No significant CTL cross-priming by dendritic cell-derived exosomes during murine lymphocytic choriomeningitis virus infection.

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells in culture, but are also present in serum. They contain a wide array of protein ligands on their surface, which has led to the hypothesis that they might mediate intercellular communication. Indeed, data support that exosomes can transfer Ags to dendritic cells (DC), and, interestingly, that these DC can subsequently induce T cell priming or tolerance. We have investigated whether this concept can be expanded to antiviral immunity. We isolated exosomes from supernatant of cultured bone marrow-derived DC (BMDC) that were infected with lymphocytic choriomeningitis virus (LCMV) or loaded with an immunodominant LCMV peptide, and characterized them by flow cytometry upon binding to beads. We then incubated the exosome preparations with BMDC and looked at their potential to activate LCMV gp33-specific naive and memory CD8 T cells. We found that exosomes do not significantly contribute to CD8 T cell cross-priming in vitro. Additionally, exosomes derived from in vitro-infected BMDC did not exhibit significant in vivo priming activity, as evidenced by the lack of protection following exosome vaccination. Thus, DC-derived exosomes do not appear to contribute significantly to CTL priming during acute LCMV infection.

T-bet controls autoaggressive CD8 lymphocyte responses in type 1 diabetes.

The T-box transcription factor T-bet is known to control lineage commitment and interferon-gamma production by T helper 1 (Th1) CD4 lymphocytes. We report here that T-bet is essential for development of CD8 lymphocyte-dependent autoimmune diabetes (type 1 diabetes [T1D]) in the rat insulin promoter-lymphocytic choriomeningitis virus (LCMV) transgenic model for virally induced T1D. In the absence of T-bet, autoaggressive (anti-LCMV) CD8 lymphocytes were reduced in number and produced less IFN-gamma, but increased IL-2 compared with controls. Further analysis showed that T-bet intrinsically controls the generation, but not apoptosis, maintenance, or secondary expansion of antiviral effector/memory CD8 lymphocytes. This observation points toward a therapeutic opportunity for the treatment of T1D and other autoimmune disorders.

Minimal impact of a de novo-expressed beta-cell autoantigen on spontaneous diabetes development in NOD mice.

During an autoimmune process, the autoaggressive response spreads from the initiating autoantigen to other antigens expressed in the target organ. Based on evidence from experimental models for multiple sclerosis, such "antigenic spreading" can play an important role in the exacerbation of clinical disease. We evaluated whether pathogenesis of spontaneous diabetes in NOD mice could be accelerated in a similar way when a novel autoantigen was expressed in pancreatic beta-cells. Unexpectedly, we found that the expression of the lymphocytic choriomeningitis virus nucleoprotein only led to marginal enhancement of diabetes, although such NOD-nucleoprotein mice were not tolerant to nucleoprotein. Although the frequency of nucleoprotein-specific CD8 T-cells in the pancreatic draining lymph node was comparable with the frequency of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific T-cells, more IGRP-specific CD8 T-cells were found both systemically and in the islets where there was a fourfold increase. Interestingly, and in contrast to nucleoprotein-specific CD8 T-cells, IGRP-specific T-cells showed increased CXCR3 expression. Thus, autoreactivity toward de novo-expressed beta-cell autoantigens will not accelerate autoimmunity unless large numbers of antigen-experienced autoreactive T-cells expressing the appropriate chemokine receptors are present.

Altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in PECAM-1-deficient mice.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), a 130-kDa glycoprotein member of the Ig superfamily of transmembrane proteins, is expressed on endothelial cells, platelets, and subsets of leukocytes. It functions as a cell adhesion molecule as well as a scaffolding molecule capable of modulating cellular signaling pathways. In this study, using PECAM-1-deficient (KO) mice, as well as cells derived from these mice, we demonstrate that the absence of PECAM-1 expression is associated with an early onset of clinical symptoms during experimental autoimmune encephalomyelitis (EAE), a mouse model for the human autoimmune disease multiple sclerosis. During EAE, mononuclear cell extravasation and infiltration of the CNS occur at earlier time points in PECAM-KO mice than in wild-type mice. In vitro, T lymphocyte transendothelial migration across PECAM-KO endothelial cells is enhanced, regardless of expression of PECAM-1 on transmigrating T cells. Additionally, cultured PECAM-KO endothelial cells exhibit prolonged permeability changes in response to histamine treatment compared with PECAM-1-reconstituted endothelial cells. Lastly, we demonstrate an exaggerated and prolonged CNS vascular permeability during the development of EAE and a delay in restoration of dermal vascular integrity following histamine challenge in PECAM-KO mice.

Using regulatory APCs to induce/maintain tolerance.

It is well established that antigen-presenting cells (APCs) such as dendritic cells (DCs) possess potent immune-stimulatory function. They are considered to be the key driving element for most immune, autoimmune, and host-defense responses. However, recent evidence suggests that as much as APCs can turn things on, they also have the capability to turn things off. To summarize the evidence for such regulatory function of APCs is the purpose of this review article. We are just beginning to understand whether regulatory APC function can be mapped to a separate lineage of APCs or whether it is more commonly acquired under certain maturation conditions. Furthermore, it becomes apparent that it is important to consider differences between human and mouse DCs as well as splenic-, lymph node-, or blood-derived APCs. We believe that, in the upcoming years, better understanding of positive and negative roles of APCs in immune regulation will benefit both sides of APC therapy: their use in enhancing immunity, for example, in vaccine design and cancer, as well as their application for the treatment of autoimmunity.

Increased memory conversion of naïve CD8 T cells activated during late phases of acute virus infection due to decreased cumulative antigen exposure.

BACKGROUND: Memory CD8 T cells form an essential part of protective immunity against viral infections. Antigenic load, costimulation, CD4-help, cytokines and chemokines fluctuate during the course of an antiviral immune response thus affecting CD8 T cell activation and memory conversion. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, naïve TCR transgenic LCMV-specific P14 CD8 T cells engaged at a late stage during the acute antiviral LCMV response showed reduced expansion kinetics but greater memory conversion in the spleen. Such late activated cells displayed a memory precursor effector phenotype already at the peak of the systemic antiviral response, suggesting that the environment determined their fate during antigen encounter. In the spleen, the majority of late transferred cells exhibited a central memory phenotype compared to the effector memory displayed by the early transferred cells. Increasing the inflammatory response by exogenous administration of IFNγ, PolyI:C or CpG did not affect memory conversion in the late transferred group, suggesting that the diverging antigen load early versus later during acute infection had determined their fate. In agreement, reduction in the LCMV antigenic load after ribavirin treatment enhanced the contribution of early transferred cells to the long lasting memory pool. CONCLUSIONS/SIGNIFICANCE: Our results show that naïve CD8 cells, exposed to reduced duration or concentration of antigen during viral infection convert into memory more efficiently, an observation that could have significant implications for vaccine design.

Transforming growth factor-beta suppresses the activation of CD8+ T-cells when naive but promotes their survival and function once antigen experienced: a two-faced impact on autoimmunity.

OBJECTIVE: Transforming growth factor-beta (TGF-beta) can exhibit strong immune suppression but has also been shown to promote T-cell growth. We investigated the differential effect of this cytokine on CD8(+) T-cells in autoimmunity and antiviral immunity. RESEARCH DESIGN AND METHODS: We used mouse models for virally induced type 1 diabetes in conjunction with transgenic systems enabling manipulation of TGF-beta expression or signaling in vivo. RESULTS: Surprisingly, when expressed selectively in the pancreas, TGF-beta reduced apoptosis of differentiated autoreactive CD8(+) T-cells, favoring their expansion and infiltration of the islets. These results pointed to drastically opposite roles of TGF-beta on naïve compared with antigen-experienced/memory CD8(+) T-cells. Indeed, in the absence of functional TGF-beta signaling in T-cells, fast-onset type 1 diabetes caused by activation of naïve CD8(+) T-cells occurred faster, whereas slow-onset disease depending on accumulation and activation of antigen-experienced/memory CD8(+) T-cells was decreased. TGF-beta receptor-deficient CD8(+) T-cells showed enhanced activation and expansion after lymphocytic choriomeningitis virus infection in vivo but were more prone to apoptosis once antigen experienced and failed to survive as functional memory cells. In vitro, TGF-beta suppressed naïve CD8(+) T-cell activation and gamma-interferon production, whereas memory CD8(+) T-cells stimulated in the presence of TGF-beta showed enhanced survival and increased production of interleukin-17 in conjunction with gamma-interferon. CONCLUSIONS: The effect of TGF-beta on CD8(+) T-cells is dependent on their differentiation status and activation history. These results highlight a novel aspect of the pleiotropic nature of TGF-beta and have implications for the design of immune therapies involving this cytokine.

Immunomodulatory dendritic cells require autologous serum to circumvent nonspecific immunosuppressive activity in vivo.

In immunotherapy, dendritic cells (DCs) can be used as powerful antigen-presenting cells to enhance or suppress antigen-specific immunity upon in vivo transfer in mice or humans. However, to generate sufficient numbers of DCs, most protocols include an ex vivo culture step, wherein the cells are exposed to heterologous serum and/or antigenic stimuli. In mouse models of virus infection and virus-induced autoimmunity, we tested how heterologous serum affects the immunomodulatory capacity of immature DCs generated in the presence of IL-10 by comparing fetal bovine serum (FBS)- or normal mouse serum (NMS)-supplemented DC cultures. We show that FBS-exposed DCs induce a systemic immune deviation characterized by reduction of virus-specific T cells, delayed viral clearance, and enhanced systemic production of interleukin 4 (IL-4), IL-5, and IL-10 to FBS-derived antigens, including bovine serum albumin (BSA). By contrast, DCs generated in NMS-supplemented cultures modulated immunity and autoimmunity in an antigen-specific fashion. These cells did not induce systemic IL-4, IL-5, or IL-10 production and inhibited generation of virus-specific T cells or autoimmunity only if pulsed with a viral antigen. These data underscore the importance of using autologous serum-derived immature DCs in preclinical animal studies to accurately assess their immunomodulatory potential in future human therapeutic settings, where application of FBS is not feasible.

Regulatory T-cells in type 1 diabetes.

Type 1 diabetes is a T-cell-mediated autoimmune disease, resulting in destruction of the insulin-producing beta cells in the pancreas. Disease progression is thought to involve the action of T-cells, particularly those producing Th1-type cytokines. Given the complexity in understanding the precise etiology of autoimmune diseases, the diversity of autoantigens, and the variability that exists between individual patients, it might be very difficult to eliminate autoaggressive T-cell responses without resorting to generalized means of immunosuppression. However, recent evidence shows that autoimmune processes are composed not only of autoaggressive T-cell responses but also of autoreactive regulatory components. Enhancing regulatory T-cell responses, therefore, has become an area of intense focus as a means of treating autoimmune diseases like type 1 diabetes. This review will concentrate on two different types of regulatory T-cells, the naturally occurring ('professional') CD4+CD25+ T-cells and antigen-induced ('adaptive') CD4+ Th2-like regulatory T-cells.

Helicobacter-induced chronic active lymphoid aggregates have characteristics of tertiary lymphoid tissue.

Susceptible strains of mice that are naturally or experimentally infected with murine intestinal helicobacter species develop hepatic inflammatory lesions that have previously been described as chronic active hepatitis. The inflammatory infiltrates in some models of chronic autoimmunity or inflammation resemble tertiary lymphoid organs hypothesized to arise by a process termed lymphoid organ neogenesis. To determine whether hepatic inflammation caused by infection with helicobacter could give rise to tertiary lymphoid organs, we used fluorescence-activated cell sorting, immunohistochemistry, and in situ hybridization techniques to identify specific components characteristic of lymphoid organs in liver tissue sections and liver cell suspensions from helicobacter-infected mice. Small venules (high endothelial venules [HEVs]) in inflammatory lesions in Helicobacter species-infected livers were positive for peripheral node addressin. Mucosal addressin cell adhesion molecule also stained HEVs and cells with a staining pattern consistent with scattered stromal cells. The chemokines SLC (CCL 21) and BLC (CXCL13) were present, as were B220-positive B cells and T cells. The latter included a naïve (CD45lo-CD62Lhi) population. These findings suggest that helicobacter-induced chronic active hepatitis arises through the process of lymphoid organ neogenesis.

Antigen-driven effector CD8 T cell function regulated by T-bet.

Type 1 immunity relies on the differentiation of two major subsets of T lymphocytes, the CD4+ T helper (Th) cell and the CD8+ cytotoxic T cell, that direct inflammatory and cytotoxic responses essential for the destruction of intracellular and extracellular pathogens. In contrast to CD4 cells, little is known about transcription factors that control the transition from the CD8 naïve to effector cell stage. Here, we report that the transcription factor T-bet, known to regulate Th cell differentiation, also controls the generation of the CD8+ cytotoxic effector cell. Antigen-driven generation of effector CD8+ cells was impaired in OT-I T cell receptor transgenic mice lacking T-bet, resulting in diminished cytotoxicity and a marked shift in cytokine secretion profiles. Furthermore, mice lacking T-bet responded poorly to infection with lymphocytic choriomeningitis virus. T-bet is a key player in the generation of type 1 immunity, in both Th and T cytotoxic cells.