Cd8+ but Not Cd8− Dendritic Cells Cross-Prime Cytotoxic T Cells in Vivo

Bone marrow–derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD8+ T cells in a process referred to as cross-priming. Cross-priming is essential for the induction of CD8+ T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are capable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded β2-microglobulin–deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter associated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol transport. To determine whether a specific subset of splenic DCs is involved in this cross-presentation, we negatively and positively selected for CD8− and CD8+ DCs. Only the CD8+, and not the CD8−, DC subset demonstrates cross-priming ability. FACS® studies after injection of splenocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD8+ and the CD8− DC subsets, respectively, had one or more associated beads. These results indicate that CD8+ DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens

Tuning Notch signals in T cell development

Thesis (Ph. D.)--University of Washington, 2005.In this study, I have attempted to understand how Notch signals promote different stages of T cell maturation by examining the regulation of Notch signals on two levels. First, I have used an in vitro culture system to examine how differential signals through 2 classes of Notch ligands, Jagged and Delta, influence thymocyte differentiation. These data reveal that Notch signals inhibit B cell development and promote the maturation of immature thymocytes in two separable stages. While both classes of Notch ligands are able to inhibit B cell development, only Delta is able to promote the proliferation of immature thymocytes. Second, I have examined how Deltex, an intracellular modulator of Notch signals, regulates Notch signals by generating mice that are deficient in two of the three known Deltex homologues. Although there is considerable evidence that over-expression of Deltex in hematopoietic stem cells can inhibit Notch signals, and that Deltex is highly expressed in developing thymocytes, my data reveals that expression of Deltex in T cell progenitors is not essential for regulating the early stages of T cell maturation

T Cells Develop Normally in the Absence of both Deltex1 and Deltex2

Deltex1, Deltex2, and Deltex4 form a family of related proteins that are the mammalian homologues of Drosophila Deltex, a known regulator of Notch signals. Deltex1 is highly induced by Notch signaling in thymocytes, and overexpression of Deltex1 in T-cell progenitors can block Notch signals, suggesting that Deltex1 may play an important role in regulating Notch signals during T-cell development. A recent report found that T cells develop normally in mice carrying a targeted deletion in the Deltex1 gene (S. Storck, F. Delbos, N. Stadler, C. Thirion-Delalande, F. Bernex, C. Verthuy, P. Ferrier, J. C. Weill, and C. A. Reynaud, Mol. Cell. Biol. 25: 1437-1445, 2005), suggesting that other Deltex homologues may compensate in Deltex1-deficient T cells. We generated mice that lack expression of both Deltex1 and Deltex2 by gene targeting and further reduced expression of Deltex4 in Deltex1/Deltex2 double-deficient T-cell progenitors using RNA interference. Using a sensitive in vitro assay, we found that Notch signaling is more potent in cells expressing lower levels of Deltex proteins. Nevertheless, we were unable to detect any significant defects in thymocyte maturation in Deltex1/Deltex2 double-knockout mice. Together these data suggest that Deltex can act as a negative regulator of Notch signals in T cells but that endogenous levels of Deltex1 and Deltex2 are not important for regulating Notch signals during thymocyte development

Dexamethasone premedication suppresses vaccine-induced immune responses against cancer

Glucocorticosteroids (GCS) have an established role in oncology and are administered to cancer patients in routine clinical care and in drug development trials as co-medication. Given their strong immune-suppressive activity, GCS may interfere with immune-oncology drugs. We are developing a therapeutic cancer vaccine, which is based on a liposomal formulation of tumor-antigen encoding RNA (RNA-LPX) and induces a strong T-cell response both in mice as well as in humans. In this study, we investigated in vivo in mice and in human PBMCs the effect of the commonly used long-acting GCS Dexamethasone (Dexa) on the efficacy of this vaccine format, with a particular focus on antigen-specific T-cell immune responses. We show that Dexa, when used as premedication, substantially blunts RNA-LPX vaccine-mediated immune effects. Premedication with Dexa inhibits vaccine-dependent induction of serum cytokines and chemokines and reduces both the number and activation of splenic conventional dendritic cells (cDC) expressing vaccine-encoded antigens. Consequently, priming of functional effector T cells and therapeutic activity is significantly impaired. Interestingly, responses are less impacted when Dexa is administered post-vaccination. Consistent with this observation, although many inflammatory cytokines are reduced, IFNα, a key cytokine in T-cell priming, is less impacted and antigen expression by cDCs is intact. These findings warrant special caution when combining GCS with immune therapies relying on priming and activation of antigen-specific T cells and suggest that careful sequencing of these treatments may preserve T-cell induction

Lack of a functional MntABC system renders <i>S</i>. <i>aureus</i> more sensitive to killing by methyl viologen and human neutrophils but not murine macrophages, unless <i>S</i>. <i>aureus</i> is pre-exposed to oxidative stress.

<p>(A, B) Survival of wild-type and <i>mntC</i> mutant strains within neutrophils harvested from heparin-treated human blood (A) and INF-γ-activated murine macrophages (B). Bacteria were either untreated or pre-exposed to 1 μM methyl viologen for 1 hour. Neutrophils (A) and macrophages (B) were lysed after 90 min and 24 hours of infection, respectively, to enumerate CFU. Bars represent the mean value of triplicate samples and error bars are standard deviation. <i>P-</i>values were determined using one-way ANOVA with multiple comparisons between samples via Tukey’s post-test.</p