Enhanced T-cell activation and differentiation in lymphocytes from transgenic mice expressing ubiquitination-resistant 2KR LAT molecules.

Linker for activation of T cells (LAT) is critical for the propagation of T-cell signals upon T-cell receptor (TCR) activation. Previous studies demonstrated that substitution of LAT lysines with arginines (2KR LAT) resulted in decreased LAT ubiquitination and elevated T-cell signaling, indicating that LAT ubiquitination is a molecular checkpoint for attenuation of T-cell signaling. To investigate the role of LAT ubiquitination in vivo, we have generated transgenic mice expressing WT and ubiquitin-defective 2KR LAT. On TCR stimulation of T cells from these mice, proximal signaling and cytokine production was elevated in 2KR versus wild-type (WT) LAT mice. Enhanced cytolytic activity as well as T-helper responses were observed on LAT expression, which were further elevated by 2KR LAT expression. Despite greater T-effector function, WT or 2KR LAT expression did not have any effect on clearance of certain pathogens or tumors. Our data support the model that lack of tumor clearance is due to increased differentiation and acquisition of effector phenotype that is associated with suboptimal immunity in an immunotherapy model. Thus, our data further reinforce the role of LAT ubiquitination in TCR signaling and uncovers a novel role for LAT in driving T-cell differentiation.

Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or gamma delta T cells.

The mechanisms of broad cross-protection to influenza viruses of different subtypes, termed heterosubtypic immunity, remain incompletely understood. We used knockout mouse strains to examine the potential for heterosubtypic immunity in mice lacking IgA, all Ig and B cells, NKT cells (CD1 knockout mice), or gamma(delta) T cells. Mice were immunized with live influenza A virus and compared with controls immunized with unrelated influenza B virus. IgA(-/-) mice survived full respiratory tract challenge with heterosubtypic virus that was lethal to controls. IgA(-/-) mice also cleared virus from the nasopharynx and lungs following heterosubtypic challenge limited to the upper respiratory tract, where IgA has been shown to play an important role. Ig(-/-) mice controlled the replication of heterosubtypic challenge virus in the lungs. Acute depletion of CD4+ or CD8+ T cell subsets abrogated this clearance of virus, thus indicating that both CD4+ and CD8+ T cells are required for protection in the absence of Ig. These results in Ig(-/-) mice indicate that CD4+ T cells can function by mechanisms other than providing help to B cells for the generation of Abs. Like wild-type mice, CD1(-/-) mice and gamma(delta) (-/-) mice survived lethal heterosubtypic challenge. Acute depletion of CD4+ and CD8+ cells abrogated heterosubtypic protection in gamma(delta) (-/-) mice, but not B6 controls, suggesting a contribution of gamma(delta) T cells. Our results demonstrate that the Ab and cellular subsets deficient in these knockout mice are not required for heterosubtypic protection, but each may play a role in a multifaceted response that as a whole is more effective than any of its parts.

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge.

DNA vaccination offers the advantages of viral gene expression within host cells without the risks of infectious virus. Like viral vaccines, DNA vaccines encoding internal influenza virus proteins can induce immunity to conserved epitopes and so may defend the host against a broad range of viral variants. CD8(+) cytotoxic T lymphocytes (CTL) have been described as essential effectors in protection by influenza nucleoprotein (NP), although a lesser role of CD4(+) cells has been reported. We immunized mice with plasmids encoding influenza virus NP and matrix (M). NP + M DNA allowed B6 mice to survive otherwise lethal challenge infection, but did not protect B6-beta(2)m(-/-) mice defective in CD8(+) CTL. However, this does not prove CTL are required, because beta(2)m(-/-) mice have multiple immune abnormalities. We used acute T cell depletion in vivo to identify effectors critical for defense against challenge infection. Since lung lymphocytes are relevant to virus clearance, surface phenotypes and cytolytic activity of lung lymphocytes were analyzed in depleted animals, along with lethal challenge studies. Depletion of either CD4(+) or CD8(+) T cells in NP + M DNA-immunized BALB/c mice during the challenge period did not significantly decrease survival, while simultaneous depletion of CD4(+) and CD8(+) cells or depletion of all CD90(+) cells completely abrogated survival. We conclude that T cell immunity induced by NP + M DNA vaccination is responsible for immune defense, but CD8(+) T cells are not essential in the active response to this vaccination. Either CD4(+) or CD8(+) T cells can promote survival and recovery in the absence of the other subset.

Mechanism of protective immunity against influenza virus infection in mice without antibodies.

There is considerable interest in developing viral vaccines intended to induce T cell immunity, especially cytotoxic CD8+ T lymphocytes, when Abs are not protective or are too narrow in viral strain specificity. We have studied protective immunity in doubly inactivated (DI) mice devoid of Abs and mature B cells. When infected with influenza B virus, these mice cleared the virus in a process dependent upon CD8+ T lymphocytes. Cytotoxic activity was detected in lung lymphocytes of DI mice after primary or secondary infection, and was abrogated by depletion of CD8+ cells in vivo. Challenge experiments showed that DI mice could be protected by immunization against reinfection 1 mo later, and protection was virus specific. Depletion of CD4+ or CD8+ T cells in vivo during the challenge period partially abrogated, and depletion of both subsets completely abrogated, the protection. This indicates that both CD4+ and CD8+ T cells are required effectors in the optimal control of virus replication. Thus, when Abs fail to protect against varying challenge viruses, as is the case with variant strains of influenza and HIV, there is hope that T cells might be able to act alone.

Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell-depleted, beta2-microglobulin-deficient, and J chain-deficient mice.

Immunity that is cross-protective between different influenza A virus subtypes (termed heterosubtypic immunity) can be demonstrated readily in some animals but only rarely in humans. Induction of heterosubtypic immunity in humans by vaccines would provide public health benefit, perhaps offering some protection against pandemics or other new influenza A strains. Therefore, we studied mechanisms mediating heterosubtypic immunity in mice. Immunization with either A/H1N1 or A/H3N2 virus protected mice against mortality following heterosubtypic challenge while providing modest reductions in lung virus titers. No cross-protection was seen with distantly related type B influenza virus. Depletion of CD4+ or CD8+ T cells or both around the time of challenge had no significant effect on survival, indicating that these cells are not required at the effector stage. beta2-microglobulin knockout mice could be protected readily against heterosubtypic challenge, confirming that class I-restricted T cells are not required. In beta2-microglobulin -/- mice, depletion of CD4+ T cells partially abrogated heterosubtypic immunity, showing that they play a role in these mice. Passive transfer of Abs to naive recipients protected against subsequent challenge with homologous but not heterosubtypic virus. Because a role for secretory Abs has been suggested, we studied dependence on the J chain, which is required for polymeric Ig receptor-mediated IgA transport. J chain knockout mice were readily protected by heterosubtypic immunity, indicating that polymeric Ig receptor-mediated transport is not required. Better understanding of heterosubtypic immunity should be valuable in analyzing new vaccines, including peptide and DNA vaccines, intended to induce broadly cross-reactive immunity.

Antibody repertoire in the response to a protein antigen. Interrelated idiotypic families in the response to Ia.7.

Mouse alloantibodies to Ia.7 display a cross-reactive idiotype (CRI) recognized by xenogeneic anti-idiotypes. The CRI is expressed on serum Abs in all responding individuals and on all anti-Ia.7 mAbs, from mice of appropriate genetic types. In addition, both xenogeneic and allogeneic anti-idiotypes in this system have a striking ability to induce Ia.7-specific responses in mice never exposed to the Ag. Because of these unusual features, we have investigated the biologic and structural basis of the idiotypic sharing in this system. Ia.7-specific Ab populations induced by eight different Ab2 mAbs were analyzed for expression of each of the set of idiotopes. Two obvious explanations for the unusual properties of the system do not appear to be correct. 1) The induction of Ag-specific immunity was not caused by internal imagery; and 2) the Ab2s do not simply recognize the same idiotope, because they induce populations that were found to be distinct in idiotope expression. The biologic properties of the system are instead caused by a pattern of expression of idiotope sets on distinct but related Ab families, and a remarkable linkage of a series of different idiotopes to Ia.7-specific activity. Mouse anti-idiotypic responses failed to recognize the widely shared CRI site, even when sequential immunizations were performed. To examine the structural basis of Id sharing, light chains of three CRI+ mAbs were sequenced. They were found to be extremely homologous to each other and to the germ-line V kappa 21E gene, and they used either J1 or J2. A model containing families of distinct but related V regions is proposed for the anti-Ia.7 repertoire.

Beta 2-microglobulin-deficient mice can be protected against influenza A infection by vaccination with vaccinia-influenza recombinants expressing hemagglutinin and neuraminidase.

Immunity to viral infections includes both antibody and T cell components. The contributions of humoral and cell-mediated immune responses vary depending on virus and host factors. We have used an in vivo challenge system to examine protective immunity to influenza A(H1N1) virus infection in immunocompetent B6 (H-2b) mice, and in H-2b mice homozygous for disruption of the gene for beta 2-microglobulin, termed beta 2 mu(-/-) mice. The latter mice do not express conventional MHC class I complexes on cell surfaces and lack CD8+ class I-restricted T cells. Ten vaccinia virus recombinants, each expressing 1 of the 10 proteins of influenza virus, were used to immunize the mice. Normal mice were protected against challenge with influenza virus by vaccination with HA-VAC and NA-VAC, but not by any of the vaccinia vectors expressing one of the eight other influenza virus proteins nor by a mixture of all eight of the latter vectors. Similar results were observed in mice of H-2d or H-2k MHC haplotypes. The beta 2 mu(-/-) mice were also protected by immunization with HA-VAC and NA-VAC, demonstrating that classical CD8+ CTL responses were not required for protection. Depletion of CD4+ T cells in either normal or beta 2 mu(-/-) mice at the time of challenge had little or no effect on protection induced by HA-VAC or NA-VAC, suggesting that preformed antibody is the dominant mediator of protective immunity induced by these recombinants. Antibody responses to vaccinia virus Ag and expressed influenza virus Ag were lower in titer in beta 2 mu(-/-) mice than in normal B6 mice, suggesting an influence of MHC class I complexes, CD8+ T cells, or their products on antibody production.

Induction of antigen-specific immunity with monoclonal anti-idiotypic antibodies in vivo: differences in potency and comparison of immunochemical properties.

Anti-idiotypic (Id) antibodies provide a means other than antigen of clone-specific regulation of immune responses, and have been proposed as an alternative form of vaccine. However, the requirements for effective induction of immunity by anti-Id are not understood. Nine monoclonal anti-idiotope antibodies (anti-Id mAb) were derived in the Ia. 7 model system. While all nine anti-Id mAb induced comparable Ab3 responses in vivo as detected by ELISA, there were dramatic differences in the potency of the antigen-specific components of the responses induced by the nine anti-Id mAb. Anti-Id mAb that were indistinguishable in isotype, combining site relatedness, fine specificity on a panel of mAb, end point binding titers, competitive binding and ability to induce Ab3 differed dramatically in their ability to induce antigen-specific immunity in vivo, thus ruling out several models for explaining differences in induction.

Inheritance of idiotype expression unlinked to the H chain allotype locus. Novel features of genetic control in the Ia.7 system.

We have observed a pattern of inherited idiotype expression in three mouse strains that is unexpected from the genetics of the strains: a dominant idiotype that was expressed at high levels in two parental strains was expressed only at low levels in a heavy chain allotype congenic strain derived from them. In the C3H.SW strain, the antibody response to the class II MHC Ag I-E is of limited diversity, with dominant expression of an idiotype and the V kappa 21 L chain. The C57BL/10 strain expresses the same idiotype at high levels, whereas the CWB/12 strain, which was derived by replacing the Ig H chain Igh-Cj allele of C3H.SW with the Igh-Cb allele derived from C57B1/10, has been found to express little of this dominant idiotype. CWB/12 responds, with titers equal to those of the parental strains, to the I-E epitope responsible for dominant idiotype expression, and it expresses normal V kappa 21 levels; thus deficiencies in epitope-specific responsiveness or in V kappa 21 expression cannot explain the low Id expression in CWB/12. Furthermore, Southern blot analysis of three VH families gave no evidence of recombination within the the VH locus of CWB/12, which was Igh-Vb throughout. Black-cross analysis demonstrated that expression of the dominant idiotype segregated independently of Ig allotype, and was therefore due to genes unlinked to the H chain gene locus. To our knowledge, this pattern of Id expression is unprecedented, and indicates the need for caution in the interpretation of studies using allotype congenic strains. It also demonstrates a role for genes outside the Igh locus in the control of Id expression.

T cell-induced expression of membrane IgG by 70Z/3 B cells.

To study T cell regulation of B cell isotype differentiation, we determined the capacity of clonal T cell populations (hybridomas derived by fusing BW5147 with Con A-activated Peyer's patch (PP) and spleen T cells) to induce "downstream" isotype expression by the pre-B cell lymphoma 70Z/3. In initial studies, we found that 70Z/3 B cells cultured in the presence of LPS (1 microgram/ml) expressed membrane IgM (mIgM) but not membrane IgG (mIgG). In contrast, 70Z/3 B cells cultured with HAJ-3 T cells, a PP-derived T cell hybridoma (as well as other similarly derived PP and spleen hybridomas), or with HAJ-3 T cells plus LPS do express mIgG. Such expression occurred in spite of mitomycin C-induced blockage of cell proliferation, and is observed in 70Z/3 B cell subclones cultured with HAJ-3 T cells. For these reasons, it is not due to selective expansion of a small pre-switched mIgG-bearing 70Z/3 B cell subpopulation. In other studies it was shown that 70Z/3 B cells expressing mIgG after induction by HAJ-3 T cells continue to express mIgM and do not secrete IgG. Finally, exposure of 70Z/3 B cells to the macrophage factor IL 1 and the T cell factors IL 2, BSF-pl, and BCGF-II present in EL-4 cell supernatants did not result in mIgG expression. On the basis of these studies, we conclude that a clonal B cell population expressing mIgM can be induced by T cells to co-express mIgG. Because the B cells do not express mIgG unless exposed to T cells, this represents a T cell-induced isotype switch.