Lytic cycle T cell epitopes are expressed in two distinct phases during MHV-68 infection.

Murine herpesvirus-68 (MHV-68) is a type 2 gamma herpesvirus that productively infects alveolar epithelial cells during the acute infection and establishes long-term latency in B cells and lung epithelial cells. In C57BL/6 mice, T cells specific for lytic cycle MHV-68 epitope p56/Db dominate the acute phase of the infection, whereas T cells specific for another lytic cycle epitope, p79/Kb, dominate later phases of infection. To further understand this response, we analyzed the kinetics of Ag presentation in vivo using a panel of lacZ-inducible T cell hybridomas specific for several lytic cycle epitopes, including p56/Db and p79/Kb. Two distinct peaks of Ag presentation were observed. The first peak was at day 6 in the mediastinal lymph nodes and correlated with the initial pulmonary lytic infection. The second peak was at day 18 in both the mediastinal lymph nodes and spleen and correlated with the peak of latent infection. Interestingly, the p56 epitope was detected only in the mediastinal lymph nodes at day 6 after infection whereas the p79 epitope was predominantly presented in the spleen at day 18, suggesting that differential epitope presentation drives the switch in T cell responses to this virus. Phenotypic analysis of APCs at day 18 postinfection revealed that dendritic cells, macrophages, and B cells all presented lytic cycle epitopes. Taken together, the data suggest that there is a resurgence of lytic cycle Ags in the spleen, which explains the kinetics and specificity of the T cell response to MHV-68.     

Antibody-independent antiviral function of memory CD4+ T cells in vivo requires regulatory signals from CD8+ effector T cells

Previous studies have shown that vaccine-primed CD4(+) T cells can mediate accelerated clearance of respiratory virus infection. However, the relative contributions of Ab and CD8(+) T cells, and the mechanism of viral clearance, are poorly understood. Here we show that control of a Sendai virus infection by primed CD4(+) T cells is mediated through the production of IFN-gamma and does not depend on Ab. This effect is critically dependent on CD8(+) cells for the expansion of CD4(+) T cells in the lymph nodes and the recruitment of memory CD4(+) T cells to the lungs. Passive transfer of a CD8(+) T cell supernatant into CD8(+) T cell-depleted, hemagglutinin-neuraminidase (HN)(421-436)-immune muMT mice substantially restored the virus-specific memory CD4(+) response and enhanced viral control in the lung. Together, the data demonstrate for the first time that in vivo primed CD4(+) T cells have the capacity to control a respiratory virus infection in the lung by an Ab-independent mechanism, provided that CD8(+) T cell "help" in the form of soluble factor(s) is available during the virus infection. These studies highlight the importance of synergistic interactions between CD4(+) and CD8(+) T cell subsets in the generation of optimal antiviral immunity.     

Dynamics of immune cell recruitment during West Nile encephalitis and identification of a new CD19+B220-BST-2+ leukocyte population

West Nile virus (WNV) is an emerging neurotropic flavivirus. We investigated the dynamics of immune cell recruitment in peripheral tissues and in the CNS during WNV encephalitis in an immunocompetent mouse model. In the periphery, immune cell expansion can successfully limit viremia and lymphoid tissue infection. However, viral clearance in the periphery is too late to prevent viral invasion of the CNS. In the CNS, innate immune cells, including microglia/macrophages, NK cells, and plasmacytoid dendritic cells, greatly expand as the virus invades the brain, whereas B and T cells are recruited after viral invasion, and fail to control the spread of the virus. Thus, the onset of WNV encephalitis was correlated both with CNS viral infection and with a large local increase of innate immune cells. Interestingly, we identify a new immune cell type: CD19(+)B220(-) BST-2(+), which we name G8-ICs. These cells appear during peripheral infection and enter the CNS. G8-ICs express high levels of MHC class II, stain for viral Ag, and are localized in the paracortical zone of lymph nodes, strongly suggesting they are previously unidentified APCs that appear in response to viral infection.     

CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421-436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.     

Nonspecific recruitment of memory CD8+ T cells to the lung airways during respiratory virus infections

Previous studies have shown that heterologous viral infections have a significant impact on pre-existing memory T cell populations in secondary lymphoid organs through a combination of cross-reactive and bystander effects. However, the impact of heterologous viral infections on effector/memory T cells in peripheral sites is not well understood. In this study, we have analyzed the impact of a heterologous influenza virus infection on Sendai virus-specific CD8(+) effector/memory cells present in the lung airways. The data show a transient increase in the numbers of Sendai virus nucleoprotein 324-332/K(b)-specific CD8(+) memory T cells in the airways of the influenza-infected mice peaking around day 4 postinfection. Intratracheal transfer studies and 5-bromo-2'-deoxyuridine incorporation demonstrate that this increase is due to the recruitment of resting memory cells into the airways. In addition, the data show that these immigrating memory cells are phenotypically distinct from the resident memory T cells of the lung airways. A similar influx of nonproliferating Sendai virus nucleoprotein 324-332/K(b)-specific CD8(+) memory T cells is also induced by a secondary (homologous) infection with Sendai virus. Together, these data suggest that inflammation can accelerate memory T cell migration to nonlymphoid tissues and is a part of the normal recall response during respiratory infections.     

Colony-stimulating factor (CSF-1): its enhancement of plasminogen activator production and inhibition of cell growth in a mouse macrophage cell line

The cell-mediated immune response by the gut-associated lymphoid tissues to antigens within the intestinal tract is poorly understood. Our objective was to investigate the antigen-specific T cell proliferative response and cytotoxic T lymphocyte (CTL) response of cells from the GALT after enteric immunization with vaccinia virus. Lymphocytes able to proliferate in the presence of vaccinia virus in vitro were found in large numbers in mesenteric lymph nodes (MLN) 6 days after the injection of vaccinia virus into the lumen of the small bowel. The MLN at this time also contained vaccinia-specific CTL, but unlike the proliferating cells, which were found for several weeks after immunization, the CTL were demonstrable in the MLN for only a few days. Peyer's patches were found to contain neither antigen-stimulated proliferating cells nor CTL. The viral-specific proliferating lymphocytes from the MLN 10 days after immunization were sIg-, monoclonal antibody W3/25+, MRC OX-8- large lymphoblasts. The vaccinia-specific CTL were also large lymphoblasts, but they belonged to the W3/25-, OX-8+ subset. Thus, a strong T helper and cytotoxic T lymphoblast response is generated within the MLN after viral challenge of the gut.     

Antigen presenting cells (APCs) from thermally injured and/or septic rats modulate CD4+ T cell responses of naive rat

Regulation of immune response is marked by complex interactions among the cells that recognize and present antigens. Antigen presenting cells (APCs), the antigen presenting cell component of the innate immune response plays an important role in effector CD4+ T cell response. Thermal injury and/or superimposed sepsis in rats' leads to suppressed CD4+ T cell functions. We investigated modulations of CD4+ T cell function by APCs (purified non-T cells) from thermally injured and/or septic rats. Rats were subjected to 30% total body surface area scald burn or exposed to 37 degrees C water (Sham burn) and sepsis was induced by cecal-ligation and puncture (CLP) method. At day 3 post-injury animals were sacrificed and CD4+ T cells and APCs from mesenteric lymph nodes (MLN) were obtained using magnetic microbead isolation procedure. APCs from injured rats were co-cultured with sham rat MLN CD4+ T cells and proliferative responses (thymidine incorporation), phenotypic changes (Flow cytometry), IL-2 production (ELISA) and CTLA-4 mRNA (RT-PCR) were determined in naive rat CD4+ T cells. The data indicate that APCs from thermally injured and/or septic rats when co-cultured with CD4+ T cells suppressed CD4+ T cell effector functions. This lack of CD4+ T cell activation was accompanied with altered co-stimulatory molecules, i.e., CD28 and/or CTLA-4 (CD152). In conclusion, our studies indicated that defective APCs from thermally injured and/or septic rats modulate CD4+ T cell functions via changes in co-stimulatory molecules expressed on naive CD4+ T cells. This altered APC: CD4+ T cell interaction leads to suppressed CD4+ T cell activation of healthy animals.     

Activation, differentiation, and migration of naive virus-specific CD8+ T cells during pulmonary influenza virus infection

The low precursor frequency of individual virus-specific CD8(+) T cells in a naive host makes the early events of CD8(+) T cell activation, proliferation, and differentiation in response to viral infection a challenge to identify. We have therefore examined the response of naive CD8(+) T cells to pulmonary influenza virus infection with a murine adoptive transfer model using hemagglutinin-specific TCR transgenic CD8(+) T cells. Initial activation of CD8(+) T cells occurs during the first 3 days postinfection exclusively within the draining lymph nodes. Acquisition of CTL effector functions, including effector cytokine and granule-associated protease expression, occurs in the draining lymph nodes and differentially correlates with cell division. Division of activated CD8(+) T cells within the draining lymph nodes occurs in an asynchronous manner between days 3 and 4 postinfection. Despite the presence of Ag for several days within the draining lymph nodes, dividing T cells do not appear to maintain contact with residual Ag. After multiple cell divisions, CD8(+) T cells exit the draining lymph nodes and migrate to the infected lung. Activated CD8(+) T cells also disseminate throughout lymphoid tissue including the spleen and distal lymph nodes following their emigration from draining lymph nodes. These results demonstrate an important role for draining lymph nodes in orchestrating T cell responses during a local infection of a discrete organ to generate effector CD8(+) T cells capable of responding to infection and seeding peripheral lymphoid tissues.     

APCs in the liver and spleen recruit activated allogeneic CD8+ T cells to elicit hepatic graft-versus-host disease

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8(+) T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8(+) T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8(+) T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8(+) T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.     

NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins

We recently demonstrated that CD1d-restricted NKT cells resident in skin can inhibit CD8 T cell-mediated graft rejection of human papillomavirus E7-expressing skin through an IFN-γ-dependent mechanism. In this study, we examined the role of systemically derived NKT cells in regulating the rejection of skin grafts expressing viral proteins. In lymph nodes draining transplanted skin, Ag-specific CD8 T cell proliferation, cytokine production, and cytotoxic activity were impaired by NKT cells. NKT cell suppression was mediated via CD11c(+) dendritic cells. Inhibition of CD8 T cell function did not require Foxp3(+) regulatory T cells or NKT cell-secreted IFN-γ, IL-10, or IL-17. Thus, following skin grafting or immunization with human papillomavirus-E7 oncoprotein, NKT cells reduce the capacity of draining lymph node-resident APCs to cross-present Ag to CD8 T cell precursors, as evidenced by impaired expansion and differentiation to Ag-specific CD8 T effector cells. Therefore, in the context of viral Ag challenge in the skin, systemic NKT cells limit the capacity for effective priming of adaptive immunity.     

Protection against influenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (T(CM)). In this study, using relB(-/-) mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP(366-374)) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.     

Targeting the gut vascular endothelium induces gut effector CD8 T cell responses via cross-presentation by dendritic cells

Systemic delivery of Ag usually induces poor mucosal immunity. To improve the CD8 T cell response at mucosal sites, we targeted the Ag to MAdCAM-1, a mucosal addressin cell adhesion molecule expressed mainly by high endothelial venules (HEV) in mesenteric lymph nodes (MLN) and Peyer's patches of gut-associated lymphoid tissue. When chemical conjugates of anti-MAdCAM-1 Ab and model Ag OVA were injected i.v., a greatly enhanced proliferative response of Ag-specific OT-I CD8 T cells was detected in MLN. This was preceded by prolonged accumulation, up to 2 wk, of the anti-MAdCAM OVA conjugate on HEV of Peyer's patches and MLN. In contrast, nontargeted OVA conjugate was very inefficient in inducing OT-I CD8 T cell proliferation in MLN and required at least 20-fold more Ag to induce a comparable response. In addition, MAdCAM targeting elicits an endogenous OVA-specific CD8 T cell response, evident by IFN-gamma production and target killing. Induced response offers protection against an OVA-expressing B cell lymphoma. We propose that the augmentation of gut CD8 T cell responses by MAdCAM targeting is due to both accumulation of Ag in the HEV and conversion of a soluble Ag to a cell-associated one, allowing cross-presentation by DCs.     

The CD8+ T cell population elicited by recombinant adenovirus displays a novel partially exhausted phenotype associated with prolonged antigen presentation that nonetheless provides long-term immunity

We have previously reported that the CD8+ T cell response elicited by recombinant adenovirus vaccination displayed a delayed contraction in the spleen. In our current study, we demonstrate that this unusual kinetic is a general phenomenon observed in multiple tissues. Phenotypic analysis of transgene-specific CD8+ T cells present 30 days postimmunization with recombinant adenovirus revealed a population with evidence of partial exhaustion, suggesting that the cells had been chronically exposed to Ag. Although Ag expression could no longer be detected 3 wk after immunization, examination of Ag presentation within the draining lymph nodes demonstrated that APCs were loaded with Ag peptide for at least 40 days postimmunization, suggesting that Ag remains available to the system for a prolonged period, although the exact source of this Ag remains to be determined. At 60 days postimmunization, the CD8+ T cell population continued to exhibit a phenotype consistent with partially exhausted effector memory cells. Nonetheless, these CD8+ T cells conferred sterilizing immunity against virus challenge 7-12 wk postimmunization, suggesting that robust protective immunity can be provided by CD8+ T cells with an exhausted phenotype. These data demonstrate that prolonged exposure to Ag may not necessarily impair protective immunity and prompt a re-evaluation of the impact of persistent exposure to Ag on T cell function.     

Herpes simplex virus infection of human dendritic cells induces apoptosis and allows cross-presentation via uninfected dendritic cells

HSV efficiently infects dendritic cells (DCs) in their immature state and induces down-regulation of costimulatory and adhesion molecules. As in mice, HSV infection of human DCs also leads to their rapid and progressive apoptosis, and we show that both early and late viral proteins contribute to its induction. Because topical HSV infection is confined to the epidermis, Langerhans cells are expected to be the major APCs in draining lymph nodes. However, recent observations in murine models show T cell activation to be mediated by nonepidermal DC subsets, suggesting cross-presentation of viral Ag. In this study we provide an explanation for this phenomenon, demonstrating that HSV-infected apoptotic DCs are readily phagocytosed by uninfected bystander DCs, which, in turn, stimulate virus-specific CD8+ T cell clones.     

Modeling of influenza-specific CD8+ T cells during the primary response indicates that the spleen is a major source of effectors

The biological parameters that determine the distribution of virus-specific CD8(+) T cells during influenza infection are not all directly measurable by experimental techniques but can be inferred through mathematical modeling. Mechanistic and semimechanistic ordinary differential equations were developed to describe the expansion, trafficking, and disappearance of activated virus-specific CD8(+) T cells in lymph nodes, spleens, and lungs of mice during primary influenza A infection. An intensive sampling of virus-specific CD8(+) T cells from these three compartments was used to inform the models. Rigorous statistical fitting of the models to the experimental data allowed estimation of important biological parameters. Although the draining lymph node is the first tissue in which Ag-specific CD8(+) T cells are detected, it was found that the spleen contributes the greatest number of effector CD8(+) T cells to the lung, with rates of expansion and migration that exceeded those of the draining lymph node. In addition, models that were based on the number and kinetics of professional APCs fit the data better than those based on viral load, suggesting that the immune response is limited by Ag presentation rather than the amount of virus. Modeling also suggests that loss of effector T cells from the lung is significant and time dependent, increasing toward the end of the acute response. Together, these efforts provide a better understanding of the primary CD8(+) T cell response to influenza infection, changing the view that the spleen plays a minor role in the primary immune response.     

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.     

A dominant Th epitope in influenza nucleoprotein. Analysis of the fine specificity and functional repertoire of T cells recognizing a single determinant.

The sequence 260-283 of the nucleoprotein (NP) of influenza A virus is an epitope recognized by virus-immune lymph node cells from CBA (H-2k), B6 (H-2b), and B10.S (H-2s) mice. Further analysis shows that there are at least two Th epitopes within this sequence: the one close to the N-terminal (p260-273) is recognized by T cells from CBA and B6 mice while that close to the carboxyl-terminal (p270-283) is a dominant Th determinant in B10.S mice. The fine specificity of the recognition of this epitope by NP-specific T cell clones is also studied. When B10.S mice were infected intranasally or i.v. with live influenza virus, or immunized by different ways with various Ag preparations, P270-283 persistently emerged as a dominant T cell epitope. Immunization of B10.S mice with peptide p270-283 induces T cells with different in vivo functions including class II-restricted cytotoxicity, cognate help for Ag-specific antibody synthesis and delayed type hypersensitivity. This may have important implications for the understanding of the differentiation and classification of subsets of CD4+ T cells. The corresponding sequence of the NP of an equine influenza virus, A/Eq/Prague/56, which has a substitution (leucine to proline) at position 283, was not recognized by the lymph node cells from mice primed with either A/Okuda or A/Eq/Prague. However, the peptide, p270-283(E), representing this sequence induced T cell responses to both human and equine viruses. The data are discussed with respect to the development of viral vaccines.     

Lymph node resident rather than skin-derived dendritic cells initiate specific T cell responses after Leishmania major infection

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11c(high)CD11b(high)Gr-1-CD8-langerin- DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11c(high) CD11b(high) DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.     

Initiation of immune responses in brain is promoted by local dendritic cells

The contribution of dendritic cells (DCs) to initiating T cell-mediated immune response in and T cell homing into the CNS has not yet been clarified. In this study we show by confocal microscopy and flow cytometry that cells expressing CD11c, CD205, and MHC class II molecules and containing fluorescently labeled, processed Ag accumulate at the site of intracerebral Ag injection. These cells follow a specific pattern upon migrating out of the brain. To track their pathway out of the CNS, we differentiated DCs from bone marrow of GFP-transgenic mice and injected them directly into brains of naive C57BL/6 mice. We demonstrate that DCs migrate from brain to cervical lymph nodes, a process that can be blocked by fixation or pertussis toxin treatment of the DCs. Injection of OVA-loaded DCs into brain initiates a SIINFEKL (a dominant OVA epitope)-specific T cell response in lymph nodes and spleen, as measured by specific tetramer and LFA-1 activation marker staining. Additionally, a fraction of activated SIINFEKL-specific T cells home to the CNS. Specific T cell homing to the CNS, however, cannot be induced by i.v. injection of OVA-loaded DCs alone. These data suggest that brain-emigrant DCs are sufficient to support activated T cells to home to the tissue of DC origination. Thus, initiation of immune reactivity against CNS Ags involves the migration of APCs from nervous tissue to peripheral lymphoid tissues, similarly to that in other organs.     

Specific immunotherapy by genetically engineered APCs: the "guided missile" strategy

We tested the hypothesis that APCs genetically engineered to present an Ag and to express Fas ligand (FasL) simultaneously can target and eliminate Ag-specific T cells. Transgenic T cells specific for influenza hemagglutinin (HA) were used as targets. We prepared recombinant vaccinia virus vectors (VVV) to transfer the gene constructs individually or simultaneously into APCs. We prevented unwanted viral replication by attenuating the VVVs with psoralen-UV light treatment. For presentation of the HA Ag, APCs were transduced with cDNA for HA flanked by sequences of the lysosome-associated membrane protein that direct efficient processing and presentation of the Ag by APCs. As a "warhead" for the APCs, we transduced them with the gene for FasL, which induces apoptosis of Fas-expressing activated T cells. To protect the transduced APCs from self-destruction by FasL, we transferred cDNA for a truncated form of Fas-associated death domain, which inhibits Fas-mediated cell death. Our results show that the engineered APCs effectively expressed the genes of interest. APCs transduced with VVV carrying all three gene constructs specifically killed HA-transgenic T cells in culture. Coculture with T cells specific for an unrelated Ag (OVA) had no significant effect. Our in vitro findings show that APCs can be genetically engineered to target and kill Ag-specific T cells and represent a promising novel strategy for the specific treatment of autoimmune diseases.