Cell-intrinsic regulation of murine dendritic cell function and survival by prereceptor amplification of glucocorticoid.

Although the inhibitory effects of therapeutic glucocorticoids (GCs) on dendritic cells (DCs) are well established, the roles of endogenous GCs in DC homeostasis are less clear. A critical element regulating endogenous GC concentrations involves local conversion of inactive substrates to active 11-hydroxyglucocorticoids, a reduction reaction catalyzed within the endoplasmic reticulum by an enzyme complex containing 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) and hexose-6-phosphate dehydrogenase (H6PDH). In this study, we found that this GC amplification pathway operates both constitutively and maximally in steady state murine DC populations and is unaffected by additional inflammatory stimuli. Under physiologic conditions, 11ßHSD1-H6PDH increases the sensitivity of plasmacytoid DCs (pDCs) to GC-induced apoptosis and restricts the survival of this population through a cell-intrinsic mechanism. Upon CpG activation, the effects of enzyme activity are overridden, with pDCs becoming resistant to GCs and fully competent to release type I interferon. CD8α(+) DCs are also highly proficient in amplifying GC levels, leading to impaired maturation following toll-like receptor-mediated signaling. Indeed, pharmacologic inhibition of 11ßHSD1 synergized with CpG to enhance specific T-cell responses following vaccination targeted to CD8α(+) DCs. In conclusion, amplification of endogenous GCs is a critical cell-autonomous mechanism for regulating the survival and functions of DCs in vivo.

A diametric role for OX40 in the response of effector/memory CD4+ T cells and regulatory T cells to alloantigen.

OX40 is a member of the TNFR superfamily that has potent costimulatory properties. Although the impact of blockade of the OX40-OX40 ligand (OX40L) pathway has been well documented in models of autoimmune disease, its effect on the rejection of allografts is less well defined. In this article, we show that the alloantigen-mediated activation of naive and memory CD4(+) T cells results in the induction of OX40 expression and that blockade of OX40-OX40L interactions prevents skin allograft rejection mediated by either subset of T cells. Moreover, a blocking anti-OX40 had no effect on the activation and proliferation of T cells; rather, effector T cells failed to accumulate in peripheral lymph nodes and subsequently migrate to skin allografts. This was found to be the result of an enhanced degree of cell death among proliferating effector cells. In clear contrast, blockade of OX40-OX40L interactions at the time of exposure to alloantigen enhanced the ability of regulatory T cells to suppress T cell responses to alloantigen by supporting, rather than diminishing, regulatory T cell survival. These data show that OX40-OX40L signaling contributes to the evolution of the adaptive immune response to an allograft via the differential control of alloreactive effector and regulatory T cell survival. Moreover, these data serve to further highlight OX40 and OX40L as therapeutic targets to assist the induction of tolerance to allografts and self-Ags.

Langerhans cells regulate cutaneous injury by licensing CD8 effector cells recruited to the skin.

Langerhans cells (LCs) are a distinct population of dendritic cells that form a contiguous network in the epidermis of the skin. Although LCs possess many of the properties of highly proficient dendritic cells, recent studies have indicated that they are not necessary to initiate cutaneous immunity. In this study, we used a tractable model of cutaneous GVHD, induced by topical application of a Toll-like receptor agonist, to explore the role of LCs in the development of tissue injury. By adapting this model to permit inducible and selective depletion of host LCs, we found that GVHD was significantly reduced when LCs were absent. However, LCs were not required either for CD8 T-cell activation within the draining lymph node or subsequent homing of effector cells to the epidermis. Instead, we found that LCs were necessary for inducing transcription of IFN-γ and other key effector molecules by donor CD8 cells in the epidermis, indicating that they license CD8 cells to induce epithelial injury. These data demonstrate a novel regulatory role for epidermal LCs during the effector phase of an inflammatory immune response in the skin.

Specificity for the tumor-associated self-antigen WT1 drives the development of fully functional memory T cells in the absence of vaccination.

Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.

A LAT-Based Signaling Complex in the Immunological Synapse as Determined with Live Cell Imaging Is Less Stable in T Cells with Regulatory Capability.

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIß inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.

The host environment regulates the function of CD8+ graft-versus-host-reactive effector cells.

We have examined how the host environment influences the graft-vs-leukemia (GVL) response following transfer of donor T cells to allogeneic chimeras. Donor T cells induce significant GVL when administered in large numbers to established mixed chimeras (MC). However, when using limiting numbers of T cells, we found that late transfer to MC induced less GVL than did early transfer to freshly irradiated allogeneic recipients. Late donor T cell transfer to MC was associated with marked accumulation of anti-host CD8 cells within the spleen, but delayed kinetics of differentiation, reduced expression of effector molecules including IFN-gamma, impaired cytotoxicity, and higher rates of sustained apoptosis. Furthermore, in contrast to the spleen, we observed a significant delay in donor CD8 cell recruitment to the bone marrow, a key location for hematopoietic tumors. Increasing the numbers of T cells transferred to MC led to the enhancement of CTL activity and detectable increases in absolute numbers of IFN-gamma(+) cells without inducing graft-vs-host disease (GVHD). TLR-induced systemic inflammation accelerated differentiation of functional CTL in MC but was associated with severe GVHD. In the absence of inflammation, both recipient T and non-T cell populations impeded the full development of GVHD-inducing effector function. We conclude that per-cell deficits in the function of donor CD8 cells activated in MC may be overcome by transferring larger numbers of T cells without inducing GVHD.

A simple method for measuring immune complex-mediated, Fc gamma receptor dependent antigen-specific activation of primary human T cells.

Immune complex (IC) deposition of IgG containing autologous antigens has been observed in autoimmunity. This can lead to IC-mediated antigen uptake and presentation by antigen presenting cells (APC) driving T cell dependent inflammation. IgG receptors (FcγRs) have been suggested to be involved in this process. Since ICs have been linked to autoimmune diseases, interfering with IC mediated effects on APCs and subsequent autoimmune T cell activation via FcγR blockade may be therapeutically beneficial. However, this is currently challenging due to a lack of translatable animal models and specific human in vitro assays to study IC-driven T cell responses. Here, we developed a simple cellular assay to study IC-mediated T cell activation in vitro using human peripheral blood mononuclear cells and tetanus toxoid as a model antigen. We observed that tetanus ICs led to a strong induction of T cell proliferation and release of pro-inflammatory cytokines, which are hallmarks of chronic inflammation. This process was exacerbated when compared to tetanus toxoid challenge alone. IC-mediated T cell effects were FcγR dependent and inhibited by high-dose intravenous IgG (IVIg), a drug often used for the clinical treatments of autoimmune diseases. Similar effects were also seen using a hepatitis antigen. Consequently, we propose our assay as a rapid yet robust alternative to more labour-intense and time-consuming protocols, for example involving separate maturation of dendritic cells followed by T cell co-culture to study antigen specific primary T cell activation.

Engineered hexavalent Fc proteins with enhanced Fc-gamma receptor avidity provide insights into immune-complex interactions.

Autoantibody-mediated diseases are currently treated with intravenous immunoglobulin, which is thought to act in part via blockade of Fc gamma receptors, thereby inhibiting autoantibody effector functions and subsequent pathology. We aimed to develop recombinant molecules with enhanced Fc receptor avidity and thus increased potency over intravenous immunoglobulin. Here we describe the molecular engineering of human Fc hexamers and explore their therapeutic and safety profiles. We show Fc hexamers were more potent than IVIG in phagocytosis blockade and disease models. However, in human whole-blood safety assays incubation with IgG1 isotype Fc hexamers resulted in cytokine release, platelet and complement activation, whereas the IgG4 version did not. We used a statistically designed mutagenesis approach to identify the key Fc residues involved in these processes. Cytokine release was found to be dependent on neutrophil FcγRIIIb interactions with L234 and A327 in the Fc. Therefore, Fc hexamers provide unique insights into Fc receptor biology.

Discovery of a Potent, Orally Bioavailable PI4KIIIß Inhibitor (UCB9608) Able To Significantly Prolong Allogeneic Organ Engraftment in Vivo.

The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.

Nonhematopoietic antigen blocks memory programming of alloreactive CD8+ T cells and drives their eventual exhaustion in mouse models of bone marrow transplantation.

Allogeneic blood or BM transplantation (BMT) is the most commonly applied form of adoptive cellular therapy for cancer. In this context, the ability of donor T cells to respond to recipient antigens is coopted to generate graft-versus-tumor (GVT) responses. The major reason for treatment failure is tumor recurrence, which is linked to the eventual loss of functional, host-specific CTLs. In this study, we have explored the role of recipient antigen expression by nonhematopoietic cells in the failure to sustain effective CTL immunity. Using clinically relevant models, we found that nonhematopoietic antigen severely disrupts the formation of donor CD8+ T cell memory at 2 distinct levels that operate in the early and late phases of the response. First, initial and direct encounters between donor CD8+ T cells and nonhematopoietic cells blocked the programming of memory precursors essential for establishing recall immunity. Second, surviving CD8+ T cells became functionally exhausted with heightened expression of the coinhibitory receptor programmed death-1 (PD-1). These 2 factors acted together to induce even more profound failure in long-term immunosurveillance. Crucially, the functions of exhausted CD8+ T cells could be partially restored by late in vivo blockade of the interaction between PD-1 and its ligand, PD-L1, without induction of graft-versus-host disease, suggestive of a potential clinical strategy to prevent or treat relapse following allogeneic BMT.

Redundant role for Zap70 in B cell development and activation.

Expression of the Syk family tyrosine kinase Zap70 is strongly correlated with poor clinical outcome in chronic lymphocytic leukemia, the most common human leukemia characterized by B cell accumulation. The expression of Zap70 may reflect the specific cell of origin of the tumor or may contribute to pathology. Thus, the normal role of Zap70 in B cell physiology is of great interest. While initial studies reported that Zap70 expression in the mouse was limited to T and NK cells, more recent work has shown expression in early B cell progenitors and in splenic B cells, suggesting that the kinase may play a role in the development or activation of B cells. In this study, we show that Zap70 is expressed in all developing subsets of B cells as well as in recirculating B cells, marginal zone B cells and peritoneal B1 cells. Analysis of Zap70-deficient mice shows no unique role for Zap70 in either the development of B cells or in their in vitro and in vivo activation. However, we show that Zap70 can rescue the defective positive selection of immature B cells into the recirculating pool in Syk-deficient mice, demonstrating functional redundancy between these two kinases.