inhibitory receptors, altered transcription factor expression, epigenetic rewiring, and dysregulated metabolism. Antigen persistence is necessary for driving TEX maintenance in both the chronic viral infection setting and cancer. In addition to persistent antigen exposure, TILs within the TME encounter numerous tumor-mediated immunosuppressive metabolic by-products, suppressive cytokines, hypoxia, and cellular debris which converge to suppress T cell function and uniquely alter its transcription factor profile. These suppressed or dysfunctional T cells are incapable of mounting an optimal anti-tumor response in part due to lack of fitness in competing for glucose and oxygen. This chapter describes two different potency assays: (a) first we describe optimization of a core recall antigen-based in vitro potency assay for screening of immunopotentiating drug candidates; (b) the second assay bundles the core assay with further addition of immunosuppressive agents derived from the TME making a customizable T cell exhaustion-like assay. Our recall antigen assay is being used as a tool for function-based screening of ICB drug candidates. In this assay healthy human Peripheral Blood Mononuclear Cells (PBMCs) are stimulated with peptide(s) and grown in culture for 1 week. Day 4 supernatants are functionally assayed by ELISA for IFN-gamma secretion, and cells are assayed on day 7 by flow cytometry for CD8(+) or CD4(+) T cell expansion by using a single or cocktail of pMHC tetramers. We have shown that in roughly 30% of donor PBMCs, ICB drugs such as pembrolizumab are able to boost both IFN-gamma secretion and antigen-specific recall. One potential explanation for this effect is that the observed increase in T cell co-inhibitory receptor expression and presumed co-inhibitory receptor downstream signaling is ameliorated with ICB drugs releasing these T cells from the repressive effects of these co-inhibitory receptors. We have further enhanced our recall assay to more closely resemble the TME by including metabolites and other suppressive agents at concentrations not normally encountered by T cells in a healthy environment. We show one example of this whereby addition of adenosine to the culture results in suppression of antigen-specific T cell expansion without affecting cell viability. Further, we screened several drug candidates on their ability to counteract the negative effects of adenosine and found that one of the drugs was indeed able to restore antigen-specific T cell expansion. Hence, this TME-based recall antigen assay allows for dissection of the effects of TME-based factors on donor-specific T cell response as well as drug candidate screening

Multiple environmental antigens may trigger autoimmunity in psoriasis through T-cell receptor polyspecificity

IntroductionPsoriasis is a T-cell mediated autoimmune skin disease. HLA-C*06:02 is the main psoriasis-specific risk gene. Using a Vα3S1/Vβ13S1 T-cell receptor (TCR) from a lesional psoriatic CD8+ T-cell clone we had discovered that, as an underlying pathomechanism, HLA-C*06:02 mediates an autoimmune response against melanocytes in psoriasis, and we had identified an epitope from ADAMTS-like protein 5 (ADAMTSL5) as a melanocyte autoantigen. The conditions activating the psoriatic autoimmune response in genetically predisposed individuals throughout life remain incompletely understood. Here, we aimed to identify environmental antigens that might trigger autoimmunity in psoriasis because of TCR polyspecificity.MethodsWe screened databases with the peptide recognition motif of the Vα3S1/Vβ13S1 TCR for environmental proteins containing peptides activating this TCR. We investigated the immunogenicity of these peptides for psoriasis patients and healthy controls by lymphocyte stimulation experiments and peptide-loaded HLA-C*06:02 tetramers.ResultsWe identified peptides from wheat, Saccharomyces cerevisiae, microbiota, tobacco, and pathogens that activated both the Vα3S1/Vβ13S1 TCR and CD8+ T cells from psoriasis patients. Using fluorescent HLA-C*06:02 tetramers loaded with ADAMTSL5 or wheat peptides, we find that the same CD8+ T cells may recognize both autoantigen and environmental antigens. A wheat-free diet could alleviate psoriasis in several patients.DiscussionOur results show that due to TCR polyspecificity, several environmental antigens corresponding to previously suspected psoriasis risk conditions converge in the reactivity of a pathogenic psoriatic TCR and might thus be able to stimulate the psoriatic autoimmune response against melanocytes. Avoiding the corresponding environmental risk factors could contribute to the management of psoriasis