A novel influenza A virus mitochondrial protein that induces cell death.

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Murine cytotoxic T lymphocyte recognition of individual influenza virus proteins. High frequency of nonresponder MHC class I alleles.

We determined the MHC restriction of CTL responses to five individual influenza virus proteins. Four viral proteins failed to be recognized in conjunction with three of the five class I alleles of the H-2k and H-2d haplotypes, while the fifth was recognized only in conjunction with a single allele. This indicates that there is a significant chance that a given class I allele will be associated with low responsiveness or nonresponsiveness for a given foreign protein. This explains, at least in part, why MHC-linked nonresponsiveness is frequently detected in polyclonal antiviral CTL responses. Most importantly, these findings support the idea that responsiveness to foreign antigens is a critical factor in maintaining the high degree of MHC class I polymorphism in outbred populations.

T-cell populations specifically depleted of alloreactive potential cannot be induced to lyse H-2-different virus-infected target cells.

Mouse lymphocyte populations of one parental H-2 type (A) were specificially depleted of alloreactive potential by filtration through irradiated A X B F1 recipients, and thoracic duct cells were then stimulated with virus in an A X B F1 environment. Experiments using T cells that had previously been exposed to influenza virus in the context of A established that cross-priming for recognition of viral components expressed on H-2-different (B) target cells does not occur. Furthermore, immunologically naive T cells stimulated with vaccinia virus, subsequent to negative selection for reactivity to B, could not be shown to interact with virus-infected cells of type B. Either there is no significant T-cell repertoire for recognition of virus associated with an H-2 determinant not encountered during ontogeny, or such T cells are also alloreactive and are removed during filtration.

RMA/S cells present endogenously synthesized cytosolic proteins to class I-restricted cytotoxic T lymphocytes.

RMA/S is a mutant cell line with decreased cell surface expression of major histocompatibility complex class I molecules that has been reported to be deficient in presenting endogenously synthesized influenza virus nucleoprotein (NP) to cytotoxic T lymphocytes (CTL). In the present study we show that RMA/S cells can present vesicular stomatitis virus nucleocapsid protein, and, under some conditions, NP, to Kb-and Db-restricted CTL, respectively. Antigen presentation results from processing of cytosolic pools of endogenously synthesized proteins, and not the binding to cell surface class I molecules of antigenic peptides present in the virus inoculum or released from infected cells. Antigen processing of RMA/S differs, however, from processing by wild-type cells in requiring greater amounts of antigen, longer times to assemble or transport class I-peptide complexes, and in being more sensitive to blocking by anti-CD8 antibody. Thus, the antigen processing deficit in RMA/S cells is of a partial rather than absolute nature.

Patterns of virus-immune T-cell responsiveness. Comparison of (H-2k X H-2b) leads to H-2b radiation chimeras and negatively selected H-2b lymphocytes.

Negatively selected H-2K(b)D(b) TDL can be induced to respond strongly to vaccinia virus presented in the context of both H-2K(k) and H-2D(b) when stimulated in irradiated H-2K(k)D(b) recipients. Addition of excess (H- 2K(k)D(b) x H-2K(b)D(b))F1 TDL, which are low responders to H-2D(b)-vaccinia virus, does not obviously suppress the reactivity pattern of the H-2K(b)D(b) T cells. However, lymphocytes from chimeras made by reconstituting H- 2K(b)D(b) mice with (H-2K(k)D(k) x H-2K(b)D(b))F(l) bone marrow cells make little, if any, cytotoxic T-cell response to vaccinia virus when sensitized in H-2K(k)D(b) recipients. We have thus documented one instance where the responder phenotype of T ceils from an F(l) {arrow} parent chimera is not equivalent to that associated with the H-2 type of the parental thymus. Lymphocytes from both the chimera and the H-2K(b)D(b) parent (after negative selection) are tolerant to the H-2K(k) and I-A(k) alloantigens encountered in the recipient, but the chimera T cells are also defective in their response to a neoantigen (vaccinia virus) presented in the context of H-2K(k) which the parental T cells invariably recognize. It is thus possible that at least part of the phenomenology associated with the F(l) {arrow} parent radiation chimeras reflects deletion of repertoire in the context of H-2 antigens present during thymocyte ontogeny on other than radiation-resistant thymic epithelium.

T cells that encounter virus in the complete absence of a particular H-2 antigen are nonresponsive when stimulated again in the context of that H-2 antigen.

Immunologically naive BALB/c (H-2d) and C57BL/6J (B6) (H-2b) T-cell populations can, after filtration to remove alloreactive precursor lymphocytes, be induced to respond to vaccinia virus presented in the context of H-2Kk when stimulated in an appropriate recipient. Exposure to vaccinia virus 6 wk previously completely abrogated the capacity of BALB/c T cells to interact with H-2Kk-vaccinia virus. This is also true for negatively selected B6 thoracic duct lymphocytes taken at 14 or 18 d, but not at 6 wk after immunization: the discrepancy is thought to reflect the progressive emergence of new T cells in the latter group. No evidence could be found for the operation of suppression, and the results are considered to indicate that T cells that interact with virus in the absence of the relevant H-2 antigen are tolerized. Whereas stimulation to effector function is H-2 restricted, induction of immune paralysis may be unrestricted. The capacity of T-cell populations to respond to virus presented in the context of allogeneic H-2 determinants thus depends upon previous antigenic experience.

Vaccinia-specific cytotoxic T-cell responses in the context of H-2 antigens not encountered in thymus may reflect aberrant recognition of a virus-H-2 complex.

BALB/c (H-2Kd-Dd) spleen and lymph node populations were specifically depleted of alloreactive potential by filtration through H-2 different, irradiated recipients. These negatively selected T cells were then stimulated with vaccinia virus in mice expressing the foreign H-2 determinants encountered previously in the filter environment. Strong virus-immune cytotoxic T-cell responses were seen in the context of H-2Kk and H-2Ks, but not 2H-2Kb. The T cells generated were not cross-reactive for the H-2Kk and H-2Kd alleles, and responsiveness was independent of concurrent presence of effector populations operating at H-2D. These findings are consisent with the idea that recognition is mediated via a complex receptor, part of which is specific for virus and part for self H-2. The capacity to interact with allogeneic, virus-infected cells may then reflect aberrant recognition of a virus-H-2-antigen complex by this single, large binding site. For instance, the T cell which would normally recognize H-2Kd-virus x, or H-2Dd-minor histocompatibility antigen Z, may now show specificity for H-2Kk-vaccinia virus. Implications for both the selective role of the thymus and for mechanisms of tolerance are discussed.

Generation of both cross-reactive and virus-specific T-cell populations after immunization with serologically distinct influenza A viruses.

Specificity of cytotoxic T-cell function was investigated for a range of different influenza viruses. T cells from mice immunized with A or B strain influenza viruses, or with vaccinia virus, showed reciprocal exclusion of cytotoxicity. Extensive cross-reactivity was, however, found for lymphocyte populations from mice infected with a variety of serologically distinct influenza A viruses, though serum antibodies did not cross-react when tested in a radioimmunoassay using comparable target cells as immunoadsorbents. This apparent lack of T-cell specificity was recognized for immune spleen cells generated after intraperitoneal inoculation of high titers of virus, and for mediastinal lymph node populations from mice with pneumonia due to infection with much less virus. The phenomenon could not be explained on the basis of exposure to the chicken host component, which is common to A and B strain viruses. However, not all of the virus-immune T-cell clones are cross-reactive. Competitive-inhibition experiments indicate that a considerable proportion of the lymphocyte response is restricted to the immunizing virus. Even so, the less specific component is significant. Also, exposure to one type A virus was found to prime for an enhanced cell-mediated immunity response after challenge with a second, serologically different A strain virus.

Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) recognize class I major histocompatibility complex molecules complexed to peptides of eight to nine residues generated from cytosolic proteins. We find that CTL recognize, in vitro and in vivo, cells synthesizing a 10-residue peptide consisting of an initiating methionine followed by nine residues corresponding to a naturally processed determinant from influenza virus nucleoprotein (NP) (residues 147-155). Addition of two COOH-terminal residues corresponding to NP residues 157 and 158 severely reduced presentation of the endogenously produced peptide to CTL in vitro and in vivo. Extension of NH2 and COOH terminal flanking residues to include residues corresponding to NP residues 137-146 and 159-168 failed to increase the antigenicity of this peptide. Its presentation was greatly enhanced, however, by further extending the NH2 and COOH termini to include all of the additional residues of NP. These findings indicate first, that a naturally processed viral ligand (with an NH2-terminal Met) of a class I molecule contains sufficient information to access intracellular class I molecules, and second, that flanking residues can influence the processing and presentation of antigens to CTL.

Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation.

The E3/19K glycoprotein of adenovirus functions to diminish recognition of adenovirus-infected cells by major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) by binding intracellular class I molecules and preventing them from reaching the plasma membrane. In the present study we have characterized the nature of the interaction between E3/19K and the H-2Kd (Kd) molecule. An E3/19K molecule genetically engineered to terminate six residues from its normal COOH terminus (delta E19), was found to associate with Kd in a manner indistinguishable from wild-type E3/19K. Unlike E3/19K, however, delta E19 was transported through the Golgi complex to the plasma membrane, where it could be detected biochemically and immunocytochemically using a monoclonal antibody specific for the lumenal domain of E3/19K. Importantly, delta E19 also differed from E3/19K in being unable to prevent the presentation of Kd-restricted viral proteins to CTLs. This is unlikely to be due to delta E19 having a lower avidity for Kd than E3/19K, since delta E19 was able to compete with E3/19K for Kd binding, both physically, and functionally in nullifying the E3/19K blockade of antigen presentation. These findings indicate that the ability of E3/19K to block antigen presentation is due solely to its ability to retain newly synthesized class I molecules in the endoplasmic reticulum.

Trimming of antigenic peptides in an early secretory compartment.

Major histocompatibility complex (MHC) class I molecules bind peptides of 8-10 residues in the endoplasmic reticulum (ER) and convey them to the cell surface for inspection by CD8-expressing T cells (TCD8+). Antigenic peptides are predominantly derived from a cytosolic pool of polypeptides. The proteolytic generation of peptides from polypeptides clearly begins in the cytosol, but it is uncertain whether the final proteolytic steps occur before or after peptides are transported into the ER by the MHC-encoded peptide transporter (TAP). To study the trimming of antigenic peptides in the secretory pathway in the absence of cytosolic processing, we used an NH2-terminal signal sequence to target to the ER of TAP-deficient cells, "tandem" peptides consisting of two defined TCD8+ determinants arranged from head to tail. We find that in contrast to cytosolic proteases in TAP-expressing cells, which are able to liberate antigenic peptides from either end of a tandem peptide, proteases (probably aminopeptidases) present in an early secretory compartment preferentially liberate the COOH-terminal determinant. These findings demonstrate that proteolytic activities associated with antigen processing are not limited to the cytosol, but that they also exist in an early secretory compartment. Such secretory aminopeptidases may function to trim TAP-transported peptides to the optimal size for binding to class I molecules.

TAP-independent, beta 2-microglobulin-dependent surface expression of functional mouse CD1.1.

CD1 molecules consist of beta 2-microglobulin (beta 2m) noncovalently complexed to a non-major histocompatibility complex (MHC)-encoded monomorphic integral membrane protein homologous to MHC class I alpha chains. Little is known about the requirements for cell surface expression and T cell recognition of CD1. We inserted the mouse CD1.1 gene into vaccinia virus to create a recombinant virus expressing CD1.1 under the control of a viral promoter. Using this recombinant virus to infect normal or mutant cell lines, we found that the expression of molecules reactive with the CD1.1-specific monoclonal antibody 3C11 requires the expression of beta 2m but was not affected by the absence of the MHC-encoded peptide transporter (TAP). Consistent with these results, IL-2 production by the mCD1.1-specific T cell hybridoma DN32.D3 was induced by thymocytes from normal mice or mice with a homozygous deletion of the TAP1 gene, but not by thymocytes from mice with a homozygous deletion of the beta 2m gene. These results indicate that expression of functional mCD1.1 occurs in a beta 2m-dependent, TAP-independent manner.

Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants.

In studying the subdominant status of two cysteine-containing influenza virus nuclear protein (NP) determinants (NP39-47 and NP218-226) restricted by H-2Kd, we found that the antigenicity of synthetic peptides was enhanced 10-100-fold by treatment with reducing agents, despite the fact that the affinity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226. We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus. The major modifications of cysteine-containing synthetic peptides are cysteinylation and dimerization occurring through cysteine residues. We demonstrate that both of these modifications occur in cells synthesizing a cytosolic NP218-226 minigene product and, further, that T cells specific for cysteinylated NP218-226 are induced by influenza virus infection in mice, demonstrating that this modification occurs in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

Class I molecules retained in the endoplasmic reticulum bind antigenic peptides.

We isolated major histocompatibility complex (MHC)-specific viral peptides from cells infected with influenza virus in the continuous presence of the drug brefeldin A, which blocks exocytosis of newly synthesized MHC class I molecules. MHC-specific peptides were also isolated from cells expressing mouse Kd class I MHC molecules whose cytoplasmic domain was substituted by that of the adenovirus E3/19K glycoprotein. This molecule was retained in an intracellular pre-Golgi complex compartment as demonstrated by immunocytochemical and biochemical means. Since we show that intracellular association of antigenic peptides with such retained class I molecules is necessary for their isolation from cellular extracts, this provides direct evidence that naturally processed peptides associate with class I MHC molecules in an early intracellular exocytic compartment.

Presentation of numerous viral peptides to mouse major histocompatibility complex (MHC) class I-restricted T lymphocytes is mediated by the human MHC-encoded transporter or by a hybrid mouse-human transporter.

The major histocompatibility complex-encoded transporter associated with antigen processing (TAP) is required for the efficient presentation of cytosolic antigens to class I-restricted T cells. TAP is thought to be formed by the interaction of two gene products, termed TAP1 and TAP2. We find that TAPs consisting either of human subunits, or mouse TAP1 and human TAP2, facilitate the presentation of numerous defined viral peptides to mouse class I-restricted T cells. As human and mouse TAP2 and TAP1 differ in 23 and 28% of their residues, respectively, this indicates that TAP1 and TAP2 can form a functional complex with partners considerably different from those they coevolved with. Moreover, these findings indicate that widely disparate TAPs facilitate delivery of the same peptides to class I molecules. These findings suggest that TAP polymorphism does not greatly influence the types of peptides presented to the immune system.

Immunoproteasomes shape immunodominance hierarchies of antiviral CD8(+) T cells at the levels of T cell repertoire and presentation of viral antigens.

Vertebrates express three cytokine-inducible proteasome subunits that are incorporated in the place of their constitutively synthesized counterparts. There is increasing evidence that the set of peptides generated by proteasomes containing these subunits (immunoproteasomes) differs from that produced by standard proteasomes. In this study, we use mice lacking one of the immunoproteasome subunits (LMP2) to show that immunoproteasomes play an important role in establishing the immunodominance hierarchy of CD8(+) T cells (T(CD8+)) responding to seven defined determinants in influenza virus. In LMP2(-/)- mice, responses to the two most dominant determinants drop precipitously, whereas responses to two subdominant determinants are greatly enhanced. Adoptive transfer experiments with naive normal and transgenic T(CD8+) reveal that the reduced immunogenicity of one determinant (PA(224-233)) can be attributed to decreased generation by antigen presenting cells (APCs), whereas the other determinant (NP(366-374)) is less immunogenic due to alterations in the T(CD8+) repertoire, and not, as reported previously, to the decreased capacity of LMP2(-/)- APCs to generate the determinant. The enhanced response to one of the subdominant determinants (PB1F2(62-70)) correlates with increased generation by LMP2(-)(/)- virus-infected cells. These findings indicate that in addition to their effects on the presentation of foreign antigens, immunoproteasomes influence T(CD8+) responses by modifying the repertoire of responding T(CD8+).

Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype.

Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed.

Induction of virus-specific modifications recognized by cytotoxic T cells is not altered by prior substitution of target cells with trinitrophenol.

Cytotoxic thymus-derived lymphocytes generated after interaction with trinitrophenyl (TNP)-substituted or virus-infected cells only lyse H-2 compatible target cells modified with the component used to immunize (TNP or virus). Prior saturation of TNP-reactive sites inhibits neither the infectivity of influenza A viruses, nor the capacity of infected cells to develop antigenic changes recognized by influenza-immune T cells. The two antigens are distinct entities on the cell membrane and do not obviously compete to form interactions with H-2 molecules.

Recognition of cloned vesicular stomatitis virus internal and external gene products by cytotoxic T lymphocytes.

It has generally been assumed that most if not all CTL specific for vesicular stomatitis virus (VSV)-infected cells recognize the viral glycoprotein (G), an integral membrane protein abundantly expressed on infected cell surfaces. Using recombinant vaccinia viruses containing copies of cloned VSV genes to examine CTL recognition of VSV, we have confirmed that G is recognized by VSV-specific CTL. More interestingly, however, we have also found that nucleocapsid protein (N), an internal virion protein, can be detected on infected cell surfaces using mAb, and serves as a major target antigen for VSV-specific CTL. In contrast to the highly serotype-specific recognition of G, N is recognized by a major population of CTL able to lyse cells infected with either the Indiana or New Jersey VSV serotypes. Using target cells expressing a cloned MHC class I gene, we could directly show that CTL recognition of N occurs in the context of the MHC Ld molecule.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1-infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1-infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1-specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.