Characterization of mouse hepatitis virus-specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain.

The cytotoxic T lymphocyte (CTL) activity of spleen cells from BALB/c (H-2d) mice immunized with the neurotropic JHM strain of mouse hepatitis virus (JHMV) was stimulated in vitro for 7 days. CTL were tested for recognition of target cells infected with either JHMV or vaccinia virus recombinants expressing the four virus structural proteins. Only target cells infected with either JHMV or the vaccinia virus recombinant expressing the JHMV nucleocapsid protein were recognized. Cytotoxic T cell lines were established by limiting dilution from the brains of mice undergoing acute demyelinating encephalomyelitis after infection with JHMV. Twenty of the 22 lines recognized JHMV-infected but not uninfected syngeneic target cells, indicating that they are specific for JHMV. All T-cell lines except one were CD8+. The specificity of the CTL lines was examined by using target cells infected with vaccinia virus recombinants expressing the JHMV nucleocapsid, spike, membrane, and hemagglutinin-esterase structural proteins. Seventeen lines recognized target cells expressing the nucleocapsid protein. Three of the JHMV-specific T-cell lines were unable to recognize target cells expressing any of the JHMV structural proteins, indicating that they are specific for an epitope of a nonstructural protein(s) of JHMV. These data indicate that the nucleocapsid protein induces an immunodominant CTL response. However, no CTL activity specific for the nucleocapsid protein could be detected in either the spleens or cervical lymph nodes of mice 4, 5, 6, or 7 days after intracranial infection, suggesting that the CTL response to JHMV infection within the central nervous system may be induced or expanded locally.     

Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope.

The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T-lymphocyte-mediated protective immunity.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

An epitope in the V1 domain of the simian immunodeficiency virus (SIV) gp120 protein is recognized by CD8+ cytotoxic T lymphocytes from an SIV-infected rhesus macaque.

Cytotoxic T-lymphocyte (CTL) responses against the external envelope glycoprotein (gp120) of the simian immunodeficiency virus (SIV) were studied in a rhesus macaque infected with SIVmac/239. CD8+ T cells enriched from concanavalin A-stimulated peripheral blood mononuclear cells lysed autologous target cells infected with recombinant vaccinia virus vectors expressing the SIVmac/239 or SIVsm/H4 envelope protein, which share approximately 80% identity in amino acid sequence. A CD8+ CTL line derived by limiting dilution culture of the concanavalin A-stimulated lymphocytes was also specific for the envelope proteins of both SIV isolates. Mapping studies revealed that this cell line recognized an epitope between amino acids 113 and 121 (CNKSETDRW) in the V1 domain of gp120. Amino acid substitutions are observed at positions 116 and 120 among viruses of the SIVsm/mac/human immunodeficiency virus type 2 group, and thus synthetic peptides representing these variants were tested for the ability to sensitize target cells for lysis by the CTL line. Autologous target cells sensitized with a synthetic peptide representing the SIVmac/239 sequence were efficiently killed. In contrast, recognition of target cells was reduced or abolished when peptides representing the amino acid substitutions at position 116 or 120 of other SIVmac, SIVsm, SIVmne, or SIVstm strains were tested. Further studies of CTL responses against this epitope could provide insights into mechanisms of variability within the gp120 V1 domain and its importance in evasion of immunity in infected or vaccinated monkeys.     

A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6.

A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli. The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides. The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced. Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone.     

The cytolytic activity of pulmonary CD8+ lymphocytes, induced by infection with a vaccinia virus recombinant expressing the M2 protein of respiratory syncytial virus (RSV), correlates with resistance to RSV infection in mice.

Previous studies demonstrated that the pulmonary resistance to respiratory syncytial virus (RSV) challenge induced by immunization with a recombinant vaccinia virus expressing the M2 protein of RSV (vac-M2) was significantly greater 9 days after immunization than at 28 days and was mediated predominantly by CD8+ T cells. In this study, we have extended these findings and sought to determine whether the level of CD8+ cytotoxic T-lymphocyte (CTL) activity measured in vitro correlates with the resistance to RSV challenge in vivo. Three lines of evidence documented an association between the presence of pulmonary CTL activity and resistance to RSV challenge. First, vac-M2 immunization induced pulmonary CD8+ CTL activity and pulmonary resistance to RSV infection in BALB/c (H-2d) mice, whereas significant levels of pulmonary CTL activity and resistance to RSV infection were not seen in BALB.K (H-2k) or BALB.B (H-2b) mice. Second, pulmonary CD8+ CTL activity was not induced by infection with other vaccinia virus-RSV recombinants that did not induce resistance to RSV challenge. Third, the peak of pulmonary CTL activity correlated with the peak of resistance to RSV replication (day 6), with little resistance being observed 45 days after immunization. An accelerated clearance of virus was not observed when mice were challenged with RSV 45 days after immunization with vac-M2. The results indicate that resistance to RSV induced by immunization with vac-M2 is mainly mediated by primary pulmonary CTLs and that this resistance decreases to very low levels within 2 months following immunization. The implications for inclusion of CTL epitopes into RSV vaccines are discussed in the context of these observations.     

CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.     

A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

Background

A subset of the virus-specific CD8+ cytotoxic T lymphocytes (CTL) isolated from the lungs of mice infected with human respiratory syncytial virus (RSV) is impaired in the ability to secrete interferon γ (IFNγ), a measure of functionality. It was suggested that the impairment specifically suppressed the host cellular immune response, a finding that could help explain the ability of RSV to re-infect throughout life.

Results

To determine whether this effect is dependent on the virus, the route of infection, or the type of infection (respiratory, disseminated, or localized dermal), we compared the CTL responses in mice following intranasal (IN) infection with RSV or influenza virus or IN or intradermal (ID) infection with vaccinia virus expressing an RSV CTL antigen. The impairment was observed in the lungs after IN infection with RSV, influenza or vaccinia virus, and after a localized ID infection with vaccinia virus. In contrast, we observed a much higher percentage of IFNγ secreting CD8+ lymphocytes in the spleens of infected mice in every case.

Conclusion

The decreased functionality of CD8+ CTL is specific to the lungs and is not dependent on the specific virus, viral antigen, or route of infection.     

Replication-defective viruses modulate immune responses.

By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.     

The 22,000-kilodalton protein of respiratory syncytial virus is a major target for Kd-restricted cytotoxic T lymphocytes from mice primed by infection.

Recombinant vaccinia viruses containing the 22-kilodalton protein (matrixlike or 22K protein) or phosphoprotein gene from respiratory syncytial virus were constructed. These recombinant viruses expressed proteins which were immunoprecipitated by appropriate respiratory syncytial virus antibodies and comigrated with authentic proteins produced by respiratory syncytial virus infection. The new recombinant viruses (and others previously described containing the attachment glycoprotein, fusion, or nucleoprotein genes of respiratory syncytial virus) were used to infect target cells for cultured polyclonal cytotoxic T lymphocytes generated from the spleens of BALB/c or DBA/2 mice primed by intranasal infection with respiratory syncytial virus. Respiratory syncytial virus-specific cytotoxic T lymphocytes (CTL) showed strong Kd (but not Dd)-restricted recognition of the 22K protein. As previously reported, the fusion protein and nucleoprotein were both seen by CTL, but recognition of these proteins was comparatively weak. There was no detectable recognition of other respiratory syncytial virus proteins tested (including phosphoprotein). 22K protein-specific splenic memory CTL persisted for at least 11 months after infection of BALB/c mice. Priming BALB/c mice with recombinant vaccinia virus containing the 22K protein gene induced respiratory syncytial virus-specific memory CTL at lower levels than that previously reported following infection with a similar recombinant containing the fusion protein gene. These data identify the 22K protein as a major target antigen for respiratory syncytial virus-specific CTL from H-2d mice primed by respiratory syncytial virus infection.     

Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL.

T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell-dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc-IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN-gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro.     

Production of transmembrane and secreted forms of tumor necrosis factor (TNF)-alpha by HIV-1-specific CD4+ cytolytic T lymphocyte clones. Evidence for a TNF-alpha-independent cytolytic mechanism.

Candidate AIDS vaccines consisting of recombinant forms of the HIV-1 envelope glycoprotein induce, in seronegative human volunteers, an env-specific T cell response that includes CD4+, MHC class II-restricted CTL capable of lysing HIV-1-infected target cells. In this study, we have analyzed the production of the cytokines TNF-alpha and lymphotoxin (LT) by a set of env-specific CD4+ human CTL clones. TNF-alpha and LT are of interest because of their potential role in target cell destruction by CD4+ CTL. Our studies focused on the possibility that a cell surface form of TNF-alpha expressed by CTL after physiologic activation with target APC might participate in the cytolytic reactions mediated by these clones. We found that, upon interaction with target cells expressing env epitopes in the context of the appropriate MHC class II molecules, CD4+ CTL released TNF-alpha with kinetics that were rapid, compared with other cytokines, and that were generally similar to the kinetics of target cell destruction. LT secretion was not detected during the time course of the cytolytic reactions. A novel flow cytometric assay was used to show that physiologic activation of CD4+ CTL with target APC induced expression by the CTL of cell surface forms of TNF-alpha. Immunoprecipitations from activated, surface-iodinated CTL clones revealed two forms of surface TNF-alpha, a 26-kDa form, representing the transmembrane precursor of secreted TNF-alpha, as well as the 17-kDa secreted form bound to the cell surface. For a subset of CD4+ CTL, we found that treatment of CTL with cyclosporin A inhibited Ag-induced production of both transmembrane and secreted forms of TNF-alpha but had no effect on cytolysis. Thus, although transmembrane and secreted TNF-alpha produced by HIV-1-specific CD4+ CTL may have important effects in vivo, the rapid destruction of target APC by the set of CD4+ CTL clones described here occurs through a TNF-alpha-independent mechanism.     

Herpes simplex virus (HSV)-specific human T-cell clones recognize HSV glycoprotein D expressed by a recombinant vaccinia virus.

Human cytotoxic T-cell (CTL) clones that lyse autologous cells infected with herpes simplex virus (HSV) type 1 or 2 were generated by stimulating lymphocytes with a recombinant vaccinia virus (recombinant vaccinia-gD-1 virus) that expresses HSV type 1 glycoprotein D (gD-1). Furthermore, CTL clones generated with HSV type 1 or with cloned gD-1 lysed autologous cells infected with the recombinant vaccinia-gD-1 virus. Our findings thus showed that gD serves as a target antigen for human CTLs and that a recombinant vaccinia-gD virus activates HSV-specific human CTL.     

Effect of the V3 loop deletion of envelope glycoprotein on cellular responses and protection against challenge with recombinant vaccinia virus expressing gp160 of primary human immunodeficiency virus type 1 isolates

The magnitude and breadth of cytotoxic-T-lymphocyte (CTL) responses induced by human immunodeficiency virus type 1 (HIV-1) envelope protein from which the hypervariable V3 loop had been deleted (DeltaV3) were evaluated in the HLA-A2/K(b) transgenic mice. It was demonstrated that vaccines expressing the DeltaV3 mutant of either HIV-1(IIIB) or HIV-1(89.6) envelope glycoprotein induced broader CD8(+) T-cell activities than those elicited by the wild-type (WT) counterparts. Specifically, the differences were associated with higher responses to conserved HLA-A2-restricted CTL epitopes of the envelope glycoprotein and could be correlated with an increased cell surface occupancy by the epitope-HLA-A2 complexes in target cells expressing the DeltaV3 mutant. Using recombinant vaccinia virus expressing heterologous gp160 of primary HIV-1 isolates in a murine challenge system, we observed that the extent of resistance to viral transmission was higher in animals immunized with the DeltaV3 than the WT envelope vaccine. The protection was linked to the presence of envelope-specific CD8(+) T cells, since depletion of these cells by anti-CD8 antibody treatment at the time of challenge abolished the vaccine-induced protection. The results from our studies provide insights into approaches for boosting the breadth of envelope-specific CTL responses.     

Molecular basis for cytolytic T-lymphocyte recognition of the murine cytomegalovirus immediate-early protein pp89

The murine cytomegalovirus protein pp89, which is encoded by gene ieI, is a nonstructural regulatory protein expressed in the immediate-early phase of the viral replication cycle and located mainly in the nucleus of infected cells. Protection of BALB/c (H-2d) mice against a lethal murine cytomegalovirus challenge infection is achieved by vaccination with a recombinant vaccinia virus, MCMV-ieI-VAC, expressing pp89 as the only murine cytomegalovirus gene product. The protection is entirely mediated by T lymphocytes of the CD8+ subset. In the present report, we analyzed the molecular basis of the recognition of pp89 by BALB/c CD8+ cytolytic T lymphocytes. A series of internal and terminal deletion mutants of gene ieI was constructed and cloned in vaccinia virus, and the antigenicity and immunogenicity of the fragments of pp89 expressed by the recombinants were studied. A region of only one-sixth of the protein, from amino acids 154 to 249 and encoded by the fourth exon of gene ieI, was sufficient for both the recognition in vitro of the protein by pp89-specific cytotoxic T lymphocytes and the induction in vivo of pp89-specific cytotoxic T lymphocytes. By using synthetic peptides, the sequence between residues 161 and 179, which is located within the defined domain, was identified as an epitope presented to BALB/C cytotoxic T lymphocytes by the class I major histocompatibility antigen Ld.     

Interleukin-4-mediated downregulation of cytotoxic T lymphocyte activity is associated with reduced proliferation of antigen-specific CD8+ T cells

During virus infection, exogenous IL-4 strongly downregulates expression of antiviral cytokines and cytotoxic T lymphocyte (CTL) responses. In this study, we have employed a T cell receptor (TCR) transgenic system to more closely investigate the effect of IL-4 on CTL activity. This system involves mice transgenic for an H2-Kb restricted TCR recognising an ovalbumin (OVA)-specific peptide (OT-I mice), and recombinant vaccinia viruses expressing the gene for OVA (VV-OVA), or OVA together with IL-4 (VV-OVA-IL-4). Spleen cells from OT-I mice were adoptively transferred to irradiated C57BL/6 mice infected with VV-OVA or VV-OVA-IL-4. Five days following transfer, markedly stronger CTL activity was detected in VV-OVA- than in VV-OVA-IL-4-infected recipients. The reduction in CTL activity was associated with a reduction in the number of OVA-specific CD8+ T cells. Proliferation of cells from VV-OVA-IL-4-infected recipients was dramatically reduced, and this is a likely explanation for the IL-4-mediated reduction in the total number of OVA-specific cells and the reduced cytotoxic activity. On a per cell basis, the production of IFNgamma and cytotoxic activity of OVA-specific CD8+ cells was not influenced by IL-4. Taken together, our results indicate that the reduction in CTL activity by exogenous IL-4 is due to a reduced number of antigen-specific effectors, and does not involve a downregulation of effector function of these cells.     

HIV结构蛋白与痘苗病毒共表达产物的免疫原性研究

目的 将艾滋病病毒外膜蛋白(env)与干扰素(IFNα-2b)融合基因表达的融合蛋白作为免疫原免疫小鼠,观察小鼠的免疫功能状态和细胞免疫应答.方法 将IFNα-2b基因片段插入到env基因的下游,经脂质体转染,筛选重组痘苗病毒,经SDS-PAGE和Western blot鉴定表达产物.用重组病毒vJ1 6env/IFNα-2b免疫小鼠,以生理盐水和野生型痘苗病毒作为对照,检测小鼠脾淋巴细胞对ConA、LPS及IgG的反应性;用流式细胞仪测定小鼠脾淋巴细胞CD4 、CD8T细胞计数与CTL.结果 与对照组比较,实验组脾淋巴细胞对ConA、LPS及IgG的反应性显著增高(P＜0.05);CD4T淋巴细胞计数和CTL活性也显著增高(P＜0.05);CD8T淋巴细胞计数呈增高趋势,但未达到显著意义的程度.结论 重组痘苗病毒v J16env/IFNα-2b能增强小鼠的免疫功能和诱导细胞免疫.     

Virus-specific and bystander CD8 T cells recruited during virus-induced encephalomyelitis

Neurotropic coronavirus-induced encephalitis was used to evaluate recruitment, functional activation, and retention of peripheral bystander memory CD8+ T cells. Mice were first infected with recombinant vaccinia virus expressing a non-cross-reactive human immunodeficiency virus (HIV) epitope, designated p18. Following establishment of an endogenous p18-specific memory CD8+ T-cell population, mice were challenged with coronavirus to directly compare recruitment, longevity, and activation characteristics of both primary coronavirus-specific and bystander memory populations trafficking into the central nervous system (CNS). HIV-specific memory CD8+ T cells were recruited early into the CNS as components of the innate immune response, preceding CD8+ T cells specific for the dominant coronavirus epitope, designated pN. Although pN-specific T-cell numbers gradually exceeded bystander p18-specific CD8+ T-cell numbers, both populations peaked concurrently within the CNS. Nevertheless, coronavirus-specific CD8+ T cells were preferentially retained. By contrast, bystander CD8+ T-cell numbers declined to background numbers following control of CNS virus replication. Furthermore, in contrast to highly activated pN-specific CD8+ T cells, bystander p18-specific CD8+ T cells recruited to the site of inflammation maintained a nonactivated memory phenotype and did not express ex vivo cytolytic activity. Therefore, analysis of host CD8+ T-cell responses to unrelated infections demonstrates that bystander memory CD8+ T cells can comprise a significant proportion of CNS inflammatory cells during virus-induced encephalitis. However, transient CNS retention and the absence of activation suggest that memory bystander CD8+ T cells may not overtly contribute to pathology in the absence of antigen recognition.     

IL-4 diminishes perforin-mediated and increases Fas ligand-mediated cytotoxicity In vivo

CTL have evolved two major mechanisms for target cell killing: one mediated by perforin/granzyme secretion and the other by Fas/Fas ligand (L) interaction. Although cytokines are integral to the development of naive CTL into cytolytic effectors, the role of cytokines on mechanisms of CTL killing is just emerging. In this study, we evaluate the effects of IL-4 in Fas(CD95)/FasL(CD95L)-mediated killing of Fas-overexpressing target cells. Recombinant vaccinia viruses (vv) were constructed to express respiratory syncytial virus M2 Ag alone (vvM2) or coexpress M2 and IL-4 (vvM2/IL-4). MHC-matched Fas-overexpressing target cells (L1210Fas+) were used to measure both perforin- and FasL-mediated killing pathways. In contrast to Fas-deficient (L1210Fas-) target cells, effectors from vvM2/IL-4-immunized mice were able to lyse L1210Fas+ target cells with similar magnitude as vvM2-infected mice. Addition of EGTA/Mg2+ revealed that effectors from vvM2/IL-4-infected mice primarily lyse targets by a Ca2+-independent Fas/FasL pathway. Analysis of FasL expression by flow cytometry showed that IL-4 increased cell surface FasL expression on CD4+ and CD8+ splenocytes, with peak expression on day 4 after infection. These data demonstrate that IL-4 increases FasL expression on T cells, resulting in a shift of the mechanism of CTL killing from a dominant perforin-mediated cytolytic pathway to a dominant FasL-mediated cytolytic pathway.