Down-regulation of human sialyltransferase gene expression during in vitro human keratinocyte cell line differentiation

We studied cytokines and anti-cytokine autoantibodies (Aabs) during T.b.brucei infections in IFN-gamma-/-, IFN-gammaR-/- and wild-type mice. Increased serum levels of IFN-gamma, TNF-gamma and IL-4 with decreased Aabs to these cytokines were recorded early during infections in all mice (except IFN-gamma in IFN-gamma-/- mice). Later, these responses were reversed, and surprisingly Aabs reacting to IFN-gamma in the IFN-gamma -/- mice were detected. To examine the possibility that an IFN-? immunoreactive molecule might be expressed due to infections and upon gene deletion, anti-IFN-gamma antibody was inoculated and resulted in abrogation of such Aabs. The scenario was different for IL-10 and TGF- since IFN-gammaR-/- and wild-type mice showed low cytokines and high Aabs early during infections, but later high cytokines and low Aabs were registered. Interestingly, IFN-gamma-/- mice exhibited reversed levels of both IL-10 and TGF-beta, and also of their Aabs. Fab fragments of purified serum immunoglobulins showed binding and neutralizing effects in biological assays. Pre-absorption of the Fab fragments with a cytokine inhibited the binding and neutralization effects of this cytokine, but not of other cytokines. These results highlight an important role for autoimmunity in cytokine regulation, and that genomic deletion of IFN-gamma modulates cytokines and their Aab responses in experimental African trypanosomiasis.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

Use of intranasal IL-12 to target predominantly Th1 responses to nasal and Th2 responses to oral vaccines given with cholera toxin

We have investigated the effects of IL-12 and cholera toxin (CT) on the immune response to tetanus toxoid (TT) given by intranasal or oral routes. CT inhibited IL-12-induced IFN-gamma secretion both in vivo and in vitro. Intranasal administration of IL-12 to mice nasally immunized with the combined vaccine of TT and CT resulted in increased TT-specific IgG2a and IgG3 Abs, while IgG1 and IgE Ab responses were markedly reduced. This shift of the CT-induced immune response toward Th1 type was associated with TT-specific CD4+ T cells secreting IFN-gamma and reduced levels of Th2-type cytokines (i.e., IL-4, IL-5, IL-6, and IL-10). In contrast, intranasal IL-12 enhanced the CT-induced serum IgG1 and IgE Ab responses in mice given the combined vaccine orally. IFN-gamma secretion by TT-specific CD4+ T cells was also enhanced; however, Th2-type cytokine responses were predominant. Mucosal secretory IgA responses to oral or nasal vaccines were not affected by intranasal IL-12. Thus, intranasal IL-12 delivery influences Th cell subset development in mucosal inductive sites that are dependent on the route of vaccine delivery.     

Disruption of the murine IL-4 gene blocks Th2 cytokine responses

Murine T-helper clones are classified into two distinct subsets (Th1 and Th2) on the basis of their patterns of lymphokine secretion. Th1 clones secrete interleukin-2 (IL-2), tumour necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), whereas Th2 clones secrete IL-4, IL-5 and IL-10 (ref. 1). These subsets are reciprocally regulated by IL-4, IL-10 and IFN-gamma and differentially promote antibody or delayed-type hypersensitivity responses. To evaluate whether IL-4 is required for mounting Th2 responses, we generated IL-4-mutant mice (IL-4-/-) and assessed the cytokine secretion pattern of T cells both from naive and Nippostrongylus brasiliensis infected mice. CD4+ T cells from naive IL-4-/- mice failed to produce Th2-derived cytokines after in vitro stimulation. The levels of Th2 cytokines IL-5, IL-9 and IL-10 from CD4+ T cells obtained after nematode infection were significantly reduced. The reduced IL-5 production in IL-4-/- mice correlated with reduced helminth-induced eosinophilia, which has been shown to be dependent on IL-5 in vivo. We conclude that IL-4 is required for the generation of the Th2-derived cytokines and that immune responses dependent on these cytokines are impaired.     

Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

IFN-gamma-secreting Th cells regulate both the frequency and avidity of epitope-specific CD8+ T lymphocytes induced by peptide immunization: an ex vivo analysis

CD8+ T lymphocytes are involved in protective immune responses to infected or tumor cells. In this report, we examined the regulation of antigen-specific CD8+ T cell frequency and avidity by distinct Th cell subsets. Peptide-specific CD8+ T cells were induced by immunization of mice with a MHC class I-restricted epitope, co-injected with a MHC class II-restricted epitope to recruit Th cells. CD8+ T cell responses were assessed directly ex vivo for lytic activity and IFN-gamma secretion using the enzyme-linked immunospot (ELISPOT) assay. Co-immunization in incomplete Freund's adjuvant (IFA) with three different helper peptides induced IFN-gamma- and IL-2-secreting Th cells, in the absence of IL-4 secretion, suggesting preferential Th1 profiles. Such immunization resulted in the increase of antigen-specific CD8+ T cell frequency, which was detected in blood as efficiently as in lymph nodes and spleen, and elicited high-avidity CD8+ T cells. We investigated whether these effects were dependent upon a particular Th profile. When alum was used instead of IFA, the production of IL-2 by Th cells was still significant, while the production of IFN-gamma was undetectable. Such Th cell activation failed to support an increase of antigen-specific CD8+ T cell frequency. Altogether, these results document in vivo the regulatory role played by Th cells in CD8+ T cell activation and may be relevant for the design of efficient vaccination schedules.     

The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals

Interleukin (IL)-4-deficient mice were used to assess susceptibility to systemic or gastrointestinal Candida albicans infections, as well as parameters of innate and elicited T helper immunity. In the early stage of systemic infection with virulent C. albicans, an unopposed interferon (IFN)-gamma response renders IL-4-deficient mice more resistant than wild-type mice to infection. Yet, IL-4-deficient mice failed to efficiently control infection in the late stage and succumbed to it. Defective IFN-gamma and IL-12 production, but not IL-12 responsiveness, was observed in IL-4-deficient mice that failed to mount protective T helper type 1 cell (Th1)-mediated acquired immunity in response to a live vaccine strain of the yeast or upon mucosal immunization in vivo. In vitro, IL-4 primed neutrophils for cytokine release, including IL-12. However, late treatment with exogenous IL-4, while improving the outcome of infection, potentiated CD4(+) Th1 responses even in the absence of neutrophils. These findings indicate that endogenous IL-4 is required for the induction of CD4(+) Th1 protective antifungal responses, possibly through the combined activity on cells of the innate and adaptive immune systems.     

Mechanisms for induction of acquired host immunity by neutrophil peptide defensins

Human neutrophil peptide (HNP) defensins were studied to determine their potential effects on adaptive mucosal immunity. Intranasal delivery of HNPs plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody (Ab) responses. However, OVA-specific IgA Abs were not induced in mucosal secretions or in serum. CD4(+) T cells of intranasally immunized mice displayed higher OVA-specific proliferative responses and elevated production of interferon gamma, interleukin (IL) 5, IL-6, and IL-10 when compared with control groups receiving OVA alone. In vitro, HNPs also enhanced both proliferative responses and T helper (Th) cytokine secretion profiles of CD3epsilon-stimulated spleen- and Peyer's patch-derived naive CD4(+) T cells. HNPs modulated the expression of costimulatory molecules by lipopolysaccharide- or CD3epsilon-stimulated splenic and Peyer's patch B or T cell populations, respectively. These studies show that defensins enhance systemic IgG, but not IgA, Ab responses through help provided by CD4(+) Th1- and Th2-type cytokines and foster B and T cell interactions to link innate immunity with the adaptive immune system.     

Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues

Female rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-gamma and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.     

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.     

Induction of systemic and mucosal immune responses to human immunodeficiency virus type 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular and intranasal routes

Induction of mucosal and cell-mediated immunity is critical for development of an effective vaccine against human immunodeficiency virus (HIV). We compared intramuscular and intranasal immunizations with a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant for augmentation of the systemic and mucosal immune responses to HIV-1 in a murine model. Vaccination via the two routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Intestinal secretory IgA production and cytolytic activity of the mesenteric lymph node cells are preferentially elicited by intranasal immunization, and QS-21 augmented these activities as well. This adjuvant augmented production of interleukin-2 (IL-2) and gamma interferon (IFN-gamma) associated with decrease in IL-4 synthesis by antigen-restimulated splenocytes. The serum immunoglobulin subtype profile showed a dominant IgG2a response and less strong IgG1 and IgE production in a QS-21 dose-dependent manner. As expected, enhancements of humoral and cell-mediated immune responses by QS-21 were abrogated by treatment with anti-IL-2 and anti-IFN-gamma monoclonal antibodies. These results suggest that the intranasal route of DNA immunization is more efficient than the intramuscular route in inducing mucosal immunity mediated by sIgA and mesenteric lymphocytes. Furthermore, QS-21 is able to act as a mucosal adjuvant in DNA vaccination and demonstrates its immunomodulatory property via stimulation of the Th1 subset. This study emphasizes the importance of the route of immunization and the use of an adjuvant for effective DNA vaccination against HIV-1.     

Psychosocial influences on immune responses to HSV-1 infection in BALB/c mice

The effects of differential housing (one or four mice/cage) on T-helper (Th) cell markers of cellular and humoral immune responses were examined. Differentially housed male BALB/cJ mice were infected with herpes simplex virus (HSV)-1 (Patton strain), and in vitro cytokine production [interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma] by splenocytes and popliteal lymph node cells and serum antibody titers (IgM and IgG) were evaluated. Differential housing of male BALB/c mice influenced the magnitude, but not the kinetics, of some, but not all, immune responses to HSV-1. Splenocytes from individually housed mice produced more IL-2, IFN-gamma, IL-4, and IL-10 than splenocytes from group-housed mice; in popliteal lymph node cells, only IFN-gamma and IL-10 production was influenced by housing. Although the social environment influenced cytokine production, there were no concomitant changes in circulating IgM or IgG antibody titers. These results do not support the hypothesis that dominant Th cell responses are the primary targets of this psychosocial manipulation, or that a reciprocal relationship exists between Th1 and Th2 cell-derived cytokines.     

Validity of alcohol consumption records: comparison of clinical history and telephone survey]

PROBLEM: Systemic immune responses during normal pregnancy are suggested to deviate toward secretion of T helper (Th)2-like cytokines. METHOD OF STUDY: Blood samples from 11 healthy primigravidae in their first, second, and third trimesters, and 8 weeks postpartum were examined. Peripheral blood mononuclear cells were separated and the numbers of interferon (IFN)-gamma- and interleukin (IL)-4-secreting cells were identified by using the enzyme-linked immunospot test. RESULTS: In all three trimesters of pregnancy, and also postpartum, the numbers of IFN-gamma- and IL-4-secreting cells were significantly higher compared with nonpregnant controls (Mann-Whitney; P < 0.001). The numbers of IFN-gamma- and IL-4-secreting cells gradually increased as the pregnancy progressed compared with postpartum (Kruskas-Wallis; P < 0.01 and P < 0.05, for IFN-gamma and IL-4, respectively). The ratios of IFN-gamma/IL-4 during pregnancy and postpartum were unchanged, and also when compared with nonpregnant controls. CONCLUSION: The results indicate a systemic up-regulation of both Th1- and Th2-like immune responses during normal pregnancy.     

Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice

IL-1 has a number of effects on T cell growth but a specific role for IL-1 in T cell responses in vivo has not been elucidated. In this study the role of IL-1 in Th1/Th2 responses was examined in mice deficient for the IL-1 type 1 receptor (IL-1RI-/-) during cutaneous Leishmania major infection or following immunization with keyhole limpet hemocyanin (KLH). After inoculation of L. major stationary phase promastigotes into the hind footpad, both IL-1RI-/- and wild-type (WT) mice developed small lesions which resolved spontaneously. Lymph node cells from infected IL-1RI-/- mice produced significantly more IL-4 and IL-10 than those from WT mice following antigenic stimulation in vitro. Splenocytes from IL-1RI-/- and WT mice showed similar levels of antigen-induced proliferation. In contrast, splenocyte cultures from the IL-1RI-/- mice contained significantly more IL-4 than those from WT mice. Similar results were also obtained after immunization with KLH. While lymph node cells from both IL-1RI-/- and WT mice displayed similar levels of KLH-specific proliferation, those from IL-1RI-/- mice produced significantly more IL-4 than those from WT mice. Conversely, antigen-stimulated lymph node cells from WT mice secreted significantly greater amounts of IFN-gamma as compared with those from IL-1RI-/- mice. These data indicate that while IL-1 is not required for mounting an immune response or antigen-dependent proliferation, it appears to be required for normal regulation of Th1/Th2 responses and may function to negatively regulate IL-4 expression.     

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.     

CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin

We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased. In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice. By contrast, no difference was observed for systemic responses between CD8-/- and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice. In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.     

Role of gamma interferon in Helicobacter pylori-induced gastric inflammatory responses in a mouse model

The immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contribution of gamma interferon (IFN-gamma) to the immune responses, especially to the induction of gastric inflammation and to protection from H. pylori infection, was investigated with IFN-gamma gene knockout (IFN-gamma-/-) mice. We first examined the colonizing abilities of eight H. pylori strains with a short-term infection test in order to select H. pylori strains which could colonize the mouse stomach. Only three strains (ATCC 43504, CPY2052, and HPK127) colonized C57BL/6 wild-type mice, although all of the strains except for ATCC 51110 could colonize IFN-gamma-/- mice. The number of H. pylori organisms colonizing the stomach in wild-type mice was lower than that in IFN-gamma-/- mice. Oral immunization with the CPY2052 sonicate and cholera toxin protected against infection with strain CPY2052 in both types of mouse. These findings suggested that IFN-gamma may play a protective role in H. pylori infection, although the degree of its protective ability was estimated to be low. In contrast, in a long-term infection test done to examine the contribution of IFN-gamma to gastric inflammation, CPY2052-infected wild-type mice developed a severe infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium 15 months after infection, whereas CPY2052-infected IFN-gamma-/- mice showed no inflammatory symptoms. This result clearly demonstrated that IFN-gamma plays an important role in the induction of gastric inflammation caused by H. pylori infection.     

Protective role of gamma interferon during the recall response to influenza virus

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-gamma). We assessed the contribution of IFN-gamma to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-gamma-/- mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-gamma-/- mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-gamma-/- mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-gamma-/- mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-gamma-/- mice was not dramatically affected, and the percentage of CD4(+) and CD8(+) T cells was similar to that of wild-type mice. The T cells from IFN-gamma-/- mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-gamma-/- mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-gamma during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs.