No abstract

The immune-mediated tissue destruction of graft- vs -host disease (GvHD) remains a major barrier to greater use of hematopoietic stem cell transplantation (HSCT). Mesenchymal stem cells (MSCs) have intrinsic immunosuppressive qualities and are being actively investigated as a therapeutic strategy for treating GvHD. We characterized Cymerus™ MSCs, which are derived from adult, induced pluripotent stem cells (iPSCs), and show they display surface markers and tri-lineage differentiation consistent with MSCs isolated from bone marrow (BM). Administering iPSC-MSCs altered phosphorylation and cellular localization of the T cell-specific kinase, Protein Kinase C theta (PKCθ), attenuated disease severity, and prolonged survival in a humanized mouse model of GvHD. Finally, we sevaluated a constellation of pro-inflammatory molecules on circulating PBMCs that correlated closely with disease progression and which may serve as biomarkers to monitor therapeutic response. Altogether, our data suggest Cymerus iPSC-MSCs offer the potential for an off-the-shelf, cell-based therapy to treat GvHD.

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB–DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.

Cross-protection between serologically distinct strains of influenza A virus (IAV) is mediated by memory CD8 T cells that recognize epitopes from conserved viral proteins. Early viral control begins with activation of tissue-resident memory CD8 T cells (T RM ) cells at the site of viral replication. These CD8 T cells do not act in isolation, as protection against disseminated infection is reinforced by multiple waves of effector cells (T EFF ) that enter the lungs with different kinetics. To define how a protective CTL response evolves, we compared the functional properties of antiviral CD8 T cells in the respiratory tract and local lymphoid tissues. When analyzed 30 dpi, large numbers of antiviral CD8 T cells in the lungs and mediastinal lymph nodes (MLNs) expressed canonical markers of T RM cells (CD69 and/or CD103). The check point inhibitor PD-1 was also highly expressed on NP-specific CD8 T cells in the lungs, while the ratios of CD8 T cells expressing CD69 and CD103 varied according to antigen specificity. We next used in vitro experiments to identify conditions that induce a canonical T RM phenotype and found that that naïve and newly activated CD8 T cells maintain CD103 expression during culture with transforming growth factor-beta (TGFβ), while central memory CD8 T cells (T CM ) do not express CD103 under similar conditions. In vivo experiments showed that the distribution of antiviral CTLs in the MLN changed when immune mice were treated with reagents that block interactions with PD-L1. Importantly, the lymphoid T RM cells were poised for early proliferation upon reinfection with a different strain of IAV and defenses in the lungs were augmented by a transient increase in numbers of T EFF cells at the site of infection. As the interval between infections increased, lymphoid T RM cells were replaced with T CM cells which proliferated with delayed kinetics and contributed to an exaggerated inflammatory response in the lungs.

The transmembrane receptor, Notch1 plays an important role during the differentiation of CD4 T cells into T helper (Th) subsets in the presence of appropriate cytokines, including differentiation into Th1 cells. MicroRNAs have also been shown to be important regulators of immune responses, including negatively regulating cytokine production by Th1 cells. The miR-29 family of microRNAs can act to inhibit tbx21 and ifng transcription, two important pro-inflammatory genes that are abundantly expressed in Th1 cells. Here we show that Notch1 may prime CD4 T cells to be responsive to Th1-polarizing cues through its early repressive effects on the miR-29 family of microRNAs. Using a combination of cell lines and primary cells, we demonstrate that Notch1 can repress miR-29a, miR-29b, and miR-29c transcription through a mechanism that is independent of NF-κB. We further show that this repression is mediated by canonical Notch signaling and requires active Mastermind like (MAML) 1, but this process is superseded by positive regulation of miR-29 in response to IFNγ at later stages of CD4 T cell activation and differentiation. Collectively, our data suggest an additional mechanism by which Notch1 signaling may fine-tune Th1 cell differentiation.