Anti-fibrillin-1 autoantibodies in systemic sclerosis are GM and KM allotype-restricted.

GM and KM allotypes--genetic markers of immunoglobulin (Ig) gamma and kappa chains, respectively--have been shown to play an important role in genetic predisposition to some autoimmune diseases. To determine their role in susceptibility to systemic sclerosis (SSc; scleroderma) and in the generation of anti-fibrillin-1 antibodies, 148 SSc patients and 191 controls were typed for several GM and KM allotypes by a standard hemagglutination inhibition method. IgG and IgM antibodies to fibrillin-1 were measured by radioimmunoassay. GM and KM phenotypes were not significantly associated with SSc. However, these determinants significantly influenced the production of anti-fibrillin-1 antibodies in SSc patients. In Caucasians, GM1,3,17 23 5,13,21 and GM3 23 5,13 phenotypes were associated with the presence and absence of IgG autoantibodies, respectively. The production of these autoantibodies was also associated with KM allotypes, KM1,3 heterozygosity being associated with response and homozygosity for the KM3 allele with nonresponse to fibrillin-1. In African-Americans, the KM1 homozygotes were associated with the absence of anti-fibrillin-1 antibodies and the KM3 homozygotes with the presence of autoantibodies. In this ethnic group, the GM1,17 5,13 phenotype was associated with the absence of IgM autoantibodies. This represents the first description of genetic control of autoimmunity to fibrillin-1 in scleroderma.

Enzymatically mediated engineering of multivalent MHC class II-peptide chimeras.

We previously reported the genetic engineering of the first soluble, bivalent major histocompatibility complex (MHC) class II-peptide ligand for T-cell receptor (TCR). This ligand binds stably and specifically to cognate T-cells and exhibits immunomodulatory effects in vitro and in vivo. The increase in valence of MHC class II-peptide ligands was shown to parallel their avidity for cognate TCRs and potency in stimulating cognate T-cells. We describe a new enzymatic method to increase the valence of MHC-peptide ligands by cross-linking the N-glycan moieties of dimeric MHC II-peptide units through a flexible, bifunctional polyethylene glycol linker. Using this method, we generated covalently stabilized tetravalent and octavalent MHC II-peptide ligands which bound stably and specifically to cognate TCR and preserved their structural integrity in blood and lymphoid organs for 72 h. Depending on the TCR/CD4 occupancy and degree of TCR/CD4 co-clustering, the multivalent MHC II-peptide ligands polarized efficiently the antigen-specific CD4(+) T-cells toward type 2 cell differentiation or induced T-cell anergy and apoptosis. The enzymatically mediated engineering of multivalent MHC-peptide ligands for cognate TCRs may provide rational grounds for the development of new therapeutic agents endowed with strong modulatory effects on antigen-specific T-cells.

Association of fibrillin 1 single-nucleotide polymorphism haplotypes with systemic sclerosis in Choctaw and Japanese populations.

OBJECTIVE: Previously, we demonstrated with the use of microsatellite markers that a 2-cM haplotype on chromosome 15q containing the fibrillin 1 gene (FBN1) was strongly associated with systemic sclerosis (SSc) in the Choctaw, a population with high SSc prevalence. In this study, all 69 known FBN1 exons were sequenced to ascertain the presence of changes that might show associations with SSc in the Choctaw and Japanese SSc patients and controls. METHODS: Screening of FBN1 exons was accomplished by polymerase chain reaction-based fluorescence sequencing of genomic DNA using single-nucleotide polymorphism (SNP) haplotypes, and their frequencies were determined with a new algorithm that recognizes past recombination events between sites. Haplotype phylogenies were inferred using the median-joining network analysis. RESULTS: Five SNPs were identified in FBN1. They are located in the 5'-untranslated region (SNP-1), exon 15 (SNP-2), intron 17 (SNP-3), exon 27 (SNP-4), and intron 27 (SNP-5). Only SNP-1 (T-->C) demonstrated an association with SSc in the Choctaw. Eleven FBN1 SNP haplotypes were ascertained in the Choctaw population, 2 of which (SNPs 5 and 6) were found only in the SSc patients. These same FBN1 SNP haplotypes were associated with SSc in the Japanese. CONCLUSION: A SNP in the 5'-untranslated region of FBN1 (SNP-1, C allele) was strongly associated with SSc in the Choctaw. Furthermore, this polymorphism is present on 2 unique FBN1 haplotypes found only in Choctaw SSc patients. The same 2 haplotypes demonstrate associations with SSc in the Japanese. These data extend the earlier microsatellite studies and are consistent with the hypothesis that FBN1 or a nearby gene on chromosome 15q is involved in SSc susceptibility in the Choctaw and the Japanese.

CpG motifs of DNA vaccines induce the expression of chemokines and MHC class II molecules on myocytes.

Determining how an immune response is initiated after in vivo transfection of myocytes with plasmids encoding foreign antigens is essential to understand the mechanisms of intramuscular (i. m.) genetic immunization. Since myocytes are facultative antigen-presenting cells lacking MHC class II and co-stimulatory molecules, it was assumed that their unique role upon DNA vaccination is to synthesize and secrete the protein encoded by the plasmid. Here we describe that i. m. injection of unmethylated CpG motifs induced the expression of chemokines (monocyte chemotactic protein-1) and MHC class II molecules on myocytes. Our results indicate that immunostimulatory DNA sequences (CpG motifs) of DNA vaccines augment synthesis of chemokine by myocytes with subsequent recruitment of inflammatory cells secreting IFN-gamma, a potent cytokine that up-regulates the expression of MHC class II molecules on myocytes. A myoblast cell line triple transfected with plasmids encoding MHC class II molecules and an immunodominant CD4 T cell epitope of influenza virus presented the endogenously synthesized peptide and activated specific T cells. These findings suggest that one mechanism for the immunogenicity of DNA vaccines consists in the presentation of peptides to CD4 T cells by in vivo plasmid-transfected myocytes.

Antigen-specific downregulation of T cells by doxorubicin delivered through a recombinant MHC II--peptide chimera.

As the number of drugs with potential therapeutic use for T-cell-mediated diseases increases, there is a need to find methods of delivering such drugs to T cells. The major histocompatibility complex (MHC)--peptide complexes are the only antigen-specific ligands for the T-cell receptor (TCR) expressed on T cells, and they may be an appropriate drug delivery system. We engineered a soluble bivalent MHC class II-peptide chimera on the immunoglobulin scaffold (I-E(d)alpha beta/Fc gamma 2a/HA110-120, DEF) that binds stably and specifically to CD4 T cells recognizing the HA110-120 peptide. Doxorubicin, a powerful antimitogenic anthracycline, was enzymatically assembled on the galactose residues of a DEF chimera. The DEF-gal-Dox construct preserved both the binding capacity to hemagglutinin (HA)-specific T cells, and the drug toxicity. Brief exposure of HA-specific T cells to DEF-gal-Dox construct in vitro was followed by drug internalization in the lysosomes, translocation to the nucleus, and apoptosis. Administration of DEF-gal-Dox to mice expressing the TCR-HA transgene reduced the frequency of TCR-HA T cells in the spleen and thymus by 27% and 42%, and inhibited HA proliferative capacity by 40% and 60%, respectively. It has not been demonstrated previously that pharmacologically active drugs able to modulate T-cell functions can be delivered to T cells in an antigen-specific manner by soluble, bivalent MHC II-peptide chimeras.

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras.

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I-, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of alpha-chain in the case of MHC I, or beta-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal 1 to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.

T-cell tolerance and autoimmune diabetes.

Herein we describe the major signaling events that occur in T-cells upon T-cell receptor (TCR) engagement, and the mechanisms responsible for the induction of T-cell anergy that may ultimately lead to the development of immunospecific therapies in T-cell mediated autoimmune diseases. A new type of antigen presenting molecule (dimeric MHC class-II/peptide, DEF) endowed with antigen-specific immunomodulatory effects such as induction of Th2 polarization and T-cell anergy is also described as a potential antidiabetogenic agent. According to our preliminary results, the MHC II/peptide-based approach may provide rational grounds for further development of antigen-specific immunotherapeutic agents such as human-like MHC lI/peptide chimeras endowed with efficient down-regulatory effects in CD4 T-cell-mediated autoimmune diseases such as Type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, and rheumatoid arthritis.

Maintenance of size and function of influenza virus hemagglutinin specific transgenic T-cell clone during life.

Immunization induces less protective immunity against infectious diseases in old compared to young subjects. We have studied the effect of age on the in vitro and in vivo function of murine transgenic T cells expressing a receptor for influenza hemagglutinin 110-120 peptide. During aging the transgenic T cells undergo the age-associated shift from naive to memory phenotype but maintain, despite thymic involution, their number as well as their cytokine production and proliferative responses induced by the hemagglutinin 110-120 peptide in vitro. The maintenance of the size and functions of transgenic T cells during the aging may be related to low expression of CTLA-4 molecules known to exhibit a negative regulatory effect subsequent to interaction with costimulatory molecules as well as of stimulation of T cells by unknown cross reactive endogenous factors but not by nominal antigen since innate immunity prevents natural infection with influenza virus of murine species. This suggests that the impaired immunity induced by immunization in old subjects reflects defects in the development and maintenance of T cell memory and not in the expression of effector activity.

Autoantibodies to fibrillin 1 in systemic sclerosis: ethnic differences in antigen recognition and lack of correlation with specific clinical features or HLA alleles.

OBJECTIVE: We previously reported the presence of autoantibodies to the extracellular matrix protein, fibrillin 1, in sera from patients with systemic sclerosis (SSc). These autoantibodies appeared to be highly disease-specific but had significantly different frequencies among ethnic groups. The aims of this study were 3-fold: 1) to determine whether sera from SSc patients of different ethnic backgrounds recognized different antigenic epitopes of fibrillin 1, 2) to determine whether sera from patients with polymyositis/dermatomyositis (PM/DM) with or without interstitial lung disease (ILD) also produced these antibodies, and 3) to determine any correlation of anti-fibrillin 1 antibodies with specific clinical features of SSc, other autoantibodies, or HLA class II alleles in a prospectively studied cohort of SSc patients with early (<5 years' duration) disease (the Genetics versus Environment In Scleroderma Outcome Study [GENISOS] cohort). METHODS: Three recombinant peptides accounting for the N-terminal end, proline-rich C region, and epidermal growth factor-like calcium-binding (EGF-cb) domains of fibrillin 1 were used in a radioimmunoassay to screen sera from a large group of SSc and PM/DM patients and ethnically matched controls. RESULTS: The majority of Choctaw American Indians, Japanese, and African Americans with SSc produced IgM and/or IgG autoantibodies to one or more recombinant fibrillin 1 proteins, while <50% of Caucasians with SSc showed seroreactivity. There were striking ethnic differences in fibrillin 1 antigenic epitope recognition among these ethnic groups. African American SSc sera recognized primarily the N-terminal end, and Caucasian sera mostly recognized the EGF-cb repeats and the proline-rich C region. In contrast, most Choctaw American Indian and Japanese SSc sera appeared to recognize 2 or 3 epitopes, respectively. PM/DM patient sera did not recognize any of the fibrillin 1 epitopes regardless of the presence of ILD. In the prospective, multiethnic GENISOS cohort, the presence of anti-fibrillin 1 antibodies did not correlate with any major clinical manifestations, other autoantibodies, or HLA class II alleles. CONCLUSION: There are striking ethnic differences in antigenic epitope specificity of anti-fibrillin 1 antibodies in patients with SSc, and the majority of SSc patients, except for Caucasians, produce antibodies to fibrillin 1. The antifibrillin response thus far remains specific for scleroderma syndromes, but it does not correlate with any major clinical features, other autoantibodies, or HLA class II alleles.

A targeted mutation in the IL-4Ralpha gene protects mice against autoimmune diabetes.

Autoimmune insulin-dependent diabetes mellitus (IDDM) occurs spontaneously in mice-bearing transgenes encoding the influenza hemagglutinin under the control of the rat insulin promoter and a T cell receptor specific for an hemagglutinin peptide associated with I-E(d). Such "double transgenic" mice expressing wild-type or targeted IL-4Ralpha genes were examined for the onset of IDDM. Eight of 11 mice homozygous for wild-type IL-4Ralpha were hyperglycemic by 8 weeks of age, whereas only 1 of 16 mice homozygous for the targeted allele were hyperglycemic at this time. Most 1L-4Ralpha-/- mice remained normoglycemic to 36 weeks of age. Although only 10% of double transgenic mice homozygous for the wild-type IL-4Ralpha allele survived to 30 weeks, 80% of mice homozygous for the targeted allele did so. Heterozygous mice displayed an intermediate frequency of diabetes. Even as late as 270 days of age, mice homozygous for the targeted allele had no insulitis or only peri-insulitis. Thus, the inability to respond to IL-4 and/or IL-13 protects mice against IDDM in this model of autoimmunity.

Fine specificity of anti-fibrillin-1 autoantibodies in primary pulmonary hypertension syndrome.

Autoantibodies to fibrillin-1 (Fbn-1) have been found in systemic sclerosis (SSc), calcinosis, Raynaud's esophagael dysmotility, sclerodectyly, and telaengectasia (CREST) and mixed connective tissue disease (MCTD) diseases. The purpose of this study was to determine whether patients with primary pulmonary hypertension (PPH) and appetite-suppressant-associated PPH have anti-Fbn-1 autoantibodies. In addition we assessed the human leucocyte antigen (HLA) class II alleles (DRB1, 3, 4, 5 and DQB1) in these patients in order to determine whether the response is genetically restricted. The frequency of anti-Fbn-1 autoantibodies in patient groups was compared with that of a control group of 88 healthy patients, and HLA was correlated similarly with a group of 51 healthy subjects. Anti-Fbn-1 autoantibodies were found at high frequency in PPH: in 70 of 75 adults with PPH (93%), in 28 of 33 children with PPH (84.8) and in 12 of 18 (67%) patients with appetite-suppressant-associated PPH. Utilization of two Fbn-1 fusion proteins allowed us to determine the dominant determinant region, recognized by anti-Fbn-1 autoantibodies, which may be located on the N-terminal fragment of the Fbn-1 protein. No significant immunogenetic correlations were found when the PPH patient groups were compared with normal controls. This novel category of autoantibodies is found in diseases characterized by endothelial and extracellular matrix protein alterations and fibrosis.

Escape from self-tolerance leads to neonatal insulin-dependent diabetes mellitus.

Double transgenic (dTg) mice expressing the hemagglutinin (HA) of influenza virus under the insulin promoter and the TCR specific for the immunodominant CD4 T cell epitope of HA (HA110-120) develop insulin-dependent diabetes mellitus (IDDM). In order to gain information on the breaking down of neonatal self-tolerance we studied the occurrence of IDDM after birth. Our results showed that newborn mice develop fulminant IDDM characterized by occurrence of insulitis as early as 3 days after birth, followed by hyperglycemia by 7 days, and significant hypoinsulinemia by 28 days. The neonatal breakdown of self-tolerance of T cells positively selected in the thymus is supported by the facts that: (i) peripheral HA110-120 specific T cells from neonates are fully functional and proliferated upon stimulation with the nominal peptide, and (ii) peptide-specific T cells were accumulated in the pancreas of dTg mice as early as 3 days after birth. Our results demonstrate that diabetes occurring in young dTg mice is due to early activation of self-reactive T cells immediately after birth. Accumulation of specific T cells in the target organ leads to destruction of pancreatic beta-cells and IDDM. These mice may provide a useful model to evaluate new strategies for the prevention of diabetes.

Antigen-specific signaling by a soluble, dimeric peptide/major histocompatibility complex class II/Fc chimera leading to T helper cell type 2 differentiation.

Interaction between a T cell receptor (TCR) and various ligands, i.e. , anti-TCR antibodies, superantigens, peptides, or altered peptide ligands in the context of major histocompatibility complex (MHC) molecules can trigger different T helper cell (Th) effector functions. Herein, we studied the T cell response induced by a soluble, dimeric peptide/MHC class II chimera, namely hemagglutinin (HA)110-120/I-E(d)alphabeta/Fcgamma2a (DEF). We have previously demonstrated that the soluble DEF molecule binds stably and specifically to HA110-120-specific TCRs expressed by a T cell hybridoma. Administration of DEF in vivo induced differentiation of resting and activated peptide-specific T cells toward a Th2 response, as indicated by the increase of interleukin (IL)-4, IL-10, and specific immunoglobulin (Ig)G1 antibodies and decrease of IL-2, specific IgG2a antibodies, and cytotoxic T lymphocyte activity. In contrast to HA110-120 peptide presented by the DEF molecule to T cells, the nominal synthetic peptide induced a predominant Th1 response, and the PR8 virus-derived HA110-120 peptides induced a mixed Th1/Th2 response. Independent of antigen processing, soluble DEF was almost 2 logs more potent in stimulating cognate T cells than the nominal peptide. Polarization of cognate T cells toward the Th2 response occurred upon interaction of soluble DEF with TCR and CD4 molecules followed by early activation of p56(lck) and ZAP-70 tyrosine kinases, and negative signaling of the signal transducer and activator of transcription (STAT)4 pathway of Th1 differentiation. DEF-like molecules may provide a new tool to study the mechanisms of signaling toward Th2 differentiation and may also provide a potential immunotherapeutic approach to modulate autoreactive T cells toward protective Th2 immune responses.

Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin-1, in patients with scleroderma and other connective tissue diseases.

A duplication in the fibrillin-1 gene has been implicated as the cause of the tight skin 1 (tsk1) phenotype, an animal model of scleroderma or systemic sclerosis (SSc). In addition to the production of abnormal fibrillin-1 protein, the tsk1 mouse also produces autoantibodies to fibrillin-1. Among a population of Choctaw Native Americans with the highest prevalence of SSc yet described, a chromosome 15q haplotype containing the fibrillin-1 gene has been strongly associated with SSc. With a recombinant human fibrillin-1 protein, autoantibodies to fibrillin-1 were detected in the sera of Native American SSc patients that correlated significantly with disease. Abs to fibrillin-1 also were detected in sera from Japanese, Caucasian, and African-American SSc patients. Compared with other ethnic groups, Japanese and Native American SSc patients had significantly higher frequencies of anti-fibrillin-1 Abs. Sera from patients with diffuse SSc, calcinosis, Raynaud's, esophageal dysmotility, sclerodactyly, and telangiectasias syndrome and mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from controls or patients with other non-SSc connective tissue diseases (lupus, rheumatoid arthritis, and Sjögren's syndrome). Ab specificity for fibrillin-1 was demonstrated by the lack of binding to a panel of other purified autoantigens. The results presented demonstrate for the first time the presence of high levels of anti-fibrillin-1 Abs in a significant portion of patients with SSc.

Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK) mice.

Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.

Expression of granulocyte colony-stimulating factor in recurrent glial tumors is inversely correlated with tumor progression.

We have previously reported that in vivo-expression of granulocyte colony-stimulating factor (G-CSF) is a characteristic feature of reactive and neoplastic astrocytes. The aim of the present study was to analyze the expression of G-CSF protein in primary and recurrent astroglial, oligodendroglial and mixed glial-differentiated tumors. G-CSF expression was present in all GFAP-positive tumors excepting glioblastomas. G-CSF expression was significantly reduced in recurrent tumors more dedifferentiated than their primary counterparts. G-CSF expression was absent in all GFAP-negative tumors such as oligodendrogliomas. Our results demonstrate that G-CSF is a highly sensitive differentiation marker of neoplastic astrocytes, which is lost during tumor progression.

Antineoplastic efficacy of doxorubicin enzymatically assembled on galactose residues of a monoclonal antibody specific for the carcinoembryonic antigen.

We have developed a novel procedure to couple enzymatically the antineoplastic agent doxorubicin (Dox) on the galactose residues of a monoclonal antibody specific for the tumor-associated carcinoembryonic antigen. The synthesis of the immunoconjugate consists of covalent attachment of the NH2 terminus of Dox to oxidized galactose residues of desialylated monoclonal antibody, followed by concurrent stabilization of Schiff bases by mild reduction with pyridine borane. The immunoconjugate preserved both antibody specificity and drug cytotoxicity. At equimolar concentrations, the immunoconjugate was 8 times more cytotoxic against two carcinoembryonic antigen-expressing carcinoma cell lines, LoVo and SW-480, than Dox alone. The intracellular drug accumulation was 8-8.5 times higher than that obtained with free Dox, and >50% of the drug delivered by the conjugate was retained for 24 h in the tumor cells. Only 4 days after treatment with a single dose of immunoconjugate carrying 2.5 ng of Dox, LoVo and SW-480 tumor transplants on the chorioallantoic membrane of embryonated hen eggs showed reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues. In contrast, the same amount of free drug induced insignificant changes in tumor progression and tumor-induced angiogenesis. Enzymatically mediated, glycosidic coupling of antineoplastic agents to antibodies specific for tumor-associated antigens may represent a novel platform for the development of more efficient anticancer agents with reduced side effects.

Spontaneous occurrence of anti-fibrillin-1 autoantibodies in tight-skin mice.

Tight-skin (TSK) mouse represents an experimental for systemic sclerosis, displaying cutaneous hyperplasia, connective tissue alterations in the internal organs and developing autoantibodies against several scleroderma target autoantigens. TSK mouse syndrome is associated with a mutation in fibrillin-1 (Fbn-1), the major component of 10 nm microfibrils. Here, we have investigated whether TSK mouse develops autoimmunity to Fbn-1 similar to scleroderma target autoantigens. Our results show that anti-Fbn-1 IgG autoantibodies are present in high titer in many TSK mice. Specificity of these antibodies was confirmed by competitive inhibition assays and Western blotting analysis using recombinant human Fbn-1 protein. TSK mouse autoantibodies recognize a conserved epitope present in the C region of Fbn-1. These results indicate the presence of Fbn-1 specific T and B cells in TSK mouse repertoire.

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.