Vaccination protects beta 2 microglobulin deficient mice from immune mediated mortality but not from persisting viral infection

Intracranial (i.c.) infection of immunocompetent mice with lymphocytic choriomeningitis virus (LCMV) results in immunopathological lethal meningitis mediated by CD8+ cytotoxic T lymphocytes (CTL). Vaccination of immunocompetent mice elicits a CD8+ CTL response that can protect the mice from lethal meningitis. beta 2 microglobulin-deficient (beta 2m-/-) mice are deficient in CD8+ CTL, exhibit CD4+ CTL, and, after i.c. LCMV infection, undergo a less severe meningitis with decreased mortality and additionally develop a wasting disease. Both wasting disease and mortality in beta 2m-/- mice are mediated by CD4+ T cells. We studied the effects of vaccination and challenge dose on weight loss, mortality and viral clearance after i.c. LCMV infection in beta 2m-/- mice. Unvaccinated beta 2m-/- mice had significant weight loss and mortality at doses of 200 and 10(3) p.f.u. LCMV, while a dose of 10(6) p.f.u. LCMV elicited significant mortality but less weight loss. Vaccination with u.v.-inactivated LCMV in complete Freund's adjuvant or with vaccinia virus expressing the LCMV glycoprotein or nucleoprotein genes protected beta 2m-/- mice from mortality but not weight loss after 200 p.f.u. LCMV challenge. Although protected from mortality, beta 2m-/- mice were unable to clear LCMV from their brains or spleens. Therefore, we show that vaccination can protect against lethal immune-meningitis in the face of persistent infection.     

A functional and kinetic comparison of antiviral effector and memory cytotoxic T lymphocyte populations in vivo and in vitro

To analyze the critical parameters for effective antiviral cytotoxic T lymphocyte (CTL) activity in vivo, control of lymphocytic choriomeningitis virus (LCMV) infection in the spleen was studied after adoptive transfer of different spleen cell populations into preinfected recipients. The quantitative, qualitative and kinetic requirements for virus control were defined and related to in vitro assays to compare the antiviral protective function of CTL from naive, acutely infected and memory mice. Treatment of mice with an established but limited LCMV infection by adoptive transfer of spleen cells from acutely LCMV-infected mice led to complete virus elimination mainly mediated by donor-derived CD8+ T cell-mediated, perforin-dependent cytotoxicity. Since virus is continuously spreading and the number of infected target cells rapidly increases, the time until target cell lysis is achieved was critical: if release of viral progeny was not prevented early, additional time to perform effector function did not improve overall virus control. When the function of various cell populations was compared in this model, we found that CTL from naive and memory mice perform considerably less well than CTL from acutely infected mice. In vitro studies indicated that this is probably due to the fact that they can not fulfill the limiting time requirements for immediate antiviral protection: while CTL from acutely infected mice can perform lytic effector function immediately, memory CTL require a considerable reactivation time before they can lyse infected target cells. This reactivation does not necessarily involve cell division. These findings illustrate how critical time limitations are for CTL to mediate early control of a dynamic virus infection in vivo.     

Minimal bystander activation of CD8 T cells during the virus-induced polyclonal T cell response

Acute infections with viruses such as lymphocytic choriomeningitis virus (LCMV) are associated with a massive polyclonal T cell response, but the specificities of only a small percentage of these activated T cells are known. To determine if bystander stimulation of T cells not specific to the virus plays a role in this T cell response, we examined two different systems, HY-specific T cell receptor (TCR)-transgenic mice, which have a restricted TCR repertoire, and LCMV-carrier mice, which are tolerant to LCMV. LCMV infection of HY-transgenic C57BL/6 mice induced antiviral CTLs that lysed target cells coated with two of the three immunodominant epitopes previously defined for LCMV (glycoprotein 33 and nucleoprotein 397). Although LCMV-induced cytotoxic T lymphocytes (CTLs) from C57BL/6 mice could lyse uninfected H-2(k) and H-2(d) allogeneic targets, LCMV-induced CTLs from HY mice lysed only the H-2(k)-expressing cells. The HY mice generated both anti-H-2(k) and anti-H-2(d) CTL in mixed leukocyte reactions, providing evidence that the generation of allospecific CTLs during acute LCMV infection is antigen specific. During the LCMV infection there was blastogenesis of the CD8+ T cell population, but the HY-specific T cells (as determined by expression of the TCR-alpha chain) remained small in size. To examine the potential for bystander stimulation under conditions of a very strong CTL response, T cell chimeras were made between normal and HY mice. Even in the context of a normal virus-induced CTL response, no stimulation of HY-specific T cells was observed, and HY-specific cells were diluted in number by day 9 after infection. In LCMV-carrier mice in which donor and host T cells could be distinguished by Thy1 allotypic markers, adoptive transfer of LCMV-immune T cells into LCMV-carrier mice, whose T cells were tolerant to LCMV, resulted in activation and proliferation of donor CD8 cells, but little or no activation of host CD8 cells. These results support the hypothesis that the massive polyclonal CTL response to LCMV infection is virus-specific and that bystander activation of non-virus-specific T cells is not a significant component of this response.     

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.     

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

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

Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design

The role of CD4+ and CD8+ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4+-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4+ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.     

Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.     

On the key role of secondary lymphoid organs in antiviral immune responses studied in alymphoplastic (aly/aly) and spleenless (Hox11(-)/-) mutant mice

The role of the spleen and of other organized secondary lymphoid organs for the induction of protective antiviral immune responses was evaluated in orphan homeobox gene 11 knockout mice (Hox11(-/-)) lacking the spleen, and in homozygous alymphoplastic mutant mice (aly/aly) possessing a structurally altered spleen but lacking lymph nodes and Peyer's patches. Absence of the spleen had no major effects on the immune response, other than delaying the antibody response by 1-2 d. In aly/aly mice, the thymus-independent IgM response against vesicular stomatitis virus (VSV) was delayed and reduced, whereas the T-dependent switch to the protective IgG was absent. Therefore, aly/aly mice were highly susceptible to VSV infection. Since aly/aly spleen cells yielded neutralizing IgM and IgG after adoptive transfer into recipients with normally structured secondary lymphoid organs, these data suggest that the structural defect was mainly responsible for inefficient T-B cooperation. Although aly/aly mice generated detectable, but reduced, CTL responses after infection with vaccinia virus (VV) and lymphocytic choriomeningitis virus (LCMV), the elimination of these viruses was either delayed (VV) or virtually impossible (LCMV); irrespective of the dose or the route of infection, aly/aly mice developed life-long LCMV persistence. These results document the critical role of organized secondary lymphoid organs in the induction of naive T and B cells. These structures also provide the basis for cooperative interactions between antigen-presenting cells, T cells, and B cells, which are a prerequisite for recovery from primary virus infections via skin or via blood.     

Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4

Antiviral immune responses of mice lacking interleukin-2 (IL-2) or IL-4 or both IL-2 and IL-4 (IL-2/4) were compared by using different viruses. Primary cytotoxic T-lymphocyte (CTL) responses against lymphocytic choriomeningitis virus (LCMV) were only moderately reduced in mice lacking IL-2 and were normal in mice lacking IL-4. Mice deficient in both interleukins exhibited variable and more strongly reduced but nevertheless in vivo protective LCMV-specific CTL responses. Similar results were obtained with vaccinia virus. Upon virus-specific restimulation in vitro, spleen cells from IL-2- and IL-2/4-deficient mice failed to generate CTL responses against virus-infected target cells, whereas the response of mice deficient in only IL-4 was comparable to that of control mice. The addition of IL-2 during in vitro restimulation completely restored the responses of both IL-2 and IL-2/4-deficient mice. T-helper-cell-independent immunoglobulin M and T-helper-cell-dependent immunoglobulin G antibody responses against vesicular stomatitis virus glycoprotein were within normal ranges for the various mutant mice. After LCMV infection, specific antibody responses against LCMV nucleoprotein were reduced four- to eightfold. These results show that mice lacking IL-2/4 have an overall tendency to exhibit more severely reduced CTL responses than IL-2- or IL-4-deficient mice. Nevertheless, and surprisingly, in vivo protective immune responses were mounted in the absence of IL-2/4, suggesting that besides a minor contribution from IL-4, other interleukins compensate in vivo for the lack of IL-2 in IL-2-deficient mice.     

The T/B cell interaction involved in induction of the mouse IgG2ab suppression is restricted by major histocompatibility complex class I, but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/ B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2ab suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igha haplotype, when neonatally transferred into histocompatible Igha/b heterozygotes, are able to induce a specific and total suppression of the IgG2ab allotype. Nevertheless, only transfer of IgG2ab-primed Igha T splenocytes induces this suppression in Ighb/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8+ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4+CD8+ T cell cooperation and operated via the recognition by the involved TCR of C gamma 2ab-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Ighb mice genetically deficient for MHC class I (beta 2-microglobulin%, or beta 2m%) or class II (I-A beta%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2ab T cells transferred into Ighb H-2b I-A beta% mice carry out the suppression process normally, while in Ighb H-2b beta 2m% recipients, their suppression induction capacity is significantly inhibited. Moreover, the C gamma 2ab 103-118 peptide, identified as the sole C gamma 2ab-derived peptide able to amplify the anti-IgG2ab T cell reactivity in Igha H-2b mice, is also able to stabilize the H-2Db, but not the H-2Kb class I molecules at the surface of RMA-S (TAP2-, H-2b) cells. These results indicate that, despite the CD4+/CD8+ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2ab+ B cells for suppression establishment.     

B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.     

IL-12 is not required for induction of type 1 cytokine responses in viral infections

To investigate the physiological role of IL-12 in viral infections in terms of T cell cytokine responses involved in virus-specific Ig isotype induction and in antiviral protection, immune responses elicited upon infection of IL-12-deficient mice with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV) were studied. Infection of IL-12-deficient mice with LCMV induced a virus-specific type 1 cytokine response as determined by in vitro cytokine secretion patterns as well as by in vivo intracellular cytokine staining of LCMV-specific CD4+ TCR transgenic T cells that had clonally expanded in LCMV-infected IL-12-deficient recipient mice. In addition, LCMV- and VSV-specific IgG responses exhibited normal serum IgG2a/IgG1 ratios, demonstrating again virus-specific CD4+ T cell induction of type 1 phenotype in IL-12-deficient mice upon viral infection. LCMV and VSV immune mice were found to be protected against challenge immunization with recombinant vaccinia viruses expressing either the LCMV- or the VSV-derived glycoprotein, respectively. This protection is known to be mediated by T cell-secreted type 1 cytokines IFN-gamma and TNF-alpha. In contrast, IL-12-deficient mice showed impaired abilities to control infection with the facultative intracellular bacterium Listeria monocytogenes at early time points after infection. However, at later time points of infection, IL-12-deficient mice were able to clear infection. These findings may indicate that viruses are able to induce type 1 T cell responses in the absence of IL-12 as opposed to some bacterial or parasitical infections that are crucially dependent on the presence of IL-12 for the induction of type 1 immune responses.     

Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy

Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic beta-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP x Tag2) mice (137 +/- 18 d) as opposed to control RIP-Tag2 mice (88 +/- 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP-specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP x Tag2) mice further prolonged survival (168 +/- 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.     

Beta2-microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition, but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains

Mice lacking beta2-microglobulin (beta2m- mice) express greatly reduced levels of MHC class I molecules, and cells from beta2m- mice are therefore highly sensitive to NK cells. However, NK cells from beta2m- mice fail to kill beta2m- normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of beta2m- NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to beta2m- NK cells, we made the following observations: First, beta2m- NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to beta2m- concanavalin A (Con A)-activated T cell blasts. Secondly, beta2m- NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than beta2m+ NK cells. Thirdly beta2m- NK cells were not only tolerant to beta2m- Con A blasts but also to Con A blasts from H-2Kb-/Db- double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of beta2m- NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in beta2m- mice, which may instead operate through mechanisms involving triggering pathways.     

3DinSight: an integrated relational database and search tool for the structure, function and properties of biomolecules

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, beta2-microglobulin-deficient mice (beta2m(-/-)) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. Beta2m(-/-) mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all beta2m(-/-) recipients were predominantly CD3+alphabetaTCR+CD4+ cells (15-20% of all splenocytes). In contrast, CD8+ cells engrafted in very small numbers (1-5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4+ T cells induced and maintained GVHD in mH-mismatched beta2m(-/-) mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic beta2m(-/-) cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Cytotoxic T cell responses to DNA vaccination: dependence on antigen presentation via class II MHC

This study was designed to test whether cytotoxic T cell (CTL) responses to DNA vaccination are dependent upon MHC class II-restricted priming of CD4+ T cells. Because DNA vaccination may directly transfect dendritic cells, and dendritic cells may be capable of directly stimulating CD8+ T cell responses, such priming might be unnecessary. To test this hypothesis, C57BL/6 mice were immunized intramuscularly or intradermally with DNA encoding either whole OVA, a class I (Kb)-restricted peptide epitope of OVA (amino acids 257-264, SIINFEKL), or this class I-restricted epitope plus the adjacent class II (I-Ab)-restricted epitope of OVA (amino acids 265-280, TEWTSSNVMEERKIKV). Very low to negligible CTL responses were observed in mice vaccinated with the SIINFEKL construct, whereas mice vaccinated with the SIINFEKLTEWTSSNVMEERKIKV or with the complete OVA construct made equally robust CTL responses. These responses were sensitive to blocking by anti-CD8 mAb and were shown to be SIINFEKL-specific by using SIINFEKL peptide-pulsed EL-4 cells as targets. To ensure that the generation of these CTL responses was indeed dependent upon CD4+ T cell help, mice were depleted of either CD4+ or CD8+ cells before immunization. Depletion of CD4+ cells completely abrogated the CTL response to OVA DNA, as did depletion of CD8+ cells. Thus, we conclude that the CTL response to both intramuscular and intradermal DNA vaccination is highly dependent upon the generation of CD4+ T cell help via a class II MHC-dependent pathway. These results will be relevant for the construction of minimal-epitope vaccines for DNA immunization.     

Helicobacter pylori infection in immunized mice lacking major histocompatibility complex class I and class II functions

The role of major histocompatibility complex (MHC) class I- and class II-restricted functions in Helicobacter pylori infection and immunity upon oral immunization was examined in vivo. Experimental challenge with H. pylori SS1 resulted in significantly greater (P 相似文献   

Consumer protection in managed care: finding the balance

CD8+ cytotoxic T lymphocytes (CTL) have an established role in anti-viral immunity, but whether CTL function efficiently in the brain remains unclear. In particular, virus-infected neurons, which express only low levels of MHC class I antigens and are resistant to the induction of apoptosis, could constitute a relatively intractable CTL target. We have used immune lymphocytes adoptively transferred into the CSF to protect naive mice against an intracerebral infection with influenza A/WSN, a virus that infects neurons in the brain parenchyma and causes a lethal encephalitis. After in vitro restimulation, heterotypically immune spleen cells protected against A/WSN encephalitis in an H-2-restricted, CD8-dependent, CD4-independent manner. Adoptively transferred CTL clones were also protective. Homotypically immune spleen cells additionally mediated CD8-independent, H-2-unrestricted protection, probably due to the generation of A/WSN-specific plasma cells from memory B cells during in vitro restimulation. Thus after in vitro restimulation, either CTL or B cells adoptively transferred into the CSF protected against an acutely lethal intracerebral virus infection.     

Antigen expressed on tumor cells fails to elicit an immune response, even in the presence of increased numbers of tumor-specific cytotoxic T lymphocyte precursors

We have used T-cell receptor (TCR) transgenic mice to analyze the interaction of tumors with the immune system. We show that the tumor cell line Lewis lung-lymphocytic choriomeningitis virus (LL-LCMV), genetically manipulated to express an H-2 Db-restricted epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV33-41), can grow progressively in TCR transgenic mice, where approximately 50% of CD8+ T cells are specific for LCMV33-41. TCR transgenic T cells were not deleted in tumor-bearing mice, and their surface phenotype and cytokine secretion patterns remained typical of naive T cells. Also, TCR transgenic T cells from tumor-bearing mice had undiminished capacity to proliferate to antigen in vitro. Tumor-protective immune responses could be elicited in TCR transgenic mice by immunization with LCMV33-41 peptide-loaded dendritic cells. Tumor resistance correlated with the switch of TCR transgenic T cells from a CD44low to a CD44high phenotype and increased capacity to produce IFNgamma in vitro. Results similar to those obtained in TCR transgenic mice were also obtained using an adoptive transfer system, where small numbers of TCR transgenic T cells were injected into normal C57BL/6 hosts. These data indicate that even large tumors may not induce specific immunization, tolerance, or anergy to tumor antigens, and that high numbers of tumor-specific CTL precursors are not sufficient to provide tumor resistance.     

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.