Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.     

Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745-1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.     

CD45RA(bright)/CD11a(bright) CD8+ T cells: effector T cells

An important aspect of peripheral T cell development is the differentiation from naive into memory cells. To distinguish naive from memory cells, CD45RA and CD11a are commonly used: CD45RA+ or CD11a(dim) T cells are regarded as naive, while CD45RA- or CD11a(bright) T cells are thought to be of memory type. There is, however, a CD8+ T cell subset which is CD45RA+ and at the same time CD11a(bright). It increases with age and in patients with systemic viral infections, though its functional role in the immune response is unknown. In the present study, we give evidence that this subset is related to memory-like T cells as it produces IFN-gamma and tumor necrosis factor-alpha, contains high levels of perforin, and expresses CD95 in the same way as memory-type CD45RA-/CD11a(bright) CD8+ T cells. Since it contains a high percentage of CD28- and CD57+ cells, is increased in size and granularity, and is transiently expressed following in vitro stimulation of naive CD8+ T cells, we speculate that this subset mainly represents recently activated effector T cells that are able to interact with CD80 and CD86 (B7-1 and B7-2 respectively) negative tissue cells.     

CD8+ T cells in intracellular bacterial infections of mice

In the normal course of an immune response, both CD4+ and CD8+ T cells respond to each of the bacterial pathogens we have discussed and both responses may be required for the most potent immunity to infection. In this discussion, we have focused on the ability of these organisms to prime CD8+ T-cell responses in vivo and the ability of CD8+ T cells as sole mediators of acquired immunity, to protect against infection. It is clear that the vacuolar location of bacterial pathogens such as Salmonella or Mycobacteria does not prevent in vivo priming of CD8+ T-cell responses to these pathogens. However, vacuolar localization may affect the potency of CD8+ T-cell responses under experimental conditions that assess the capacity of CD8+ T cells as the sole mediators of acquired immunity. In the case of cytoplasmic L. monocytogenes, clear evidence exists that antigen-specific CD8+ T cells, in the absence of immune CD4+ T cells, can provide substantial acquired immunity to naive mice. Similar clear experimental results with Salmonella and Mycobacteria are lacking. Such results would provide stronger support for vaccines that elicit CD8+ T-cell responses to these vacuolar pathogens. Although our discussion has focused on only three specific organisms, we suggest that detection of an in vivo CD8+ T-cell response to a bacterial antigen does not ensure that the response will be protective against infection in a vaccine setting. In the case of Salmonella and Mycobacteria, this issue remains unresolved.     

CD8+ T cells clear influenza virus by perforin or Fas-dependent processes

Influenza virus infection is controlled in CD4-depleted mice that are also defective for the expression of either Fas (Fas-/-) or perforin (P-/-). Virus-immune P+/+ and P-/- CD8+ T cells can thus function in, respectively, a Fas-/- or Fas+/+ lung environment. The obvious question is whether the P-/- CD8+ set is effective in Fas-/- mice, a conclusion that would tend to favor cytokine secretion as the mode of virus clearance. Short term chimeras were made with P-/- bone marrow, P+/+ or P-/- T cells, and Fas+/+ or Fas-/- irradiated recipients. While the P+/+ CD8+ population cleared the virus from Fas+/+ and Fas-/- respiratory epithelium, the P-/- effectors were operational only if there was the potential for Fas to be expressed on radiation-resistant lung cells. Target cell destruction mediated via the Fas or perforin pathways is clearly the primary mechanism used by CD8+ T cells to terminate this viral pneumonia.     

Reduction of respiratory syncytial virus titer in the lungs of mice after intranasal immunization with a chimeric peptide consisting of a single CTL epitope linked to a fusion peptide

In the work described here, the effect of intranasal immunization of BALB/c mice with synthetic chimeric peptides consisting of a cytotoxic T-cell epitope (amino acids 81-95) from the M2 protein of respiratory syncytial virus (RSV) and a fusion peptide (amino acids 113-131) from the F1 protein of measles virus on response to challenge with RSV has been assessed. Three intranasal immunizations with the chimeric peptides without adjuvant induce peptide- and RSV-specific cytotoxic T-cell responses (CTL) at 1 or 3 weeks after the third immunization. The CTL responses significantly declined at 6 weeks after immunization. Furthermore, viral load in the lungs following challenge with RSV was significantly reduced in mice immunized with the F/M2:81-95 chimeric peptide compared to control animals at 1 or 3 weeks after immunization and no reduction of RSV titers was detectable 6 weeks after immunization. The CTL activity induced by F/M2:81-95 was therefore short-lived (less than 6 weeks) but was significantly correlated with the reduction in viral load in the lungs.     

CD8+ T cells are the effectors of the contact dermatitis induced by urushiol in mice and are regulated by CD4+ T cells

Nitric oxide (NO) reduces platelet aggregation in vitro. However, repeated measurements of platelet aggregation in infants and small children are impossible due to the large blood samples required. Instead, the expression of different platelet receptors mediating platelet adhesion (CD 36 and CD 42b), activation (CD 42b and CD 61) and aggregation (CD 41a) was measured repeatedly by flow cytometry. First, the expression of platelet receptors was quantified in platelet suspensions of 20 healthy volunteers after incubation with different concentrations of NO (0, 25, 100 and 640 ppm) and compared to changes in platelet aggregation and intrathrombocytic cGMP levels. It was then studied in 21 infants and children before, during and up to 3 days after cardiopulmonary bypass surgery. Seven of these patients required NO inhalation postoperatively. The in vitro experiments showed a reduced expression of the CD 41a, CD 42b and CD 61 receptors with increasing doses of NO, predominantly affecting the CD 41a receptor (-11% at 100 ppm and -20% at 640 ppm). This significant effect is in keeping with the observed NO-induced inhibition of platelet aggregation (-44% at 100 ppm) and the rise in platelet cGMP levels (+69% at 100 ppm). In patients without inhaled NO, the expression of CD 41a was slightly attenuated during cardiopulmonary bypass surgery (-15%) but increased significantly afterwards (2 h: +31%, 1st day: +129%, 2nd day: +120%, 3rd day: +111%). Comparable results were obtained regarding the other adhesion molecules CD 36, CD 42b and CD 61. In patients with inhaled NO the same pattern was observed and analysis of variance did not reveal any significant difference between both groups of patients. CONCLUSIONS: NO (> or = 100 ppm) decreases the expression of different platelet adhesion molecules and platelet aggregation, presumably via an increase in intracellular cGMP. However, due to the low dose range used in the clinical setting (1-40 ppm) this is clinically not relevant. Immediately after cardiopulmonary bypass surgery the expression of these adhesion molecules is reduced, but recovers on the 1st postoperative day.     

Rejection of cardiac xenografts by CD4+ or CD8+ T cells

We recently showed that brief complement inhibition induces accommodation of hamster cardiac transplants in nude rats. We have reconstituted nude rats carrying an accommodated xenograft with syngeneic CD4+ or CD8+ T cells to investigate the cellular mechanism of xenograft rejection. We show that CD4+ T cells can initiate xenograft rejection (10 +/- 1.7 days) by promoting production of IgG xenoreactive Abs (XAb). These XAb are able to activate complement as well as to mediate Ab-dependent cell-mediated cytotoxicity. Adoptive transfer of these XAb into naive nude rats provoked hyperacute xenograft rejection (38 +/- 13 min). The rejection was significantly (p < 0.001) delayed by cobra venom factor (CVF; 11 +/- 8 h in four of five cases) but was still more rapid than in control nude rats (3.3 +/- 0.5 days). CVF plus NK cell depletion further prolonged survival (>7 days in four of five cases; p < 0.01 vs CVF only). CD8+ T cell-reconstituted nude rats rejected their grafts later (19.4 +/- 5.8 days) and required a larger number of cells for transfer as compared with CD4+ T cell-reconstituted nude rats. However, second xenografts were rejected more rapidly than first xenografts in CD8+ T cell-reconstituted nude rats (9 +/- 2 days), indicating that the CD8+ T cells had been activated. This study demonstrates that CD4+ and CD8+ T cells can both reject xenografts. The CD4+ cells do so at least in part by generation of helper-dependent XAb that act by both complement-dependent and Ab-dependent cell-mediated cytotoxicity mechanisms; the CD8+ cells do so as helper-independent cytotoxic T cells.     

Partial activation of CD8+ T cells by a self-derived peptide

T cells are normally activated when the peptide for which they are specific is presented to them in the context of the appropriate major histocompatibility complex (MHC) (class I and Class II for CD8+ and CD4+ T cells, respectively). An increasing body of evidence indicates that structural homologues of the immunogenic peptide can partially activate or antagonize CD4+ T cells. CD8+ T cells may also be partially antagonized by such peptides, and self-derived peptides of this type may play a role in CD8+ T cell selection in the thymus. Activated CD8+ T cells lyse their targets by perforin-dependent granule exocytosis and by inducing apoptosis mediated by CD95 (also known as Fas or APO1) with its ligand (CD95L). Here we show that a clone of Kd-restricted CD8+ T cells specific for influenza haemagglutinin, which can also be activated in a crossreactive manner by a peptide derived from a myeloma tumour immunoglobulin heavy-chain variable region (IgVH) to kill by both routes, kills only by the CD95-CD95L pathway when stimulated by the corresponding germline IgVH peptide. As this germline IgVH peptide differs from the tumour peptide only at a single position buried in the MHC-binding groove, this indicates that CD95-CD95L-mediated killing can be triggered independently of the perforin-mediated pathway, and can be selectively affected by changes in MHC conformation.     

Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8+, but not of CD4+, T cells and accelerated by increasing the frequency of LCMV-specific CD8+ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8+ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8+ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P0/0 and P0/0 x TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-alpha and interferon (IFN)-gamma produced by CD8+ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-gamma-producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-gamma-producing CD8+ T cells. Thus, in the absence of IFN-gamma and/or TNF/LT-alpha, exhaustion of virus-specific T cells was not hampered.     

Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice

The pathogenetic effector functions of hepatitis B virus (HBV)-specific CD4+, Th1 cells were analyzed in two inbred lineages of HBV transgenic mice, one of which overexpresses the HBV large envelope protein rendering the hepatocytes hypersensitive to the cytopathic effects of IFN-gamma, and another that expresses all of the HBV proteins and replicates the virus in the liver. Transfer of HBV envelope-specific Th1 cells resulted in recognition of viral Ag expressed by hepatic nonparenchymal cells, cytokine release, and a transient necroinflammatory liver disease in both lineages. The liver disease was very severe in the IFN-gamma-sensitive lineage, and it was less severe in the lineage that replicates the HBV genome; nonetheless, in this lineage the Th1 cytokines produced by these cells suppressed viral replication in the liver. These results demonstrate that CD4+ T cells with a Th1 functional phenotype can perform pathogenetic and antiviral effector functions in vivo. This suggests that CD4+ T cells can contribute directly to disease pathogenesis and inhibit viral replication during HBV infection.     

Nitrofurantoin-induced hepatotoxicity mediated by CD8+ T cells

Nitrofurantoin is a synthetic nitrofuran commonly used for the treatment and prophylaxis of urinary tract infections. We describe the case of a 75-yr-old woman who was taking nitrofurantoin as prophylaxis against recurrent urinary tract infections, and who subsequently developed pulmonary and hepatic toxicity. We postulate that a breakdown product of the drug or the drug itself complexed to an endogenous peptide is presented by the class I HLA antigen on the hepatocyte cell membrane, inducing cytotoxic T cell activation and subsequently, hepatocyte death.     

Selective induction of CD8+ cytotoxic T lymphocyte effector function by staphylococcus enterotoxin B

Upon encounter with its antigenic stimulus, CTL characteristically proliferate, produce cytokines, and lyse the Ag-presenting cell in an attempt to impede further infection. Superantigens are extremely efficient immunostimulatory proteins that promote high levels of proliferation and massive cytokine production in reactive T cells. We compared the activation of murine influenza-specific CD8+ CTL clones stimulated with either influenza peptide or the superantigen staphylococcus enterotoxin B (SEB). We found that influenza peptide/MHC and SEB appeared equally capable of eliciting proliferation and IFN-gamma production. However, while influenza peptide/MHC elicited both perforin- and Fas ligand (FasL)/Fas (CD95L/CD95)-mediated cytolytic mechanisms, SEB was unable to trigger perforin-mediated cytolysis or serine esterase release. Examination of intracellular Ca2+ mobilization events revealed that the ability to trigger intracellular Ca2+ flux was not comparable between influenza peptide and SEB. SEB stimulated only a small rise in levels of intracellular Ca2+, at times indistinguishable from background. These findings indicate that the short-term cytolytic potential of superantigen-activated CD8+ CTL clones appears to be restricted to FasL/Fas (CD95L/CD95) mediated cytolysis.     

Characterization of circulating CD4+ CD8+ lymphocytes in healthy individuals prompted by identification of a blood donor with a markedly elevated level of CD4+ CD8+ lymphocytes

During flow cytometric analysis of lymphocytes from healthy donors, we identified a donor (donor A) with 22% CD4+ CD8+ cells (versus values of < 4% for 65 other controls). To determine if CD4+ CD8+ cells from donor A and other controls were similar, we first defined the phenotypic profile of control CD4+ CD8+ cells. Enriched CD4+ CD8+ cell populations for 10 controls were prepared by a two-step positive selection scheme with anti-CD4-coated magnetic beads and anti-CD8-coated culture flasks; the selected population averaged 69% CD4+ CD8+ cells and 31% CD4+ CD8- cells. For all 10 controls, two subsets of CD4+ CD8+ cells, CD4dim CD8bright and CD4bright CD8dim, were observed. Phenotypic profiles of these two CD4+ CD8+ subsets were defined by pairing anti-CD8 with other monoclonal antibodies, and the profiles were compared with each other and with those of CD4+ CD8-, CD4- CD8bright, and CD4- CD8dim cells. CD8bright and CD4bright CD8dim cells differed in their proportions of CD62-L+ cells and in their levels of CD11a and CD2 expression. Both CD4+ CD8+ subsets resembled CD4+ CD8- cells in CD45RA, CD45RO, and CD25 expression; the comparable CD- CD8+ cells in CD62-L expression; and CD4- CD8bright cells in CD11b, CD11b, CD16/56, and CD28 expression. CD38 expression in both CD4+ CD8+ subsets was decreased compared with those of other cell subsets. Whereas control CD4+ CD8+ cells averaged 33% CD4dim CD8bright, CD4+ CD8+ cells from donor A were > 90% CD4dim CD8bright. Donor A CD4dim CD8bright cells exhibited proportional decreases in CD25 and CD62-L expression and increases in CD11b and CD54 expression compared with those of control CD4dim CD8bright cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and antigen specificity of CD8+ cytotoxic T lymphocytes in beta 2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells

beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able to develop functional TCR(+)-alpha beta, CD8+ CTLs in response to tumor cell injection. The i.p. injection of beta 2-m-/- mice with tumor results in the massive accumulation of highly lytic CD8+ CTLs in the peritoneum and causes the local recruitment of CD8+ T cells into lymph nodes and spleens of immune animals. The accumulation of CD8+ CTLs in peritoneum is accompanied by the rejection of tumor cells and the survival of animals. The deficiency in MHC class I expression in beta 2-m/- mice is reflected in the delayed tumor rejection and CD8+ cell accumulation during the primary anti-tumor response in comparison with normal mice. The secondary response, however, is identical in normal and MHC class I-deficient mice. The rejection of tumor cells appears to be MHC class I directed because no rejection of tumors, no accumulation of CD8+ CTLs, and no survival of animals were observed when syngeneic tumor cells were used for injection with the notable exception of anti-minor Ag response. The Ag specificity of CD8+ CTLs in beta 2-m-/- mice is demonstrated using a panel of tumor target cells and class I transfectants. Although no substantial differences were found in the number and specificity of peritoneal CD8+ CTLs in beta 2-m-/- and normal mice using tumor rejection studies, the analysis of TCR-V beta phenotype using the panel of mAbs revealed the reduction in proportion of TCR-V beta 5 and TCR-V beta 6 used by CD8+ cell population from beta 2-m-/- mice. Development of lytic and H-2-directed CD8+ cells in regional lymph nodes was also observed after footpad immunization of beta 2-m-/- mice with TNP-labeled C57BL/6 splenocytes, suggesting anti-minor Ag reaction.     

Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.     

CD8+ T cells in atopic disease

Refinements in radiographic techniques have resulted in increased use of radiographic studies in the evaluation of patients with head and neck cancer over the past 20 years. To assess the impact of such studies, we compared tumor clinical stages based solely on physical-examination findings with those obtained with the addition of CT findings. This study was accomplished through case review of 81 head and neck cancer patients who underwent CT after preliminary TNM-stage assignment as determined on the basis of physical examination alone. In this cohort, 44 patients (54%) had a change in assigned clinical stage. We reviewed individual anatomic sites to determine where CT was found to be most useful in modifying tumor stage. Changes in tumor and nodal stage were found across all major sites of the head and neck. Tumors of the hypopharynx were the most likely to change stage (90%) on the basis of CT findings, whereas tumors of the glottic larynx were least likely to undergo a change in stage (16%). The therapeutic implications of these findings are discussed in the context of the published literature.     

Cellular mechanisms involved in protection against influenza virus infection in transgenic mice expressing a TCR receptor specific for class II hemagglutinin peptide in CD4+ and CD8+ T cells

Mice transgenic for a TCR that recognizes peptide110-120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.