Viral immune evasion due to persistence of activated T cells without effector function

We examined the regulation of virus-specific CD8 T cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. Our study shows that within the same persistently infected host, different mechanisms can operate to silence antiviral T cell responses; CD8 T cells specific to one dominant viral epitope were deleted, whereas CD8 T cells responding to another dominant epitope persisted indefinitely. These virus-specific CD8 T cells expressed activation markers (CD69(hi), CD44(hi), CD62Llo) and proliferated in vivo but were unable to elaborate any antiviral effector functions. This unresponsive phenotype was more pronounced under conditions of CD4 T cell deficiency, highlighting the importance of CD8- CD4 T cell collaboration in controlling persistent infections. Importantly, in the presence of CD4 T cell help, adequate CD8 effector activity was maintained and the chronic viral infection eventually resolved. The persistence of activated virus-specific CD8 T cells without effector function reveals a novel mechanism for silencing antiviral immune responses and also offers new possibilities for enhancing CD8 T cell immunity in chronically infected hosts.     

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.     

Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8+, but not of CD4+, T cells and accelerated by increasing the frequency of LCMV-specific CD8+ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8+ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8+ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P0/0 and P0/0 x TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-alpha and interferon (IFN)-gamma produced by CD8+ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-gamma-producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-gamma-producing CD8+ T cells. Thus, in the absence of IFN-gamma and/or TNF/LT-alpha, exhaustion of virus-specific T cells was not hampered.     

T-lymphocyte downregulation after acute viral infection is not dependent on CD95 (Fas) receptor-ligand interactions

Infection of mice with lymphocytic choriomeningitis virus (LCMV) causes a major expansion of CD8+ T cells followed by a period of immune downregulation that coincides with the induction of lymphocyte apoptosis in the mouse spleen. CD95 (Fas) and its ligand are important for regulating peripheral T-lymphocyte numbers and can mediate apoptosis of mature T lymphocytes. We infected CD95- and CD95L-deficient mice (lpr and gld, respectively) with LCMV to determine if the immune downregulation that occurred following resolution of the LCMV infection was due to a CD95-dependent apoptotic mechanism. Lymphocytes from LCMV-infected lpr and gld mice were capable of normal T-cell expansion and cytolytic function but were, in contrast to activated cells from normal virus-infected mice, relatively more resistant to T-cell receptor-induced apoptosis in vitro. However, in vivo there were significant numbers of apoptotic cells in the spleens of lpr and gld mice recovering from the infection, and the T-cell number and cytolytic activity decreased to normal postinfection levels. Thus, CD95 is not required for the immune downregulation of the CD8+-T-lymphocyte response following acute LCMV infection.     

A role for perforin in downregulating T-cell responses during chronic viral infection

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf -/-) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf -/- mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral "exhaustion" of activated CD8 T cells occurred less efficiently in perf -/- mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf -/- mice; approximately 50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.     

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.     

Contributions of antibody and T cell subsets to protection elicited by immunization with a replication-defective mutant of herpes simplex virus type 1

Replication-defective mutants of herpes simplex virus 1 (HSV-1) elicit immune responses in mice that reduce acute and latent infection after corneal challenge and are protective against development of disease. To understand the basis for the protective immunity induced by this new form of immunization, we investigated the contribution of various components of the immune response to protection against corneal infection and disease. Passive transfer of sera from mice immunized with the replication-defective mutant virus, d301, its parental HSV-1 strain, or uninfected cell lysate was used to examine the role of antibody. Despite posttransfer neutralizing antibody titers equivalent to those in control mice directly immunized with mutant virus, recipients of immune serum showed no reductions in primary replication in the eye, keratitis, or latent infection of the nervous system. However, immune serum protected mice from encephalitis and death. To examine the contribution of T cell subsets to protection, mice were immunized once with mutant virus and then were depleted in vivo of CD4+ or CD8+ T cells prior to corneal challenge. CD4 depletion resulted in higher titers of challenge virus in the eye at 3 to 4 days after challenge compared to control mice. Latent infection of the nervous system was increased by depletion of CD4+ T cells but not by depletion of CD8+ T cells keratitis developed only in a portion of the CD8+ T cell-depleted mice, suggesting that an immunopathologic potential of CD4+ T cells is held in check when immune CD8+ T cells are also present. Taken together, these data support a role for antibody induced by immunization with a replication-defective virus principally in protecting the central nervous system from disease, roles for CD4+ T cells in reducing primary replication in the eye and protecting against latent infection of the nervous system, and a role for CD8+ T cells in regulating the immunopathologic activity of CD4+ T cells.     

Protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory T cell populations

A basic principle of immunology is that prior immunity results in complete protection against a homologous agent. In this study, we show that memory T cells specific to unrelated viruses may alter the host's primary immune response to a second virus. Studies with a panel of heterologous viruses, including lymphocytic choriomeningitis (LCMV), Pichinde (PV), vaccinia (VV), and murine cytomegalo (MCMV) viruses showed that prior immunity with one of these viruses in many cases enhanced clearance of a second unrelated virus early in infection. Such protective immunity was common, but it depended on the virus sequence and was not necessarily reciprocal. Cell transfer studies showed that both CD4 and CD8 T cell populations from LCMV-immune mice were required to transfer protective immunity to naive hosts challenged with PV or VV. In the case of LCMV-immune versus naive mice challenged with VV, there was an enhanced early recruitment of memory phenotype interferon (IFN) gamma-secreting CD4(+) and CD8(+) cells into the peritoneal cavity and increased IFN-gamma levels in this initial site of virus replication. Studies with IFN-gamma receptor knockout mice confirmed a role for IFN-gamma in mediating the protective effect by LCMV-immune T cell populations when mice were challenged with VV but not PV. In some virus sequences memory cell populations, although clearing the challenge virus more rapidly, elicited enhanced IFN-gamma-dependent immunopathogenesis in the form of acute fatty necrosis. These results indicate that how a host responds to an infectious agent is a function of its history of previous infections and their influence on the memory T cell pool.     

Resistance to and recovery from lethal influenza virus infection in B lymphocyte-deficient mice

In the adaptive immune response to most viruses, both the cellular and humoral arms of the immune system play complementary roles in eliminating virus and virus-infected cells and in promoting recovery. To evaluate the relative contribution of CD4+ and CD8+ effector T lymphocytes in virus clearance and recovery, we have examined the host response to lethal type A influenza virus infection in B lymphocyte-deficient mice with a targeted disruption in the immunoglobulin mu heavy chain. Our results indicate that naive B cell-deficient mice have a 50- 100-fold greater susceptibility to lethal type A influenza virus infection than do wild type mice. However, after priming with sublethal doses of influenza, immune B cell-deficient animals show an enhanced resistance to lethal virus infection. This finding indicates that an antibody-independent immune-mediated antiviral mechanism accounts for the increased resistance to lethal virus challenge. To assess the contribution of influenza-specific CD4+ and CD8+ effector T cells in this process, defined clonal populations of influenza-specific CD4+ and CD8+ effector T cells were adoptively transferred into lethally infected B cell-deficient mice. Cloned CD8+ effectors efficiently promoted recovery from lethal infection, whereas cloned CD4+ T cells conferred only partial protection. These results suggest that memory T lymphocytes can act independently of a humoral immune response in order to confer resistance to influenza infection in immune individuals. The potential implications of these results for vaccination against human influenza infection are discussed.     

Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.     

Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes

Intravaginal (IVAG) inoculation of wild-type herpes simplex virus type 2 (HSV-2) in mice causes epithelial infection followed by lethal neurological illness, while IVAG inoculation of attenuated HSV-2 causes epithelial infection followed by development of protective immunity against subsequent IVAG challenge with wild-type virus. The role of T cells in this immunity was studied by in vivo depletion of these cells with monoclonal antibodies. Three groups of mice were used for each experiment: nonimmune/challenged mice, immune/challenged mice, and immune depleted mice [immune mice depleted of a T-cell subset(s) shortly before challenge with HSV-2]. Mice were assessed for epithelial infection 24 h after challenge, virus protein in the vaginal lumen 3 days after challenge, and neurological illness 8 to 14 days after challenge. Monoclonal antibodies to CD4, CD8, or Thy-1 markedly reduced T cells in blood, spleen, and vagina, but major histocompatibility complex class II antigens were still partially upregulated in the vaginal epithelium after virus challenge, indicating that virus-specific memory T-cell function was not entirely eliminated from the vagina. Nevertheless, immune mice depleted of CD4+ and CD8+ T cells, Thy-1+ T cells, or CD8+ T cells alone had greater viral infection in the vaginal epithelium than nondepleted immune mice, indicating that T cells contribute to immunity against vaginal HSV-2 infection. All immune depleted mice retained substantial immunity to epithelial infection and were immune to neurological illness, suggesting that other immune mechanisms such as virus-specific antibody may also contribute to immunity.     

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.     

Transfer of lymphocytic choriomeningitis disease in beta 2-microglobulin-deficient mice by CD4+ T cells

In this study we have investigated the role of CD4+, MHC class II-restricted cytotoxic T lymphocytes (CTLs) in the disease caused by lymphocytic choriomeningitis virus (LCMV) in beta 2-microglobulin deficient (beta 2m-) mice. Intracranial (i.c.) infection with LCMV resulted in death of six out of 11 beta 2m- mice. Mice that survived showed a marked loss in body weight. Death and loss of body weight could be prevented by immunosuppressing the mice with irradiation or cyclosporine prior to i.c. injection of LCMV. This treatment also prevented induction of virus-specific, MHC class II-restricted CTL following peripheral inoculation with LCMV. In vivo depletion of CD4+ cells with antibody also prevented death following i.c. injection whereas in vivo depletion of CD8+ cells had no effect. Disease could be transferred to recipient beta 2m- mice by adoptive transfer of beta 2m- derived immune spleen cells. Transfer of non-immune spleen cells did not result in illness. In vitro treatment of immune spleen cells with anti-CD4 antibody and complement eliminated class II-restricted CTL activity and also prevented mortality of recipients after adoptive transfer. Treatment with anti-CD8 antibody had no effect. We were unable to transfer LCM disease to beta 2m- recipients by adoptive transfer of immune spleen cells from C57BL/6 mice. These results suggest that, unlike normal mice, the pathology of LCM disease in beta 2m- mice is dependent upon virus-specific, CD4+CD8-, MHC class II-restricted T cells.     

Cooperation of B cells and T cells is required for survival of mice infected with vesicular stomatitis virus

To define the role of T cells and B cells in resistance to vesicular stomatitis virus (VSV) infection, knockout mice with different specific immune defects on an identical background were infected i.v. and the outcome of infection was compared; in this way a more complete picture of the relative importance of various host defence mechanisms could be obtained. Compared to T and B cell-deficient SCID mice which all succumbed from encephalitis within 5-9 days of infection, T cell-deficient nude mice generally lived longer, but within a period of approximately 1 month after challenge all died. In contrast, B cell-deficient mice were highly susceptible even to low doses of virus and mortality could be prevented by transfer of naive B cells prior to challenge as well as by immune serum given after challenge. Analysis of MHC class I- and class II-deficient mice revealed that CD8+ T cells could exert some antiviral activity, but CD4+ T cells sufficed for survival and were required for optimal resistance. Consistent with this it was found that in nude mice a lethal outcome could be prevented by transfer of CD8-depleted cells from B cell-deficient mice. Thus our results clearly demonstrate that while antibodies are pivotal for survival in the early phase of VSV infection, T cells are required for long-term survival, with CD4+ T cells being more effective in controlling this infection than CD8+ T cells.     

Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells

Measles causes a profound immune suppression which is responsible for the high morbidity and mortality induced by secondary infections. Dendritic cells (DC) are professional antigen-presenting cells required for initiation of primary immune responses. To determine whether infection of DC by measles virus (MV) may play a role in virus-induced suppression of cell-mediated immunity, we examined the ability of CD1a+ DC derived from cord blood CD34+ progenitors and Langerhans cells isolated from human epidermis to support MV replication. Here we show that both cultured CD1a+ DC and epidermal Langerhans cells can be infected in vitro by both vaccine and wild type strains of MV. DC infection with MV resulted within 24-48 h in cell-cell fusion, cell surface expression of hemagglutinin, and virus budding associated with production of infectious virus. MV infection of DC completely abrogated the ability of the cells to stimulate the proliferation of naive allogeneic CD4+ T cell as early as day 2 of mixed leukocyte reaction (MLR) (i.e., on day 4 of DC infection). Mannose receptor-mediated endocytosis and viability studies indicated that the loss of DC stimulatory function could not be attributed to the death or apoptosis of DC. This total loss of DC stimulatory function required viral replication in the DC since ultraviolet (UV)-inactivated MV or UV-treated supernatant from MV-infected DC did not alter the allostimulatory capacity of DC. As few as 10 MV- infected DC could block the stimulatory function of 10(4) uninfected DC. More importantly, MV-infected DC, in which production of infectious virus was blocked by UV treatment or paraformaldehyde fixation, actively suppressed allogeneic MLR upon transfer to uninfected DC-T-cultures. Thus, the mechanisms which contribute to the loss of the allostimulatory function of DC include both virus release and active suppression mediated by MV-infected DC, independent of virus production. These data suggest that carriage of MV by DC may facilitate virus spreading to secondary lymphoid organs and that MV replication in DC may play a central role in the general immune suppression observed during measles.     

A critical role for neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers

This study demonstrates that neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons (IFNs) are of key importance in virus control both in adoptive immunotherapy of persistent infection and in the late phase of acute infection with the WE strain of lymphocytic choriomeningitis virus (LCMV). We report the following results. (i) Clearance of LCMV-WE from C57BL/6 carrier mice by adoptive transfer of memory spleen cells requires B cells and CD4(+) T cells but not necessarily CD8(+) T cells. (ii) At the doses examined, CD8(+) T cells contribute to the initial reduction of viral titers but are alone not sufficient to clear the virus because they are exhausted. (iii) In the presence of functional IFN-gamma, virus clearance correlates well with the generation of neutralizing antibodies in the treated carrier mice. (iv) In the absence of receptors for IFN-gamma, virus clearance is not achieved. (v) Adoptive immunotherapy of mice persistently infected with a distinct virus isolate, LCMV-Armstrong, revealed only low levels of neutralizing antibodies; in this case, CD8(+) T cells were needed for virus clearance in addition to B and CD4(+) T cells. (vi) After low dose infection of C57BL/6 mice with LCMV-WE, virus is eliminated below detectable levels by CD8(+) T cells, but long-term (>2 months) virus control is usually not achieved in the absence of B cells or CD4(+) T cells; reappearance of the virus is paralleled either by exhaustion of virus-specific cytotoxic T lymphocytes or lethal immunopathology. These findings are of importance for adoptive immunotherapy strategies against persistent virus infections in humans.     

Antitumor effect of CD40 ligand: elicitation of local and systemic antitumor responses by IL-12 and B7

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 has been implicated in the establishment of cell-mediated immunity as well as humoral immune responses. To examine the role of CD40L in eliciting antitumor immunity, we introduced murine CD40L gene into P815 mastocytoma (CD40L-P815). CD40L-P815 cells underwent prompt rejection when inoculated s.c. into syngenic DBA/2 mice or athymic BALB/c nu/nu mice, which was mediated by NK cells and dependent on endogenous IL-12. The primary rejection of CD40L-P815 cells in DBA/2 mice elicited CD8+ T cell-mediated protective and systemic immunity against parental tumor cells, which was induced by CD4+ T cells and endogenous B7. These results indicated a potent antitumor effect of CD40L that is mediated by potentiation of host Ag-presenting cell functions, and introduction of CD40L will be useful as a new strategy of immuno-gene therapy against tumors.     

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.     

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.