T cell receptor engagement leads to phosphorylation of clathrin heavy chain during receptor internalization

T cell receptor (TCR) internalization by clathrin-coated vesicles after encounter with antigen has been implicated in the regulation of T cell responses. We demonstrate that TCR internalization after receptor engagement and TCR signaling involves inducible phosphorylation of clathrin heavy chain (CHC) in both CD4+ and CD8+ human T cells. Studies with mutant Jurkat T cells implicate the Src family kinase Lck as the responsible enzyme and its activity in this process is influenced by the functional integrity of the downstream signaling molecule ZAP-70. CHC phosphorylation positively correlates with ligand-induced TCR internalization in both CD4+ and CD8+ T cells, and CHC phosphorylation as a result of basal Lck activity is also implicated in constitutive TCR endocytosis by CD4+ T cells. Remarkably, irreversible CHC phosphorylation in the presence of pervanadate reduced both constitutive and ligand-induced TCR internalization in CD4+ T cells, and immunofluorescence studies revealed that this inhibition affected the early stages of TCR endocytosis from the plasma membrane. Thus, we propose that CHC phosphorylation and dephosphorylation are involved in TCR internalization and that this is a regulatory mechanism linking TCR signaling to endocytosis.     

Adoptive immunotherapy induces CNS dendritic cell recruitment and antigen presentation during clearance of a persistent viral infection

Given the global impact of persistent infections on the human population, it is of the utmost importance to devise strategies to noncytopathically purge tissues of infectious agents. The central nervous system (CNS) poses a unique challenge when considering such strategies, as it is an immunologically specialized compartment that contains a nonreplicative cell population. Administration of exogenously derived pathogen-specific memory T cells (referred to as adoptive immunotherapy) to mice burdened with a persistent lymphocytic choriomeningitis virus (LCMV) infection from birth results in eradication of the pathogen from all tissues, including the CNS. In this study, we sought mechanistic insights into this highly successful therapeutic approach. By monitoring the migration of traceable LCMV-specific memory CD8+ T cells after immunotherapy, it was revealed that cytotoxic T lymphocytes (CTLs) distributed widely throughout the CNS compartment early after immunotherapy, which resulted in a dramatic elevation in the activity of CNS antigen-presenting cells (APCs). Immunotherapy induced microglia activation as well as the recruitment of macrophages and dendritic cells (DCs) into the brain parenchyma. However, DCs emerged as the only CNS APC population capable of inducing memory CTLs to preferentially produce the antiviral cytokine tumor necrosis factor-alpha, a cytokine demonstrated to be required for successful immunotherapeutic clearance. DCs were also found to be an essential element of the immunotherapeutic process because in their absence, memory T cells failed to undergo secondary expansion, and viral clearance was not attained in the CNS. These experiments underscore the importance of DCs in the immunotherapeutic clearance of a persistent viral infection and suggest that strategies to elevate the activation/migration of DCs (especially within the CNS) may facilitate pathogen clearance.     

Lung dendritic cells imprint T cell lung homing and promote lung immunity through the chemokine receptor CCR4

T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC–activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC–imprinted T cells protect against influenza more effectively than do gut and skin DC–imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC–activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC–activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4.CD4⁺ T cells orchestrate the recruitment and subsequent activation of innate and adaptive immune cells in the tissue through the production of cytokines and critically contribute to the generation of a robust immune response to invading pathogens (Reinhardt et al., 2006). A prerequisite for CD4⁺ T cell participation in host defense is their recruitment into peripheral nonlymphoid tissue both in response to pathogens and at homeostasis so that antigen-experienced T cells are positioned where pathogen reencounter is most likely to occur. The mechanisms that govern this strategic distribution of T cells into tissues are not fully defined.Organs with large epithelial surfaces such as the gut and the skin are in constant contact with the environment and are exposed to potential pathogens on a regular basis and therefore need an efficient immune response strategy to prevent infections at these sites. The unique structure and function of each organ determine its exposures and vulnerabilities to specific pathogens and make reexposure to a particular pathogen more likely in the same organ. For example, by virtue of its ecology, the gut is susceptible to infection with Salmonella and Shigella, organisms which are not pathogens in the skin. To streamline T cell memory immune responses based on the predictability of pathogen reexposure, the gut and the skin have evolved tissue-selective T cell imprinting, a process whereby DCs derived from the gut instruct T cells to home preferentially into the gut, whereas DCs derived from the skin instruct T cells to preferentially home into the skin. Gut DCs imprint the expression of α4β7 and CCR9 on T cells, and in doing so enable their entry into the small intestine in response to intestinal MAdCAM-1 and CCL25, respectively (Stagg et al., 2002; Johansson-Lindbom et al., 2003; Mora et al., 2003). Similarly, skin-derived DCs imprint T cell expression of P- and E-selectin ligands and CCR10, allowing T cell skin homing via cutaneous P- and E-selectins and CCL27, respectively (Campbell and Butcher, 2002; Dudda et al., 2004; Sigmundsdottir et al., 2007). This may explain why peripheral blood memory T cells that proliferate in response to rotavirus, a gut pathogen, are α4β7⁺ (Rott et al., 1997), whereas peripheral blood T cells specific for herpes simplex virus 2, a skin tropic virus, express high levels of CLA (Koelle et al., 2002). The unique structure and function of each organ also offer a distinct set of tissue-specific autoantigens, such that the autoantigens generated in the intestine would be different from those generated in the skin. Therefore, tissue-selective T cell imprinting might have also evolved to enable tissue-specific regulatory T cells to home efficiently to the location of their autoantigens.Tissue-selective T cell imprinting was described a decade ago for the gut and the skin and to date has not been shown for any other organ, raising the possibility that it might be a phenomenon restricted to these two organs. Like the gut and the skin, however, the lung is a large epithelial organ in continuous contact with the environment and potential pathogens. The lung also has its own unique structure and function, specific pathogen susceptibility (i.e., pneumococcus and influenza), and autoantigens and thus is poised to benefit from tissue-selective T cell imprinting. Understanding whether lung DCs imprint T cell lung homing is fundamental to understanding T cell immunity to inhaled antigens and pathogens. Whether lung DCs instruct T cells to home to the lung has implications for vaccine development and potentially opens novel therapeutic approaches for a variety of inflammatory T cell–mediated lung diseases. Here, we sought to determine whether lung DCs imprint T cell lung homing and, if so, the impact of lung imprinting on lung immunity.     

Selective reovirus infection of murine hepatocarcinoma cells during cell division. A model of viral liver infection.

Reovirus type 1, strain Lang (1/L), can infect hepatocytes in vivo only after hepatocellular damage is induced by hepatotoxins, surgical trauma, resection, or profound immunosuppression. To examine the role of cell cycle and cellular differentiation on liver cell susceptibility to reovirus infection, a murine hepatocarcinoma cell line, Hepa 1/A1, was infected with reovirus and assayed for the presence of infectious virus or reovirus antigen in cells. Despite a > 95% binding of reovirus to hepatocarcinoma cells as indicated by cytometric analysis; only 10% of hepatoma cells contained infectious virus by infectious center assay. In comparison, 100% of L cells were infected. Analysis of intracellular reovirus antigen revealed its presence in dividing but not in quiescent hepatocytes. This correlation of cellular division and cell capacity to support viral replication suggests that induction of hepatocyte proliferation may be a mechanism for liver susceptibility to reovirus infection.     

Characterization of murine lung dendritic cells: similarities to Langerhans cells and thymic dendritic cells

Dendritic cells (DC) are potent accessory cells (AC) for the initiation of primary immune responses. Although murine lymphoid DC and Langerhans cells have been extensively characterized, DC from murine lung have been incompletely described. We isolated cells from enzyme-digested murine lungs and bronchoalveolar lavages that were potent stimulators of a primary mixed lymphocyte response (MLR). The AC had a low buoyant density, were loosely adherent and nonphagocytic. AC function was unaffected by depletion of cells expressing the splenic DC marker, 33D1. In addition, antibody and complement depletion of cells bearing the macrophage marker F4/80, or removal of phagocytic cells with silica also failed to decrease AC activity. In contrast, AC function was decreased by depletion of cells expressing the markers J11d and the low affinity interleukin 2 receptor (IL-2R), both present on thymic and skin DC. AC function was approximately equal in FcR+ and FcR- subpopulations, indicating there was heterogeneity within the AC population. Consistent with the functional data, a combined two-color immunofluorescence and latex bead uptake technique revealed that lung cells high in AC activity were enriched in brightly Ia+ dendritic-shaped cells that (a) were nonphagocytic, (b) lacked specific T and B lymphocyte markers and the macrophage marker F4/80, but (c) frequently expressed C3biR, low affinity IL-2R, FcRII, and the markers NLDC-145 and J11d. Taken together, the functional and phenotypic data suggest the lung cells that stimulate resting T cells in an MLR and that might be important in local pulmonary immune responses are DC that bear functional and phenotypic similarity to other tissues DC, such as Langerhans cells and thymic DC.     

Reprogramming of antiviral T cells prevents inactivation and restores T cell activity during persistent viral infection

Failure to clear persistent viral infections results from the early loss of T cell activity. A pertinent question is whether the immune response is programmed to fail or if nonresponsive T cells can specifically be fixed to eliminate infection. Although evidence indicates that T cell expansion is permanently programmed during the initial priming events, the mechanisms that determine the acquisition of T cell function are less clear. Herein we show that in contrast to expansion, the functional programming of T cell effector and memory responses in vivo in mice is not hardwired during priming but is alterable and responsive to continuous instruction from the antigenic environment. As a direct consequence, dysfunctional T cells can be functionally reactivated during persistent infection even after an initial program of inactivation has been instituted. We also show that early therapeutic reductions in viral replication facilitate the preservation of antiviral CD4+ T cell activity, enabling the long-term control of viral replication. Thus, dysfunctional antiviral T cells can regain activity, providing a basis for future therapeutic strategies to treat persistent viral infections.     

Cross-presentation of glycolipid from tumor cells loaded with alpha-galactosylceramide leads to potent and long-lived T cell mediated immunity via dendritic cells

We report a mechanism to induce combined and long-lived CD4⁺ and CD8⁺ T cell immunity to several mouse tumors. Surprisingly, the initial source of antigen is a single low dose of tumor cells loaded with α-galactosylceramide (α-GalCer) glycolipid (tumor/Gal) but lacking co-stimulatory molecules. After tumor/Gal injection intravenously (i.v.), innate NKT and NK cells reject the tumor cells, some of which are taken up by dendritic cells (DCs). The DCs in turn cross-present glycolipid on CD1d molecules to NKT cells and undergo maturation. For B16 melanoma cells loaded with α-GalCer (B16/Gal), interferon γ–producing CD8⁺ T cells develop toward several melanoma peptides, again after a single low i.v. dose of B16/Gal. In all four poorly immunogenic tumors tested, a single dose of tumor/Gal i.v. allows mice to become resistant to tumors given subcutaneously. Resistance requires CD4⁺ and CD8⁺ cells, as well as DCs, and persists for 6–12 mo. Therefore, several immunogenic features of DCs are engaged by the CD1d-mediated cross-presentation of glycolipid-loaded tumor cells, leading to particularly strong and long-lived adaptive immunity.     

Type I IFN promotes NK cell expansion during viral infection by protecting NK cells against fratricide

Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor (Ifnar^−/−) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar^−/− NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar^−/− NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell–mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar^−/− NK cells into NK cell–deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN–dependent mechanism by which NK cells evade mechanisms of cell death after viral infection.Type I IFNs provide a potent line of antiviral defense through direct and indirect effects on cells of the immune system, leading to their activation and effector function (Biron, 2001; González-Navajas et al., 2012) and resulting in the attenuation of viral replication (Müller et al., 1994). IFN-α and IFN-β are members of the type I IFN family. All members of the type I IFN family signal through a ubiquitously expressed heterodimeric receptor that is composed of the IFN-α receptor 1 (IFNAR1) and IFNAR2 chains. Type I IFNs act directly on NK cells to promote their activation, cell cycle entry, and cytotoxic function during viral infection (Biron et al., 1984; Orange and Biron, 1996; Biron, 2001; Nguyen et al., 2002; Martinez et al., 2008; Baranek et al., 2012; Fortin et al., 2013). However, the experimental systems used in previous studies—direct infection of IFN receptor–deficient mice or WT mice with IFN neutralization—are complicated by potential differences in the degree of inflammation, effects on many cell types, and viral load. Thus, the direct influence of type I IFN on effector and long-lived antiviral NK cell responses, while eliminating pleotropic effects on other cells, has not been investigated previously.Although substantial amounts of type I IFN are produced during viral infection, this cytokine is constitutively present at basal levels and affects the development and homeostasis of various hematopoietic lineages (Honda et al., 2004; Sato et al., 2009; Gough et al., 2012). An indirect effect of type I IFN on NK cell development and maturation has been described recently (Mizutani et al., 2012; Guan et al., 2014). Because the prolific expansion and generation of memory NK cells during mouse cytomegalovirus (MCMV) infection are dependent predominantly on the proinflammatory cytokines IL-12 and IL-18 (Andoniou et al., 2005; Sun et al., 2012; Madera and Sun, 2015), it was of interest to determine whether type I IFNs play a role in these processes. Here, we use NK cells deficient in the IFNAR1 chain (Ifnar^−/−) in an adoptive cotransfer system and mixed bone marrow chimeric mice to investigate the direct influence of type I IFN signaling on NK cells responding against MCMV infection.     

Induction of high-affinity interleukin 1 receptor on human peripheral blood lymphocytes by glucocorticoid hormones

The in vitro effect of glucocorticoids (GCs) on IL-1-R expression of human PBMCs was investigated. Both physiological and pharmacological concentration ranges of GC increased the specific binding of 125I-labeled human rIL-1 alpha to PBMCs. This enhancement was specific for GC, since other steroid hormones, such as progesterone, 17 beta-estradiol, and testosterone failed to elevate the binding of 125I-IL-1 alpha to PBMCs. The effect was time dependent with maximal effect occurring 6 h after treatment and dose dependent with half-maximal effect elicited by 100 nM prednisolone. Scatchard plot analysis indicated that 125I-IL-1 alpha binding increased from approximately 100 IL-1-R per cell to 2 X 10(3) receptors per cell without a major change in affinity (Kd = 2.6 X 10(-10) M). The subpopulation of PBMCs induced by GC to express higher levels of IL-1-R consisted predominantly of B lymphocytes, but not T lymphocytes, large granular lymphocytes, or monocytes. GCs also induced the expression of IL-1-R on some other cell types, including normal human dermal fibroblasts and the human large granular lymphocyte cell line YT. Since cycloheximide and actinomycin D inhibited the induction of IL-1-R by GC, synthesis of both new RNA and protein seems to be required for IL-1-R induction. This study presents the first evidence of upregulation of the receptors for IL-1 by GC, and may account for the reported enhancement of in vitro and in vivo humoral immune responses by GCs.     

Malaria infection changes the ability of splenic dendritic cell populations to stimulate antigen-specific T cells

The capacity of splenic CD11c+ dendritic cell (DC) populations to present antigen (Ag) to T cells differs during malarial infection with Plasmodium chabaudi in mice. Both CD11c+ CD8+ and CD8- DCs presented malarial peptides on their surface during infection. However, although both DC subsets expressing malaria peptides could induce interferon-gamma production by CD4 T cells, only CD8- DCs isolated at the acute phase of infection stimulated Ag-specific T cell proliferation and interleukin (IL)-4 and -10 production from MSP1-specific T cell receptor for Ag transgenic T cells coincidental with our reported Th1 to Th2 switch at this stage in response to the pathogen. The timing of these distinct DC responses coincided with increased levels of apoptosis in the CD8+ population and an increase in the numbers of CD8- DCs in the spleen. Our data suggest that the switch in CD4 T cell responses observed in P. chabaudi-infected mice may be the result of the presentation by different DC populations modified by the malaria infection.     

Transfection of RNA encoding tumor antigens following maturation of dendritic cells leads to prolonged presentation of antigen and the generation of high-affinity tumor-reactive cytotoxic T lymphocytes.

Common tumor vaccination strategies utilizing peptide-pulsed dendritic cells (DC) are limited to targeting antigens with known epitopes in patients expressing a defined restricting allele and can result in the preferential induction of low-avidity T cells that fail to recognize tumor cells. The use of dendritic cells transfected with RNA encoding tumor antigen offers the prospect of antigen-specific immunization without requiring prior knowledge of the immunogenic epitope or restricting allele, since epitopes from the translated protein are processed by the endogenous antigen-presentation machinery. However, its use in vaccine studies has been limited by low RNA transfection efficiency and the use of immature DC as recipient cells. In this study, we report an RNA transfection strategy that routinely achieves expression in 40-50% of mature DC, which are better stimulator cells. Such RNA-transfected mature DC exhibited a prolonged duration of presentation of immunogenic epitopes compared to peptide-pulsed DC, induced greater frequencies of tumor antigen-specific CTL, and generated a CTL population that exhibited higher target avidity and increased tumor lytic capacity. These studies provide compelling in vitro data supporting the evaluation of RNA-transfected mature DC in vaccination protocols as a means to overcome several obstacles to generating anti-tumor responses in vivo.     

Human parainfluenza virus evolution during lung infection of immunocompromised individuals promotes viral persistence

The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN’s receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.     

IL-15 trans-presentation by pulmonary dendritic cells promotes effector CD8 T cell survival during influenza virus infection

We have recently demonstrated that peripheral CD8 T cells require two separate activation hits to accumulate to high numbers in the lungs after influenza virus infection: a primary interaction with mature, antigen-bearing dendritic cells (DCs) in the lymph node, and a second, previously unrecognized interaction with MHC I–viral antigen–bearing pulmonary DCs in the lungs. We demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis in the lungs; however, reconstitution with pulmonary plasmacytoid DCs and CD8α⁺ DCs promotes increased T cell survival and accumulation in the lungs. Further, our results show that the absence of DCs after influenza virus infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC–mediated rescue of virus-specific CD8 T cell responses in the lungs requires trans-presentation of IL-15 via DC-expressed IL-15Rα. This study demonstrates a key, novel requirement for DC trans-presented IL-15 in promoting effector CD8 T cell survival in the respiratory tract after virus infection, and suggests that this trans-presentation could be an important target for the development of unique antiviral therapies and more effective vaccine strategies.Clearance of a primary influenza A virus (IAV) infection is known to require killing of virus-infected host cells by activated, antigen-specific CD8 T cells in the lungs (Topham et al., 1997). Until recently, antigen-specific CD8 T cells were thought to undergo programmed activation, whereby a single, brief interaction with a mature, antigen-bearing DC in the LN was sufficient to induce a full program of activation, division, and differentiation from naive to mature, cytotoxic CD8 T cells (Kaech and Ahmed, 2001; Wong and Pamer, 2001). Increasing evidence has suggested, however, that activation of antigen-specific CD8 T cells is not as simple as previously thought, and multiple factors, including cytokine signals such as IL-2 (Wong and Pamer, 2004), IFN-α (Marrack et al., 1999; Price et al., 2000; Kolumam et al., 2005), and IL-12 (Curtsinger et al., 2003a,b; Trinchieri, 2003), and late co-stimulatory signals such as CD70 (Dolfi and Katsikis, 2007) and 4-1BBL (Bertram et al., 2002; Lin et al., 2009), can regulate and fine tune the magnitude and duration of the effector response, as well as the nature of the ensuing memory T cell population.We have recently demonstrated in a model of IAV infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDCs) and CD8α⁺ DCs, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells (McGill et al., 2008). Reconstitution of the lungs with physiological numbers of pDCs or CD8α⁺ DCs is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent on direct DC–T cell interactions, DC-expressed MHC I, and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DCs (aDCs) or alveolar macrophages (aMϕs) was unable to rescue the virus-specific CD8 T cell response (McGill et al., 2008). After IAV infection there is an abundance of IAV antigen– and MHC I–expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8α⁺ DC–mediated rescue of the T cell response in the lungs. Further, it remained unclear what mechanism was contributing to decreased numbers of IAV-specific CD8 T cells in the lungs of aDC-depleted mice: impaired DC migration from the lungs to the LN, impaired CD8 T cell proliferation within the lungs, or impaired CD8 T cell survival within the lungs. It was also unclear what mechanism pulmonary DC subsets were using to rescue this defect.The cytokine IL-15 has been demonstrated to play a key role in promoting lymphoid homeostasis, particularly with respect to CD8 T cells (Budagian et al., 2006; Kim et al., 2008). IL-15 was initially thought to signal similar to IL-2, whereby IL-15Rα formed a heterotrimeric complex with IL-2/IL15Rβ and common γ for high affinity signaling. Although this model appears to hold true in certain situations, recent reports have demonstrated a unique, alternative signaling mechanism, termed trans-presentation. In this model, IL-15Rα is required for the processing and presentation of active IL-15 in trans to cells expressing the IL-2/IL15Rβ–common γ chain complex (Sandau et al., 2004; Schluns et al., 2004; Kobayashi et al., 2005). At this time, it is unclear which cell types serve as the primary trans-presenting cells during an immune response; however, several lines of evidence have indicated that DCs may play an important role (Burkett et al., 2003, 2004). It is known that DCs express protein for both IL-15 and IL-15Rα, and that stimulation by IFN-αβ (Mattei et al., 2001) or IFN-γ (Doherty et al., 1996; Musso et al., 1999), or exposure to viral infection leads to further up-regulation of these molecules (Liu et al., 2000; Dubois et al., 2005; Budagian et al., 2006; Mattei et al., 2009). Interestingly, DCs matured in the presence of IL-15 have been demonstrated to promote enhanced antigen-specific CD8 T cell proliferation (Jinushi et al., 2003; Mattei et al., 2009) and a robust Th1 skewing in vivo (Pulendran et al., 2004).IL-15 has been best characterized for its role in maintaining memory CD8 T cell homeostasis, primarily through promoting enhanced basal proliferation (Becker et al., 2002; Goldrath et al., 2002; Schluns et al., 2002). More recently, however, there is accumulating evidence that IL-15 is also important for promoting primary effector CD8 T cell responses (Akbar et al., 1996; Bulfone-Paus et al., 1997; Vella et al., 1998; Schluns et al., 2002; Rausch et al., 2006; Yajima et al., 2006). Surface expression of both IL-15Rα and IL-2/IL15-Rβ is up-regulated after TCR activation (Vella et al., 1998), and IL-15 has been proposed to enhance activated CD8 T cell survival after challenge with staphylococcal enterotoxin A (Vella et al., 1998), Mycobacterium tuberculosis (Rausch et al., 2006), and vesicular stomatitis virus infection (Sandau et al., 2004). Collectively, these studies suggest a particularly important role for IL-15 in the generation and maintenance of an appropriate immune response; however, it remains unclear what role IL-15 plays during the effector phase of the immune response or in what context IL-15 contributes to activated CD8 T cell survival in vivo.In this study, we demonstrate a previously unrecognized role for pulmonary DC–mediated IL-15 trans-presentation in regulating virus-specific CD8 T cell responses in the lungs after IAV infection. The reduction in T cell numbers observed in the lungs of aDC-depleted mice after IAV challenge results not from impaired proliferation within the lungs but is caused by significantly increased levels of apoptosis of virus-specific CD8 T cells compared with nondepleted controls. Further, reconstitution with purified pDCs or CD8α⁺ DCs rescues the IAV-specific CD8 T cell response by promoting increased CD8 T cell survival in the lungs of aDC-depleted mice. Additionally, our results show that IAV infection induces up-regulation of both IL-15 mRNA and protein in the lungs and that depletion of aDCs at 48 h post infection (p.i.) results in a significant reduction in pulmonary IL-15 expression. Finally, this study demonstrates that pulmonary DCs prevent virus-specific CD8 T cell apoptosis through trans-presentation of IL-15, as blockade of IL-15 or IL-15Rα on the surface of pulmonary DCs before adoptive transfer, or transfer of IL-15^−/− pulmonary DC subsets ablates the rescue of the virus-specific CD8 T cell response in the lungs of aDC-depleted mice.     

Induction of leukemic-cell-specific cytotoxic T lymphocytes by autologous monocyte-derived dendritic cells presenting leukemic cell antigens

Leukemic-dendritic cells (leukemic-DCs) have certain limitations, which include difficult generation in 30-40% of patients, and low levels of expression of several key molecules. Therefore, an alternative approach using monocyte-derived DCs pulsed with tumor antigens is required. We investigated the possibility of immunotherapy for AML using leukemic-cell-specific cytotoxic T lymphocytes that were stimulated in vitro by autologous DCs pulsed with tumor antigens. To generate DCs, CD14(+) cells were isolated from peripheral blood mononuclear cells using magnetic-activated cell sorting, and cultured in the presence of GM-CSF and IL-4. On day 6, maturation of DCs was induced by addition of cytokine cocktail (TNF-alpha, IL-1beta, IL-6, and prostaglandin E(2)) for 2 days, and then the mature DCs were pulsed with whole leukemic cell lysates or apoptotic leukemic cells. There were no differences in the phenotypic expressions of mature DCs generated by pulsing with or without leukemic antigens. The mature DCs pulsed with tumor cell lysates or apoptotic leukemic cells showed a higher allostimulatory capacity for allogeneic CD3(+) T cells as compared with mature non-pulsed DCs. Autologous CD3(+) T cells stimulated by the mature pulsed DCs showed more potent cytotoxic activities against autologous leukemic cells than those stimulated by mature non-pulsed DCs. These results suggest that use of DCs pulsed with leukemic cell lysates or apoptotic leukemic cells is a feasible alternative immunotherapeutic approach to overcome the limitations of leukemic-DCs for the treatment of AML patients.     

冻融肺癌细胞对骨髓树突状细胞的免疫调节作用

背景:冷冻免疫是近年来倍受关注的课题,但冷冻对细胞免疫功能的影响一直缺乏深入的研究,特别是在细胞凋亡和树突状细胞方面.目的:观察体外培养的肺癌NCI-H446细胞经氩氦刀冻融处理后细胞形态和细胞表面免疫表型的变化,及其能否有效激发骨髓树突状细胞产生特异性抗瘤效应.设计、时间及地点:细胞免疫水平的对照实验,于2002-08/2003 04在解放军海军总医院血液实验室及解放军军事医学科学院细胞与基因治疗中心完成.材料:小细胞肺癌细胞系NCI-H446细胞购自上海科学院细胞库.方法:取体外培养的肺痛NCI-H446细胞经氩氦刀处理后,在体外树突状细胞培养过程中,加入冻融的肺癌细胞,分别观察肺癌细胞的形态结构、混合淋巴细胞反应及细胞毒T淋巴细胞杀癌细胞效应.主要观察指标:培养树突状细胞的细胞形态和细胞表面免疫表型变化,培养细胞的混合淋巴细胞反应结果,培养细胞的凋亡情况.结果:骨髓树突状细胞体外培养后,符合树突状细胞特征.在培养过程中加入冻融的肺癌细胞,不影响树突状细胞细胞表面抗原的表达,混合淋巴细胞反应增强,细胞毒T淋巴细胞可引起肺癌细胞凋亡.结论:经氩氦刀处理的肺痛细胞溶解,胞膜不完整,体外能有效激发骨髓树突状细胞产生特异性抗瘤效应.     

Activation of the D prostanoid 1 receptor suppresses asthma by modulation of lung dendritic cell function and induction of regulatory T cells

Prostaglandins (PGs) can enhance or suppress inflammation by acting on different receptors expressed by hematopoietic and nonhematopoietic cells. Prostaglandin D(2) binds to the D prostanoid (DP)1 and DP2 receptor and is seen as a critical mediator of asthma causing vasodilation, bronchoconstriction, and inflammatory cell influx. Here we show that inhalation of a selective DP1 agonist suppresses the cardinal features of asthma by targeting the function of lung dendritic cells (DCs). In mice treated with DP1 agonist or receiving DP1 agonist-treated DCs, there was an increase in Foxp3(+) CD4(+) regulatory T cells that suppressed inflammation in an interleukin 10-dependent way. These effects of DP1 agonist on DCs were mediated by cyclic AMP-dependent protein kinase A. We furthermore show that activation of DP1 by an endogenous ligand inhibits airway inflammation as chimeric mice with selective hematopoietic loss of DP1 had strongly enhanced airway inflammation and antigen-pulsed DCs lacking DP1 were better at inducing airway T helper 2 responses in the lung. Triggering DP1 on DCs is an important mechanism to induce regulatory T cells and to control the extent of airway inflammation. This pathway could be exploited to design novel treatments for asthma.     

Induction of cyclo-oxygenase-2 in non-small cell lung cancer cells by infection with DeltaE1, DeltaE3 recombinant adenovirus vectors

Infection of epithelial-derived cells by adenovirus vectors has myriad effects on cellular behavior and function. Some are relevant to the desired effect of the encoded transgene and therapeutic goals of gene therapy approach. The current experiments describe the induction of COX-2 protein and PGE-2 production by non-small cell lung cancer (NSCLC) cells following infection with a first generation (DeltaE1, DeltaE3) Ad vector. COX-2 overexpression by malignant cells has been shown to enhance cellular invasion, induce angiogenesis, regulate anti-apoptotic cellular defenses and augment immunologic resistance through production of PGE-2. Data show DeltaE1, DeltaE3, Ad5 vector infection induces dose-dependent increases in PGE-2 production by NSCLC cell lines. Data with UV/psoralen inactivated vectors and control vectors show this effect is dependent on Ad vector gene expression, but independent of the transgene expressed. Selective blockade of ERK with PD98029 abrogated induction of PGE-2 by Ad vectors. Consistent with these data, detectable increases in COX-2 protein were seen at 48 h after infection by Western blot that were paralleled by increases in the phosphorylation of ERK-1/2. UV/psoralen-inactivated vector did not induce COX-2 protein or ERK phosphorylation at 48 h. Further, an inhibitor of NF-kappa B (NFkappaB) translocation to the nucleus, SN50, had no effect on PGE-2 levels. In contrast, Ad vector infection did induce NFkappaB activity measured by NFkappaB-luciferase reporter plasmid, transfected into a NSCLC cell line. Collectively the data indicate DeltaE1, DeltaE3, Ad5 vector infection leads to ERK phosphorylation with parallel increases in COX-2 protein and PGE-2 production. These effects appear unrelated to NFkappaB and are dependent on gene expression by the vector. This information may need to be considered when defining targets for cancer gene therapy and/or the choice of viral vector.