Human placenta-derived adherent cells induce tolerogenic immune responses.

Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14(+) monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells.

Optimizing the production of suspension cells using the G-Rex "M" series.

Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo. To overcome this limitation, Wilson Wolf Manufacturing developed the G-Rex, a cell culture flask with a gas-permeable membrane at the base that supports large media volumes without compromising gas exchange. Although this culture platform has recently gained traction with the scientific community due to its superior performance when compared with traditional culture systems, the limits of this technology have yet to be explored. In this study, we investigated multiple variables including optimal seeding density and media volume, as well as maximum cell output per unit of surface area. Additionally, we have identified a novel means of estimating culture growth kinetics. All of these parameters were subsequently integrated into a novel G-Rex "M" series, which can accommodate these optimal conditions. A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium. The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.

Dominant expression of the inhibitory FcgammaRIIB prevents antigen presentation by murine plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are key regulators of the innate immune response, yet their direct role as APCs in the adaptive immune response is unclear. We found that unlike conventional DCs, immune complex (IC) exposed murine pDCs neither up-regulated costimulatory molecules nor activated Ag-specific CD4(+) and CD8(+) T cells. The inability of murine pDCs to promote T cell activation was due to inefficient proteolytic processing of internalized ICs. This defect in the IC processing capacity of pDCs results from a lack of activating FcgammaR expression (FcgammaRI, III, IV) and the dominant expression of the inhibitory receptor FcgammaRIIB. Consistent with this idea, transgenic expression of the activating human FcgammaRIIA gene, not present in the mouse genome, recapitulated the human situation and rescued IC antigenic presentation capacity by murine pDCs. The selective expression of FcgammaRIIB by murine pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. The unexpected difference between the mouse and human in the expression of activating/inhibitory FcgammaRs has implications for the role of pDCs in Ab-modulated autoimmunity and anti-viral immunity.

Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II.

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcgammaRgamma and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.

Perceiving the avidity of T cell activation can be translated into peripheral T cell regulation.

The structure recognized by regulatory T cells that enables them to discriminate self from nonself in the periphery is one of the central issues of regulatory T cell biology. A link between immunoregulation and self-nonself discrimination has emerged from experiments showing that Qa-1-restricted CD8(+) T cells selectively down-regulate target T cells activated by the intermediate avidity of their own T cell antigen receptor-ligand interactions. Because the peripheral self-reactive T cell repertoire is devoid of high-avidity T cells compared with the foreign-reactive repertoire, as a result of thymic negative selection, the selective down-regulation of intermediate but not high-avidity T cells enables the immune system to suppress autoimmunity without damaging the ongoing immune response to foreign pathogens. However, the molecular mechanism delineating how avidity of T cell activation is perceived by the regulatory T cells has not been elucidated. Here we show that a heat shock peptide (Hsp60sp), coupled with the MHC class Ib molecule Qa-1, is a surrogate target structure that is preferentially expressed at a higher level on the intermediate avidity T cells and specifically recognized by the Qa-1-restricted CD8(+) T cells. The biological significance of this observation was confirmed by the ability of Hsp60sp-loaded relevant dendritic cells to induce a Qa-1-restricted CD8(+) T cell-mediated protection from autoimmune encephalopathy in the experimental allergic encephalomyelitis model. Thus, perceiving the avidity of T cell activation can be translated into peripheral T cell regulation to discriminate self from nonself in the periphery to maintain self-tolerance.

An affinity/avidity model of peripheral T cell regulation.

We show in these studies that Qa-1-dependent CD8+ T cells are involved in the establishment and maintenance of peripheral self tolerance as well as facilitating affinity maturation of CD4+ T cells responding to foreign antigen. We provide experimental evidence that the strategy used by the Qa-1-dependent CD8+ T cells to accomplish both these tasks in vivo is to selectively downregulate T cell clones that respond to both self and foreign antigens with intermediate, not high or low, affinity/avidity. Thus, the immune system evolved to regulate peripheral immunity using a unified mechanism that efficiently and effectively permits the system to safeguard peripheral self tolerance yet promote the capacity to deal with foreign invaders.

A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm.

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.