Alveolar epithelial cell chemokine expression triggered by antigen-specific cytolytic CD8(+) T cell recognition

CD8(+) T lymphocyte responses are a critical arm of the immune response to respiratory virus infection and may play a role in the pathogenesis of interstitial lung disease. We have shown that CD8(+) T cells induce significant lung injury in the absence of virus infection by adoptive transfer into mice with alveolar expression of a viral transgene. The injury is characterized by the parenchymal infiltration of host cells, primarily macrophages, which correlates with physiologic deficits in transgenic animals. CD8(+) T cell-mediated lung injury can occur in the absence of perforin and Fas expression as long as TNF-alpha is available. Here, we show that the effect of TNF-alpha expressed by CD8(+) T cells is mediated not exclusively by cytotoxicity, but also through the activation of alveolar target cells and their expression of inflammatory mediators. CD8(+) T cell recognition of alveolar cells in vitro triggered monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) expression in the targets, which was mediated by TNF-alpha. Antigen-dependent alveolar MCP-1 expression was observed in vivo as early as 3 hours after CD8(+) T cell transfer and depended upon TNF-R1 expression in transgenic recipients. MCP-1 neutralization significantly reduced parenchymal infiltration after T cell transfer. We conclude that alveolar epithelial cells actively participate in the inflammation and lung injury associated with CD8(+) T cell recognition of alveolar antigens.     

CD8(+) T cell-mediated injury in vivo progresses in the absence of effector T cells.

Tissue injury is a common sequela of acute virus infection localized to a specific organ such as the lung. Tissue injury is an immediate consequence of infection with lytic viruses. It can also result from the direct destruction of infected cells by effector CD8(+) T lymphocytes and indirectly through the action of the T cell-derived proinflammatory cytokines and recruited inflammatory cells on infected and uninfected tissue. We have examined CD8(+) T cell-mediated pulmonary injury in a transgenic model in which adoptively transferred, virus-specific cytotoxic T lymphocytes (CTLs) produce lethal, progressive pulmonary injury in recipient mice expressing the viral target transgene exclusively in the lungs. We have found that over the 4-5 day course of the development of lethal pulmonary injury, the effector CTLs, while necessary for the induction of injury, are present only transiently (24-48 h) in the lung. We provide evidence that the target of the antiviral CD8(+) T cells, the transgene expressing type II alveolar cells, are not immediately destroyed by the effector T cells. Rather, after T cell-target interaction, the type II alveolar cells are stimulated to produce the chemokine monocyte chemoattractant protein 1. These results reinforce the concept that, in vivo, the cellular targets of specific CTLs may participate directly in the development of progressive tissue injury by activating in response to interaction with the T cells and producing proinflammatory mediators without sustained in vivo activation of CD8(+) T cell effectors.     

Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury

The progression of kidney disease to renal failure correlates with infiltration of mononuclear immune cells into the tubulointerstitium. These infiltrates contain macrophages, DCs, and T cells, but the role of each cell type in disease progression is unclear. To investigate the underlying immune mechanisms, we generated transgenic mice that selectively expressed the model antigens ovalbumin and hen egg lysozyme in glomerular podocytes (NOH mice). Coinjection of ovalbumin-specific transgenic CD8⁺ CTLs and CD4⁺ Th cells into NOH mice resulted in periglomerular mononuclear infiltrates and inflammation of parietal epithelial cells, similar to lesions frequently observed in human chronic glomerulonephritis. Repetitive T cell injections aggravated infiltration and caused progression to structural and functional kidney damage after 4 weeks. Mechanistic analysis revealed that DCs in renal lymph nodes constitutively cross-presented ovalbumin and activated CTLs. These CTLs released further ovalbumin for CTL activation in the lymph nodes and for simultaneous presentation to Th cells by distinct DC subsets residing in the kidney tubulointerstitium. Crosstalk between tubulointerstitial DCs and Th cells resulted in intrarenal cytokine and chemokine production and in recruitment of more CTLs, monocyte-derived DCs, and macrophages. The importance of DCs was established by the fact that DC depletion rapidly resolved established kidney immunopathology. These findings demonstrate that glomerular antigen–specific CTLs and Th cells can jointly induce renal immunopathology and identify kidney DCs as a mechanistic link between glomerular injury and the progression of kidney disease.     

Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma

Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 x 10(6) DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 x 10(6) and 12 x 10(6) DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1-specific CD8(+) cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8(+) T cell infiltration, whereas nonregressing lesions lacked CD8(+) T cells as well as Mage-3 mRNA expression. This study proves the principle that DC "vaccines" can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8(+) CTL-tumor cell interaction in situ as well as escape by lack of tumor antigen expression.     

Immune-mediated inflammation directly impairs pulmonary function, contributing to the pathogenesis of Pneumocystis carinii pneumonia

The clinical severity of Pneumocystis carinii pneumonia (PCP) correlates closely with the appearance of pulmonary markers of inflammation. Therefore, a model system was developed whereby physiological studies could be performed on live mice to determine the extent to which pulmonary inflammation contributes to respiratory impairment during PCP. P. carinii-infected severe combined immunodeficient mice displayed little evidence of pulmonary inflammation and exhibited normal oxygenation and dynamic lung compliance. When comparably infected littermates were immunologically reconstituted, however, an intense immune-mediated inflammatory response was observed that resulted in significant decreases in both lung compliance and oxygenation. As the pneumonia resolved pulmonary function returned toward normal. To begin to define the cell populations contributing to inflammation-associated respiratory impairment during PCP, similar studies were performed in CD4(+) T cell-depleted mice. Mice depleted of both CD4(+) and CD8(+) cells developed infection, but they demonstrated neither abnormal lung compliance nor increased respiratory rate and displayed no markers of lung injury. In contrast, mice depleted of only CD4(+) T cells exhibited severe pulmonary inflammation and injury, decreased oxygenation and lung compliance, and increased respirations. Respiratory compromise was associated with the presence of activated CD8(+) cells and neutrophils in broncho-alveolar lavage fluid. These observations provide direct experimental evidence that the host's response to P. carinii directly impairs pulmonary function and contributes to the pathogenesis of PCP. Furthermore, CD8(+) T cells likely contribute to the respiratory compromise observed during PCP.     

Active participation of CCR5+CD8+ T lymphocytes in the pathogenesis of liver injury in graft-versus-host disease

We examined the molecular pathogenesis of graft-versus-host disease-associated (GVHD-associated) liver injury in mice, focusing on the role of chemokines. At the second week after cell transfer in the parent-into-F1 model of GVHD, CD8(+) T cells -- especially donor-derived CD8(+) T cells -- infiltrated the liver, causing both portal hepatitis and nonsuppurative destructive cholangitis (NSDC). These migrating cells expressed CCR5. Moreover, macrophage inflammatory protein-1alpha (MIP-1alpha), one of the ligands for CCR5, was selectively expressed on intralobular bile duct epithelial cells, endothelial cells, and infiltrating macrophages and lymphocytes. Administration of anti-CCR5 antibody dramatically reduced the infiltration of CCR5(+)CD8(+) T lymphocytes into the liver, and consequently protected against liver damage in GVHD. The levels of Fas ligand (FasL) mRNA expression in the liver were also decreased by anti-CCR5 antibody treatment. Anti-MIP-1alpha antibody treatment also reduced liver injury. These results suggest that MIP-1alpha-induced migration of CCR5-expressing CD8(+) T cells into the portal areas of the liver plays a significant role in causing liver injury in GVHD; thus, CCR5 and its ligand may be the novel target molecules of therapeutic intervention of hepatic GVHD.     

Identification of HLA-A2-restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells.

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.     

In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma

Although dendritic cells (DCs) play an important role in sensitization to inhaled allergens, their function in ongoing T helper (Th)2 cell-mediated eosinophilic airway inflammation underlying bronchial asthma is currently unknown. Here, we show in an ovalbumin (OVA)-driven murine asthma model that airway DCs acquire a mature phenotype and interact with CD4(+) T cells within sites of peribronchial and perivascular inflammation. To study whether DCs contributed to inflammation, we depleted DCs from the airways of CD11c-diphtheria toxin (DT) receptor transgenic mice during the OVA aerosol challenge. Airway administration of DT depleted CD11c(+) DCs and alveolar macrophages and abolished the characteristic features of asthma, including eosinophilic inflammation, goblet cell hyperplasia, and bronchial hyperreactivity. In the absence of CD11c(+) cells, endogenous or adoptively transferred CD4(+) Th2 cells did not produce interleukin (IL)-4, IL-5, and IL-13 in response to OVA aerosol. In CD11c-depleted mice, eosinophilic inflammation and Th2 cytokine secretion were restored by adoptive transfer of CD11c(+) DCs, but not alveolar macrophages. These findings identify lung DCs as key proinflammatory cells that are necessary and sufficient for Th2 cell stimulation during ongoing airway inflammation.     

Rapid cytotoxic T lymphocyte activation occurs in the draining lymph nodes after cutaneous herpes simplex virus infection as a result of early antigen presentation and not the presence of virus

Localized cutaneous herpes simplex virus type 1 (HSV-1) infection leads to arming and initial expansion of cytotoxic T lymphocytes (CTLs) in the draining popliteal lymph nodes (PLNs) followed by migration and further proliferation in the spleen. To accurately characterize the sequence of events involved in the activation and generation of anti-HSV CTLs, we used T cell receptor (TCR) transgenic mice specific for the immunodominant epitope from HSV glycoprotein B (gB(498-505)). We describe the detection of the initiation of antigen presentation in the draining lymph nodes by 4-6 h after infection with HSV-1. Analysis of CD69 up-regulation revealed activation of gB-specific CD8(+) T cells by 6-8 h after infection. Furthermore, we show that T cell proliferation begins no sooner than 24 h after activation and is marked by the concurrent appearance of CTL activity in the PLNs. These events are not dependent on the presence of virus in the draining lymph nodes, and suggest a requirement for recruitment of professional antigen-presenting cells to the site of T cell activation. Consequently, we have defined the initiation of the CD8(+) T cell-mediated response to cutaneous HSV-1 infection, demonstrating that the immune response to localized viral infection depends only on the appearance of cells presenting virus-derived antigen and commences with remarkable swiftness.     

Fatal rapidly progressive interstitial pneumonitis associated with amyopathic dermatomyositis and CD8 T lymphocytes

A patient with amyopathic dermatomyositis associated with fatal rapidly progressive interstitial pneumonitis resistant to therapy is described. Pathologic examination of a transbronchial lung biopsy specimen showed diffuse alveolar damage and nonspecific interstitial pneumonia-organizing pneumonia-like findings. Bronchoalveolar lavage fluid contained many CD8+ lymphocytes, considered to be cytotoxic T cells. Analysis of bronchoalveolar lavage fluid in this case may provide prognostically and pathogenetically important information.     

Increased frequency of antigen-specific CD8+ cytotoxic T lymphocytes infiltrating an Epstein-Barr virus–associated gastric carcinoma

Gastric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor beta chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24(+) fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV(+) gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I-restricted, antigen-specific CD8(+) CTLs are specifically expanded within EBV(+) gastric carcinoma tissue.     

Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses

Therapeutic efficacy of a tumor cell-based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte-associated antigen (CTLA)-4 pathway or the CD25(+) regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25(+) Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8(+) T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25(+) Treg cells indicates that CD25(+) Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity.     

Altering the distribution of Foxp3(+) regulatory T cells results in tissue-specific inflammatory disease

CD4(+)Foxp3(+) regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4(+)CD103(hi) phenotype. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4(+)Foxp3(+) T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease.     

Chemokine receptor CXCR3 facilitates CD8(+) T cell differentiation into short-lived effector cells leading to memory degeneration

Strength of inflammatory stimuli during the early expansion phase plays a crucial role in the effector versus memory cell fate decision of CD8(+) T cells. But it is not known how early lymphocyte distribution after infection has an impact on this process. We demonstrate that the chemokine receptor CXCR3 is involved in promoting CD8(+) T cell commitment to an effector fate rather than a memory fate by regulating T cell recruitment to an antigen/inflammation site. After systemic viral or bacterial infection, the contraction of CXCR3(-/-) antigen-specific CD8(+) T cells is significantly attenuated, resulting in massive accumulation of fully functional memory CD8(+) T cells. Early after infection, CXCR3(-/-) antigen-specific CD8(+) T cells fail to cluster at the marginal zone in the spleen where inflammatory cytokines such as IL-12 and IFN-α are abundant, thus receiving relatively weak inflammatory stimuli. Consequently, CXCR3(-/-) CD8(+) T cells exhibit transient expression of CD25 and preferentially differentiate into memory precursor effector cells as compared with wild-type CD8(+) T cells. This series of events has important implications for development of vaccination strategies to generate increased numbers of antigen-specific memory CD8(+) T cells via inhibition of CXCR3-mediated T cell migration to inflamed microenvironments.     

IFN-gamma protects short-term ovarian carcinoma cell lines from CTL lysis via a CD94/NKG2A-dependent mechanism

IFN-gamma regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor-mediated recognition by CD8(+) T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-gamma treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8(+) T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-gamma-treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-gamma. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-gamma modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.     

Viral escape by selection of cytotoxic T cell-resistant variants in influenza A virus pneumonia

Antigenic variation is a strategy exploited by influenza viruses to promote survival in the face of the host adaptive immune response and constitutes a major obstacle to efficient vaccine development. Thus, variation in the surface glycoproteins hemagglutinin and neuraminidase is reflected by changes in susceptibility to antibody neutralization. This has led to the current view that antibody-mediated selection of influenza A viruses constitutes the basis for annual influenza epidemics and periodic pandemics. However, infection with this virus elicits a vigorous protective CD8(+) cytotoxic T lymphocyte (CTL) response, suggesting that CD8(+) CTLs might exert selection pressure on the virus. Studies with influenza A virus-infected transgenic mice bearing a T cell receptor (TCR) specific for viral nucleoprotein reveal that virus reemergence and persistence occurs weeks after the acute infection has apparently been controlled. The persisting virus is no longer recognized by CTLs, indicating that amino acid changes in the major viral nucleoprotein CTL epitope can be rapidly accumulated in vivo. These mutations lead to a total or partial loss of recognition by polyclonal CTLs by affecting presentation of viral peptide by class I major histocompatibility complex (MHC) molecules, or by interfering with TCR recognition of the mutant peptide-MHC complex. These data illustrate the distinct features of pulmonary immunity in selection of CTL escape variants. The likelihood of emergence and the biological impact of CTL escape variants on the clinical outcome of influenza pneumonia in an immunocompetent host, which is relevant for the design of preventive vaccines against this and other respiratory viral infections, are discussed.     

Virus-induced hepatocellular carcinomas cause antigen-specific local tolerance

T cell surveillance is often effective against virus-associated tumors because of their high immunogenicity. It is not clear why surveillance occasionally fails, particularly against hepatitis B virus– or hepatitis C virus–associated hepatocellular carcinoma (HCC). We established a transgenic murine model of virus-induced HCC by hepatocyte-specific adenovirus-induced activation of the oncogenic SV40 large T antigen (TAg). Adenovirus infection induced cytotoxic T lymphocytes (CTLs) targeted against the virus and TAg, leading to clearance of the infected cells. Despite the presence of functional, antigen-specific T cells, a few virus-infected cells escaped immune clearance and progressed to HCC. These cells expressed TAg at levels similar to HCC isolated from neonatal TAg-tolerant mice, suggesting that CTL clearance does not select for cells with low immunogenicity. Virus-infected mice revealed significantly greater T cell infiltration in early-stage HCC compared with that in late-stage HCC, demonstrating progressive local immune suppression through inefficient T cell infiltration. Programmed cell death protein-1 (PD-1) and its ligand PD-L1 were expressed in all TAg-specific CD8⁺ T cells and HCC, respectively, which contributed to local tumor-antigen-specific tolerance. Thus, we have developed a model of virus-induced HCC that may allow for a better understanding of human HCC.     

Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice

Autoimmune diabetes in nonobese diabetic (NOD) mice results from destruction of pancreatic beta cells by T lymphocytes. It is believed that CD8(+) cytotoxic T lymphocytes (CTLs) effect the initial beta-cell insult in diabetes, but the mechanisms remain unclear. Studies of NOD.lpr mice have suggested that disease initiation is a Fas-dependent process, yet perforin-deficient NOD mice rarely develop diabetes despite expressing Fas. Here, we have investigated the role of perforin and Fas in the ability of beta cell-reactive CD8(+) T cells bearing a T-cell receptor (8.3-TCR) that is representative of TCRs used by CD8(+) CTLs propagated from the earliest insulitic lesions of NOD mice, and that targets an immunodominant peptide/H-2Kd complex on beta cells, to effect beta-cell damage in vitro and in vivo. In vitro, 8.3-CTLs killed antigenic peptide-pulsed non-beta-cell targets via both perforin and Fas, but they killed NOD beta cells via Fas exclusively. Perforin-deficient 8.3-TCR-transgenic NOD mice expressing an oligoclonal or monoclonal T-cell repertoire developed diabetes even more frequently than their perforin-competent littermates. These results demonstrate that diabetogenic CD8(+) CTLs representative of CTLs putatively involved in the initiation of autoimmune diabetes kill beta cells in a Fas-dependent and perforin-independent manner.