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Vesicular stomatitis virus (VSV) induces a T helper cell-independent IgM antibody response, whereas the IgG response is strictly T helper cell dependent. Since VSV induces B cells in complete absence of T helper cells, the question arises as to whether this induction occurs in the absence of a second signal or whether it depends upon an alternative or replacing signal 2. We therefore asked whether VSV has polyclonal B cell stimulator activity and/or whether B cell induction by VSV needs costimulation via complement or tumor necrosis factor (TNF) receptor or by natural killer (NK) cell activity. In vitro B cell proliferation assays and analysis of the in vivo antibody response in CD40-deficient mice excluded that VSV has properties of a polyclonal B cell stimulator. C3 depletion by cobra venom factor and application of anti-complement receptor antibodies showed that the T-independent IgM response was largely C3-independent except under very limiting antigen doses. Immunization of TNF receptor-deficient mice showed a normal anti-VSV IgM response, and in a cytotoxicity assay on YAC target cells there was no evidence of NK cell activation by VSV. Thus, VSV seems to induce B cells without polyclonal activation and/or C3, TNF, or NK cells functioning as a replacing second signal.

Induction of T-helper cells and T-B cell interaction have been considered to critically depend upon recognition of major histocompatibility complex (MHC) class II molecules by the T cell receptor. Mice lacking either MHC class II molecules (class II(0/0) mice) or its associated invariant chain (Ii0/0 mice) provide new opportunities to test this premise. Immune responses to some protein antigens have been studied in these mice; little is known about their ability to withstand viral infections. We therefore tested CD8+ effector T cells and CD4+ T-cell-dependent B cell function during different viral infections. The vesicular stomatitis virus (VSV)-specific primary cytotoxic T cell response which is largely T-helper-dependent was diminished in Ii(0/0) and absent in class II(0/0) mice. The usually less T-helper-dependent cytotoxic vaccinia or lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cell responses were reduced up to ninefold in class II(0/0) and up to threefold in Ii(0/0) mice. In class II(0/0) mice, the T-helper-independent neutralizing IgM response against the glycoprotein of VSV was within normal ranges but, in contrast to previous results on CD4(0/0) mice, the T-helper-dependent IgG response was absent. Ii(0/0) mice exhibited a normal neutralizing IgM response; in contrast to class II(0/0) mice, they mounted a significant, though reduced specific IgG response. Similar results were obtained for antibody responses against the nucleoprotein of VSV. Although the T-helper-cell response upon infection with VSV seemed diminished only a little in Ii(0/0) mice, presentation of VSV-G to a class II-restricted specific hybridoma was greater than 300-fold reduced in the absence of Ii. This suggests that local protein concentrations reached during viral infection in the host are high enough to override the Ii deficiency of antigen-presenting cells in vivo.

Immunological memory is a hallmark of the immune system. Evolution can teach us which effector arms of immunological memory are biologically relevant against which virus. Antibodies appear to be the critical protective mechanism against cytopathic viruses. Since these viruses cause cell damage and disease directly, particularly in the absence of an immune response, mothers protect their offspring during a critical immunoincompetent period (a consequence of MHC- restricted T cell recognition) by passive transfer of neutralizing antibodies. In contrast, CTL appear to be the crucial effector mechanism against noncytopathic viruses. Since MHC polymorphism has made vertical transmission of T cell memory impossible, immunoincompetent offspring are not, and need not be, protected against such noncytopathic viruses. During the primary response and again during secondary infection, the most important function of CTL is to eliminate noncytopathic viruses, which may otherwise cause lethal immunopathology. Increased precursor frequencies of B and T cells appear to remain in the host independent of antigen persistence. However, in order to protect against cytopathic viruses, memory B cells have to produce antibody to maintain protective elevated levels of antibody; B cell differentiation into plasma cells is driven by persisting antigen. Similarly, to protect against infection with a noncytopathic virus, CTL have to recirculate through peripheral organs. Activation and capacity to emigrate into solid tissues as well as cytolytic effector function are also dependent upon, and driven by, persisting antigen. Because no convincing evidence is available yet of the existence of identifiable B or T cells with specialized memory characteristics, the phenotype of immunological memory correlates best with antigen-driven activation of low frequency effector T cells and plasma cells.

Studies with perforin-deficient mice have demonstrated that two independent mechanisms account for T cell-mediated cytotoxicity: A main pathway is mediated by the secretion of the pore-forming protein perforin by the cytotoxic T cell, whereas an alternative nonsecretory pathway relies on the interaction of the Fas ligand that is upregulated during T cell activation with the apoptosis-inducing Fas molecule on the target cell. NK cells use the former pathway exclusively. The protective role of the perforin-dependent pathway has been shown for infection with the noncytopathic lymphocytic choriomeningitis virus, for infection with Listeria monocytogenes, and for the elimination of tumor cells by T cells and NK cells. In contrast, perforin-dependent cytotoxicity is not involved in protection against the cytopathic vaccinia virus and vesicular stomatitis virus. LCMV-induced immunopathology and autoimmune diabetes have been found to require perforin-expression. A contribution of perforin-dependent cytotoxicity to the rejection of MHC class I-disparate heart grafts has also been observed. Its absence is efficiently compensated in rejection of fully allogeneic organ or skin grafts. So far, evidence for a role of Fas-dependent cytotoxicity as a T cell effector mechanism in vivo is lacking. Current data suggest that the main function of Fas may be in regulation of the immune response and apparently less at the level of an effector mechanism in host defense. Further analysis is necessary, however, to settle this point finally.

The pregnant uterus of humans and rodents contains a population of granulated lymphoid cells, which, in the mouse, are called granular metrial gland (GMG) cells and have been described to express high levels of perforin. Since there is evidence for cytolytic activity of these cells and since perforin is a crucial effector molecule for the lytic action of cytotoxic T cells and natural killer cells, we evaluated the function of perforin in the pregnant uterus by using perforin-deficient mice. Perforin-deficient female mice were found to reproduce as efficiently as normal control females when bred either with syngeneic or allogeneic males. However, perforin-deficient mice differed from normal mice in that the frequency of GMG cells was significantly higher within maternal blood spaces and within several compartments of the feto-maternal interface. Proliferating GMG cells, identified by [3H] thymidine incorporation, were observed during more advanced stages of pregnancy when compared to normal controls. In contrast to normal mice, perforin-deficient mice did not display GMG cells attached to degenerating trophoblasts; instead perforin-deficient GMG cells were often observed in association with small maternal lymphocytes. In addition, the lack of transmission of lymphocytic choriomeningitis virus from infected pregnant perforin-deficient mice to the fetuses argued against a role of perforin expression by GMG cells in prevention of virus transmission from the mother to the fetus. Our data indicate that functional perforin is not necessary for successful pregnancies. The morphological changes in the pregnant uterus of perforin-deficient mice might, however, point to a certain, as-yet undefined function of perforin in the uterus of pregnant normal mice, which is functionally compensated in perforin-deficient mice.

The neutralizing antibody response against vesicular stomatitis virus (VSV) was studied to analyze conditions necessary for induction of mature B cells. The glycoprotein of VSV (VSV-G) is expressed as repetitive epitopes that are in a rigid densely packed paracristalline form in the virus envelope; in contrast, VSV-G is present in a mobile randomly ordered form on infected cells and in micelles. We found that the rigid, paracristalline form of VSV-G spaced about 5-10 mm on VSV virons induced potent primary and secondary neutralizing B cell responses which were independent of T helper cells, whereas the more randomly distributed, mobile forms of VSV-G induced primary and secondary B cell responses that were more tightly controlled by T helper cells. These data suggest that (i) cross-linking is a critical signal for B cell activation and antibody production, and that this signal alone does not necessarily anergize or delete mature B cells, (ii) the more regularly and rigidly ordered in a distance of 5-10 nm repetitive identical antigenic determinants are, the less are primary and secondary B cell responses controlled by T cells. We therefore propose that B cells take antigen organization as a marker for foreigness.

In vitro, T cell-dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin-, the other Fas-dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the non-cytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin-dependent mechanism without measurable involvement of the Fas-dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin-dependent cytotoxicity mediated by T cells is crucial for protection against non-cytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell-dependent soluble mediators such as cytokines (IFN-gamma against vaccinia virus) and neutralizing antibodies (against VSV and SFV).

Adaptive immune surveillance by T cells against infections and tumors depends on the presence of antigenic peptides presented by major histocompatibility complex (MHC) molecules. If antigenic tumor-specific peptides or MHC class I molecules are absent, the adaptive T cell immune response fails. Natural killer (NK) cells seem to complement the specific T cells by recognizing target cells lacking MHC class I (e.g. RMA-S). The role of perforin, which is crucially involved in T cell and NK cell-mediated target cell lysis, was evaluated in mice lacking perforin with respect to their capacity to eliminate a syngeneic lymphoid tumor. Here, we show that growth of MHC class I RMA-S tumor cells in unprimed mice was controlled by NK cells through perforin-dependent cytotoxicity.

Major histocompatibility complex (MHC) class II-associated antigen presentation is mainly linked to processing of exogenous antigens upon cellular uptake by endocytosis, but has also been observed for endogenously synthesized antigens. We have studied the MHC class II-associated presentation of the endogenously synthesized membrane associated glycoprotein (GP) and the cytosolic nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) in professional antigen presenting cells (APC) of mice. Since LCMV is a noncytopathic virus and minimally affects cellular protein synthesis, it is a convenient virus for the study of antigen presentation. In contrast, most other studies assessing class II-associated presentation of endogeneously synthesized viral antigens used cytolytic viruses such as vaccinia, measles and influenza virus, which drastically interfere with host cell functions. In addition, most studies were performed using non-professional APC. We found that class II-associated presentation of endogenously synthesized membrane associated LCMV-GP was efficient and could not be inhibited by chloroquine or leupeptin. Neither the transporter associated with processing (TAP) system nor the invariant chain (Ii) were significantly involved in this process. In contrast, MHC class II-associated presentation of endogenously synthesized cytosolic LCMV-NP was not observed even in Ii-deficient APC. Thus, MHC class II loading of endogenously synthesized LCMV-GP apparently does not require processing in acidic endosomal compartments as defined by chloroquine and leupeptin insensitivity. Furthermore, although the TAP molecules transport peptides of up to 15 amino acids in length, which potentially could bind to MHC class II molecules in the endoplasmic reticulum, such a process apparently does not occur for either the glycoprotein or the nucleoprotein. Therefore, the subcellular localization of an endogenously synthesized protein influences crucially whether or not MHC class II loading can occur independently of the acidic compartments usually involved in MHC class II loading.

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) alpha chain derived from the H-2Db-lymphocytic choriomeningitis virus (LCMV)-specific cytolytic T lymphocyte (CTL) clone P14. To generate these alpha P14 mice, mice transgenic for the P14 TCR alpha chain were backcrossed to TCR alpha-deficient mice. Thymi from alpha P14 mice exhibited a marked decrease of mature CD4+8- and CD8+4- single-positive thymocytes comparable to thymi from TCR alpha-deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from alpha P14 mice generated a primary anti-LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with V beta-specific antibodies showed a diverse endogenous TCR V beta repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30-fold) allogeneic and the absence of a vesicular stomatitis virus-specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR alpha chain.

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.

It is well known that synthetic peptides are able to both induce and tolerize T cells. We have examined the parameters leading either to priming or tolerance of CD8+ cytotoxic T lymphocytes (CTL) in vivo with a major histocompatibility complex class I (H-2 Db) binding peptide derived from the glycoprotein (GP aa33-41) of lymphocytic choriomeningitis virus (LCMV). By varying dose, route, and frequency of LCMV GP peptide application, we found that a single local subcutaneous injection of 50-500 micrograms peptide emulsified in incomplete Freund's adjuvant protected mice against LCMV infection, whereas repetitive and systemic intraperitoneal application of the same dose caused tolerance of LCMV-specific CTL. The peptide-induced tolerance was transient in euthymic mice but permanent in thymectomized mice. These findings are relevant for a selective use of peptides as a therapeutic approach: peptide-induced priming of T cells for vaccination and peptide-mediated T cell tolerance for intervention in immunopathologies and autoimmune diseases.

Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1-/-) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glyco- and nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1-/- cells as by control cells. Peptide regurgitation was not involved. Since particulate, non-replicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.

During most immune responses, T cells help antigen-specific B cells to make antibodies against the antigen. One of the contributions of T cells to antibody production is the induction of isotype switching from IgM to IgG, which is the most abundant isotype in blood serum during recall responses. Other features of memory responses are faster kinetics and higher titers of antibody in the serum. What causes a primary immune response to be different from a secondary is not yet very clear and, particularly, the influence of precursor frequencies of T and B cells on memory responses still remains to be answered. To address this issue, a transgenic (tg) mouse line (ADA) was developed; it expresses the beta chain (V beta 2) of a major histocompatibility complex class II-restricted T cell receptor (TcR) specific for the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana (VSV-IND). These mice exhibit an increased precursor frequency of VSV-specific CD4+ T cells that leads to enhanced neutralizing IgG production against VSV in vivo in unprimed mice. The data indicate that increased frequency of naive specific helper T cells alone may account for features of a memory phenotype such as high titer of antibodies and isotype switching.

The role of innate, alpha/beta interferon (IFN-alpha/beta)-dependent protection versus specific antibody-mediated protection against vesicular stomatitis virus (VSV) was evaluated in IFN-alpha/beta receptor-deficient mice (IFN-alpha/beta R0/0 mice). VSV is a close relative to rabies virus that causes neurological disease in mice. In contrast to normal mice, IFN-alpha/beta R0/0 mice were highly susceptible to infection with VSV because of ubiquitous high viral replication. Adoptive transfer experiments showed that neutralizing antibodies against the glycoprotein of VSV (VSV-G) protected these mice efficiently against systemic infection and against peripheral subcutaneous infection but protected only to a limited degree against intranasal infection with VSV. In contrast, VSV-specific T cells or antibodies specific for the nucleoprotein of VSV (VSV-N) were unable to protect IFN-alpha/beta R0/0 mice against VSV. These results demonstrate that mice are extremely sensitive to VSV if IFN-alpha/beta is not functional and that under these conditions, neutralizing antibody responses mediate efficient protection, but apparently only against extraneuronal infection.

Affinity maturation of IgG antibodies in adaptive immune responses is a well-accepted mechanism to improve effector functions of IgG within 2 weeks to several months of antigen encounter. This concept has been defined mainly for IgG responses against chemically defined haptens. We have evaluated this concept in a viral system and analyzed neutralizing IgG antibody responses against vesicular stomatitis virus (a close relative of rabies virus) with a panel of monoclonal antibodies obtained early (day 6 or 12) and late (day 150) after hyperimmunization. These neutralizing IgG antibodies recognize a single major antigenic site with high affinities (Ka of 10(8)-10(10) liter.mol-1) and with rapid on-rates already on day 6 of a primary response and with no evidence for further antigen dose- and time-dependent overall improvement of affinity. This type of IgG response is probably representative for viruses or bacterial toxins which are crucially controlled by neutralizing antibodies.

To study the role of perforin in cell-mediated graft rejection, vascularized hearts were grafted to perforin-deficient C57BL/6 and control C57BL/6 recipient mice. Fully allogeneic heart grafts (BALB/c) were acutely rejected by both recipients within 6 days. Peritoneal exudate lymphocytes from control mice but not from perforin-deficient mice exhibit a strong alloreactive cytotoxic activity in vitro. Histological analysis of the rejected tissues demonstrated extensive mononuclear cell infiltrates in both recipients. Flow cytometry analysis and immunohistology of graft-infiltrating cells showed similar proportions of lymphocyte subsets (CD8 >> CD4). Collectively, these data indicate that perforin is not essential in the cell-mediated acute rejection of a fully mismatched heart allograft. However, perforin-dependent effector mechanisms appeared to be limiting in the T cell-mediated rejection of heart allografts differing only at a single major histocompatibility complex class I antigen (bm1), because these grafts survived longer (mean 87.8 days) in perforin-deficient than in control mice (mean 31.5 days).

T cell tolerance is usually established by clonal deletion of self-specific T cells in the thymus, or some times, in the periphery. Alternatively, tolerance may also be achieved by induction of clonal T cell unresponsiveness by a poorly understood mechanism called "anergy". We found that transgenic mice expressing a soluble form of vesicular stomatitis virus (VSV) glycoprotein (G) predominantly in liver and kidney exhibited normal B cell responses. VSV-G-specific T help-independent neutralizing IgM responses were within normal ranges, but no T help-dependent neutralizing IgG antibodies were generated upon immunization with recombinant VSV-G protein and recombinant vaccinia virus expressing VSV-G. This demonstrated absence of B cell tolerance but presence of T helper cell unresponsiveness. After adoptive transfer of transgenic spleen cells into thymectomized immuno-incompetent hosts, the unresponsive T helper cells regained function and switched the neutralizing IgM response to IgG, comparably to control T helper cells, within 7 days. Conversely, when naive non-transgenic spleen cells were transferred into transgenic mice, VSV-G-specific T helper cells became unresponsive within 3-4 days. These results suggest that VSV-G-specific T helper cells are rendered unresponsive within a few days in the VSV-G transgenic host also outside of the thymus and that this unresponsiveness was reversed by transfer into antigen-free recipients.

Safe vaccines should optimally induce both cell-mediated and humoral immunity. Recently, it has been shown that protective cytotoxic T cells (CTLs) can be induced not only with live vaccines, but also with recombinant viral proteins. This report shows in C57BL/6 (H-2b) mice that the recombinant nucleoprotein (N) of vesicular stomatitis virus (VSV) induced protective CTLs but no neutralizing antibodies in mice, whereas the recombinant glycoprotein (G) of VSV alone induced neutralizing antibodies but no CTLs. If the N and G of VSV were coinjected, both CTLs and a long-lasting neutralizing IgG response was measurable, demonstrating that mixed vaccines can be used to induce protective CTLs and antibodies with an efficiency comparable to live virus. In an attempt to define optimal conditions for CTL priming, the intravenous, intraperitoneal and subcutaneous route of injection were compared. Intravenous injection of recombinant VSV-N induced up to 30 times higher responses than the latter two routes. Finally, we tried to define conditions inducing only CTLs and no antibodies binding to the native protein form, or vice versa, only antibodies and no CTLs. Intravenous injection of boiled VSV-N induced a CTL response but no antibodies specific for the native VSV-N, whereas VSV-N injected subcutaneously in incomplete Freund's adjuvant induced high amounts of anti-VSV-N antibodies but virtually no CTLs. The conditions defined here permit vaccines to be designed which would function along selected and defined immunological effector pathways.

Growth of Listeria monocytogenes is mainly controlled by macrophages, which are activated by specific T cells. A potential role of CD8+ T cells by direct lysis of infected cells was investigated in perforin-deficient mice generated by homologous recombination. The absence of perforin-mediated cytotoxicity resulted in delayed clearance of Listeria from the spleen but not the liver after primary infection, overall susceptibility to Listeria however was not increased. Protection against a secondary infection was drastically impaired in perforin-deficient mice. Adoptive transfer of immune spleen cells to recipients revealed that anti-Listeria protection by CD8+ T cells from perforin-deficient versus normal mice was about 10-fold reduced in livers and about 100-fold reduced in the spleen of recipients. CD4+ T cells from immune control and perforin-deficient mice conferred comparable protection. These results indicate that the protective effect of CD8+ T cells against an intracellular bacterium mainly evident in secondary infection is mediated by a perforin-dependent pathway, presumably cytotoxicity, and less by other direct or indirect effector mechanisms.

This study investigated whether or not the localization of persisting Ag influenced the localization of memory B and/or of Ab-forming cells (AFC). As a model Ag, we used vesicular stomatitis virus, which does not measurably replicate extraneuronally in adult mice after peripheral infection. Our results show that memory B cells and AFC are induced at the site where Ag is present; induced memory B cells then recirculate throughout the lymphoid system independently of Ag localization. In contrast, AFC are induced only at the sites of Ag persistence. These triggered Ab producing cells do not recirculate through the lymphoid tissue but migrate to the bone marrow.

Expression of self antigen on lymphohemopoietic cells and in the thymus has been shown to cause tolerance by negative selection. To investigate the role of self antigen expression levels on the induction of tolerance, we generated transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) driven by the H-2Kb promoter. Two mouse lines differing in transgene expression levels were obtained and evaluated for the induction of tolerance to LCMV-GP. LCMV-GP high (GPhi) expressing animals thymically deleted self-reactive thymocytes. Low expressors (GPlo) partially deleted self-reactive mature T cells in the periphery in the absence of any obvious signs of negative selection in the thymus. Functionally, the LCMV-GP-specific cytotoxic T cell (CTL) response was absent in GPhi mice, whereas GPlo mice produced diminished LCMV-GP-specific CTL responses. Therefore limiting levels of expression of self antigen influence efficiency of negative selection, enabling potentially self-reactive T cells to escape from tolerance induction.

Antigens normally induce an immunoglobulin (Ig)G response which stays at an elevated level for several weeks or months, constituting an important part of the immunological memory. This study investigated factors influencing the level of neutralizing IgG titers against a virus and shows that within the range tested it was independent of the number of initially available and potentially responding T helper and B cells, but was regulated by the amount of specific IgG-immune complexes forming depots of persisting antigen. These findings support the notion that the efficiency of vaccines in inducing long-lasting protective IgG is regulated predominantly by the amount of persisting (and presumably follicular dendritic cell-associated) antigen-antibody complexes.

Induction of CD8+, class I-restricted T cells by non-infectious, exogenous antigens has been documented for model protein antigens such as ovalbumin and for major histocompatibility complex restricted short peptides in viral and tumor systems. However, the protective capacity of cytotoxic T cells induced by conventional proteins has not been tested in vivo so far. We, therefore, evaluated the induction of protective cytotoxic T cells against three different full-length recombinant viral proteins derived from a baculovirus expression system, i.e. the glycoprotein and nucleoprotein of lymphocytic choriomeningitis virus (LCMV) and the nucleoprotein of vesicular stomatitis virus (VSV). These viral proteins induced cytotoxic T cells in a T helper cell-independent fashion which lysed infected target cells in vitro and protected mice from viral replication, immunopathological disease and growth of a tumor expressing the same antigen as a tumor antigen. These results are surprising, since it had been shown earlier for completely inactivated nonreplicating viral vaccines and again here for beta-propiolactone-inactivated VSV or UV-light inactivated LCMV that nonreplicating viral vaccines were incapable of inducing protective cytotoxic T cells. Our data show that immunization of mice with as little as 10 micrograms of non-infectious viral proteins triggered long-lasting CD8+ T cell-mediated antiviral immunity. It was found that the protein alone was only weakly able to induce cytotoxic T cells, and that association with cellular debris functioned as an adjuvant. These findings may be relevant for our understanding of the phenomenon of cross-priming and have obvious implications for vaccine strategies.

We have characterized a prominent (15-20%) thymocyte population expressing CD4 at a high and CD8 at a low level (CD4+8lo) in mice transgenic for a T cell receptor (TCR) restricted by major histocompatibility complex (MHC) class I molecules. The results demonstrate that the CD4+8lo population is an intermediate stage between immature CD4+8+ and end-stage CD4+8- thymocytes and that the survival of these cells crucially depends on the successful interaction of the transgenic TCR with self MHC class I molecules. In addition we demonstrate that the avidity of the interaction between TCR and self MHC class I molecules determines whether CD4+8lo thymocytes are found in significant numbers in this transgenic model. Our findings support a selective and multi-step model of T cell differentiation in the thymus.

Two molecular mechanisms of T cell-mediated cytotoxicity, one perforin-based, the other Fas-based, have been demonstrated. To determine the extent of their contribution to T cell-mediated cytotoxicity, a range of effector cells from normal control or perforin-deficient mice were tested against a panel of target cells with various levels of Fas expression. All cytotoxicity observed was due to either of these mechanisms, and no third mechanism was detected. Thus, the perforin- and Fas-based mechanisms may account for all T cell-mediated cytotoxicity in short-term in vitro assays.

CD4+ T cells play an important role in regulating the immune response; their contribution to virus clearance is variable. Mice that lack CD4+ T cells (CD4-/- mice) and are therefore unable to produce neutralizing antibodies cleared viscero-lymphotropic lymphocytic choriomeningitis virus (LCMV) strain WE when infected intravenously with a low dose (2 x 10(2) PFU) because of an effective CD8+ cytotoxic T-cell (CTL) response. In contrast, infection with a high dose (2 x 10(6) PFU) of LCMV strain WE led to expansion of antiviral CTL, which disappeared in CD4-/- mice; in contrast, CD4+ T-cell-competent mice developed antiviral memory CTL. This exhaustion of specific CTL caused viral persistence in CD4-/- mice, whereas CD4+ T-cell-competent mice eliminated the virus. After infection of CD4-/- mice with the faster-replicating LCMV strain DOCILE, abrogation of CTL response and establishment of viral persistence developed after infection with a low dose (5 x 10(2) PFU), i.e., an about 100-fold lower dose than in CD(4+)-competent control mice. These results show that absence of T help enhances establishment of an LCMV carrier state in selected situations.

The neutralizing immunoglobulin M (IgM) response to vesicular stomatitis virus (VSV) has been shown to be largely T-cell independent in several T-cell-deficient models of mice. By using different antigen froms of VSV, VSV antigen doses could be graded in vivo (infectious > > UV inactivated > formalin inactivated). The present study reveals a T-cell-dependent component of the neutralizing IgM response in nude mice given intravenous injections of low doses of noninfectious UV-inactivated VSV serotype Indiana (VSV-IND) only if the mice are transfused with VSV-IND-specific helper T cells. Instead, nude mice immunized with infectious VSV, which leads to greater antigen doses in vivo, were able to mount an IgM response in the absence of T cells. These results indicate that the IgM response to low doses of VSV-IND glycoprotein (G) is T-cell dependent. Nude mice immunized with infectious VSV also made a variable but low VSV-IND-neutralizing IgG response. A VSV-IND matrix (M)-specific helper T-cell line rendered this response more consistent, much higher, and longer lasting. Thus (i) VSV-G induces a mostly T-cell-independent but partially T-cell-dependent IgM (the latter can be visualized best at low doses of antigen) and (ii) the antibody response to VSV in nude mice proceeds through steps, i.e., IgM and IgG, that are dose dependent. The results suggest that the predominant role of helper T cells may be to expand and maintain the individual steps of differentiating B cells.

It has been much debated as to whether CD4+ T-cell depletion and the pathogenesis of AIDS is the result of direct cytolytic effects of human immunodeficiency virus (HIV), T-cell apoptosis by nonspecific activation, dysregulation of cytokine production, or autoimmunity. In this context, Rolf Zinkernagel and Hans Hengartner discuss data from model infections with non-cytopathic viruses. They suggest that HIV may cause immunosuppression, not by direct cytolytic effects, but rather by a conventional virus-specific cytotoxic T-cell-mediated immunopathology.

The influence of the murine MHC gene complex H-2 class I alleles or of the genetic background and the role of virus variants on establishment of a persistent infection with lymphocytic choriomeningitis virus (LCMV) was analyzed in immunocompetent mice of H-2d haplotype by evaluation of cytotoxic T cell responses. Susceptibility to establishment of a virus carrier state increased in various H-2d mice with lower CTL response and slower CTL kinetics. Low responder BALB/c-H-2dm2, lacking H-2Ld molecules to present the major T cell epitope amino acids 118-126 of the LCMV nucleoprotein or DBA/2 mice possessing relatively few CD8+ T cells, were more susceptible than high responder BALB/c mice expressing H-2Ld. Additional critical factors were LCMV isolate and dose of infection. The rapidly replicating LCMV-DOCILE and Cl 13 Armstrong induced viral persistence readily, whereas the slowly replicating parental virus strains WE or Armstrong did not. The presented findings illustrate a model of MHC-linked or MHC-unlinked susceptibility for virus persistence and may help to explain pathogeneses of chronic virus infections in humans that are often associated with slowly progressing immunopathology.

Perforin-deficient mice have been generated by homologous recombination to determine whether the effects of CD8+ cytolytic T cells and natural killer cells are mediated by pore formation involving perforin. These mice are viable and fertile and have normal numbers of CD8+ T cells and natural killer cells which do not lyse virus-infected or allogeneic fibroblasts or natural killer target cells in vitro. The mice fail to clear lymphocytic choriomeningitis virus and they eliminate fibrosarcoma tumour cells with reduced efficiency. Perforin is therefore a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis.

The size of the Ab repertoire has been estimated to comprise theoretically somewhere between > 10(10) and approximately 10(4) specificities, dependent on the criteria used. In an attempt to estimate the anti-viral protective Ab repertoire of the mouse the B cell and Ab-forming cell (AFC) frequencies and protective neutralizing Ab levels during the course of an infection with vesicular stomatitis virus (VSV) were analyzed. Determination of AFC frequencies, limiting dilution assays, and adoptive transfer experiments to SCID mice revealed that during the acute phase (day 8) of the immune response, more than 50% of all IgG2a-producing AFCs were specific for VSV, most of them recognizing the neutralizing determinant. In a later phase (days 21 or 50), 10 to 20 times fewer VSV-specific AFCs were present, corresponding to a frequency of approximately 1:10(4) spleen cells. Finally, in a protection assay in SCID mice, adoptively transferred protective Ab concentrations were found to be approximately 1 to 10 micrograms Ab/ml mouse serum. Because during the memory phase of the anti-VSV response usually 10(4) AFC/mouse are engaged to maintain a high level of memory IgG against the neutralizing determinant on VSV and if one assumes a total number of about 10(6) AFCs/mouse, these data suggest a rather limited neutralizing anti-viral protective memory-AFC repertoire of 10(2) to 10(4) different specificities.

Various strategies for the production of safe vaccines have been used. This study compared three different inactivation procedures, i.e. treatment with formaldehyde, beta-propiolactone or UV-light using vesicular stomatitis virus (VSV) as a model antigen. All three inactivation procedures drastically impaired induction of neutralizing IgG responses, confirming previous observations [Bachmann et al. (1993) J Virol 67:3917-3922]. This reduction could be overcome using higher doses for all three preparations. Both formaldehyde and beta-propiolactone completely abrogated the induction of VSV-specific cytotoxic T cells (CTLs), whereas UV-inactivated virus was able to induce significant and long-lasting CTL responses. These results may be of practical relevance since induction of neutralizing antibodies alone is often not sufficient for protection and sometimes may even enhance immunopathological responses of vaccinees.

T lymphocytes have been implicated in the onset of many autoimmune diseases; however, the mechanisms underlying T-cell activation toward self antigens are poorly understood. To study whether T-lymphocyte costimulation can overcome the immunologic unresponsiveness observed in an in vivo model, we have created transgenic mice expressing the costimulatory mouse molecule B7-1, a ligand for the CD28 receptor, on pancreatic beta cells. We now report that triple-transgenic mice expressing both B7-1 and a viral glycoprotein on their beta cells, along with T cells expressing the viral-glycoprotein-specific transgenic T-cell receptor, all develop insulitis (lymphocytic infiltration of the pancreatic islets) and diabetes. In striking contrast, the T cells in double-transgenic mice expressing the same viral glycoprotein (but no B7) on their pancreatic beta cells and the transgenic T-cell receptor on their T cells, reported earlier, remain indifferent to the glycoprotein-expressing beta cells. In fact, all three transgenes are required to initiate immune-mediated destruction of the beta cells. Mice expressing any of the transgenes alone, or any two in combination, maintain normal islet architecture and never spontaneously develop insulitis or diabetes. Our results show that aberrant B7 expression on peripheral tissues may play an important role in the activation of self-reactive T cells and further suggest that abnormal expression of costimulatory receptors may be involved in various T-cell-mediated autoimmune diseases.

The T-helper (Th) cell epitopes in the glycoprotein (GP) of vesicular stomatitis virus serotype Indiana (VSV-IND) were analyzed with a complete panel of overlapping synthetic peptides. Three Th-cell epitopes in C57BL/6 (H-2b) mice and two epitopes in BALB/c (H-2d) mice were defined by their ability to stimulate in vitro proliferation of virus-primed, CD8+ T-cell-depleted spleen cells in a class II-restricted manner. A series of CD4+, I-Ab-restricted T-cell hybridomas from VSV-primed C57BL/6 mice were characterized by their production of interleukin-2 and interleukin-3 upon stimulation with VSV-IND or purified VSV GP in vitro. Of nine hybridomas derived from three independent fusions, five were specific for amino acids (aa) 415 to 433 (p8) of VSV-IND GP, three recognized aa 52 to 71 (p41), and one reacted against aa 316 to 335 (p17). Fluorocytometric analysis of Th hybridomas or VSV-stimulated T-cell lines with monoclonal antibodies specific for the T-cell receptor V beta chain did not reveal obvious correlations between the T-cell receptor V beta gene segment used and the epitope recognized. All three peptides recognized by H-2b mice and both epitopes recognized by H-2d mice which were characterized in primed T-cell populations were capable of activating specific Th cells in vivo as measured by the induction of antibody class switch from immunoglobulin M (IgM) to IgG. Thus, the epitopes are relevant for VSV GP-specific Th response in vivo and are able to provide functional help for the production of anti-VSV-specific neutralizing IgG antibodies.

We investigated the mechanism leading to an IgG autoantibody response in two transgenic mouse lines expressing the glycoprotein of vesicular stomatitis virus (VSV-G). Previous experiments have shown that these animals do not mount a transgene-specific IgG response upon stimulation with purified VSV-G or infection with recombinant vaccinia virus expressing VSV-G. However, infection of VSV-G transgenic animals with wild-type vesicular stomatitis virus, serotype Indiana, readily induced VSV-G-specific, neutralizing IgG autoantibodies. We have tested whether this labile state of tolerance reflected differential availability of VSV-G-specific T help. For this, we immunized transgenic mice with the self-antigen VSV-G covalently coupled to sperm-whale myoglobulin (VSV-G-SWM), to provide new T helper epitopes that are linked to the B cell epitope; co-injected uncoupled VSV-G and SWM served as control. High titers of VSV-G specific IgG autoantibodies were detected in serum of mice immunized with VSV-G-SWM but not after co-injection of uncoupled VSV-G and SWM. Transgenic animals depleted of CD4+ T cells prior to injection of VSV-G-SWM failed to mount an IgG response. Priming of transgenic mice with the foreign carrier did not accelerate the IgG autoantibody response to VSV-G-SWM, suggesting that B cells were limiting the rate of the response. Thus, self-reactive B cells could be triggered to produce IgG, if they received linked T help specific for a foreign carrier determinant provided either by a classical carrier determinant or a virus.

A synthetic peptide corresponding to an immunodominant epitope of lymphocytic choriomeningitis virus glycoprotein (LCMV GP) was used to prime or to tolerize CD8+ T cells in vivo, dependent on mode of immunization. Peptide-specific tolerance was then examined in transgenic mice expressing LCMV GP in the beta islet cells of the pancreas; these mice develop CD8+ T-cell-mediated diabetes within 8-14 days after LCMV infection. Specific peptide-induced tolerance prevented autoimmune destruction of beta islet cells and diabetes in this transgenic mouse model.

Successful interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC) molecules during thymic selection down-regulates the expression of the recombination activating genes (RAG)-1 and -2 in cortical thymocytes and thereby prevents further endogenous TCR alpha-chain gene rearrangements (Borgulya, P., Kishi, H., Uematsu, Y. and von Boehmer, H., Cell. 1992. 69: 529-537; Brändle, D., Müller, C., Rülicke, T., Hengartner, H. and Pircher, H., Proc. Natl. Acad. Sci. USA 1992. 89: 9529-9533). To address the question whether down-regulation of RAG-1 activity represents an irreversible process we have blocked TCR-MHC interactions of thymocytes with thymic stromal cells. Firstly, transgenic (Tg) mice expressing a virus-specific MHC class I (H-2Db)-restricted TCR were injected with anti-Db or anti-CD8 monoclonal antibodies and RAG-1 expression was examined by in situ hybridization on thymus sections. The results show that cortical thymocytes up-regulated RAG-1 expression within 24 h after antibody administration. Secondly, immature thymocytes from TCR Tg mice were released from the thymic microenvironment and cultured in vitro for 14 h in single-cell suspension. The amount of RAG-1 mRNA was increased sixfold in cultured cells when compared to freshly isolated thymocytes. In addition, we show that immature thymocytes from TCR transgenic mice bearing non-selective MHC molecules (H-2d) down-regulated RAG-1 expression after antigen-induced TCR engagement. Cytofluorometric analysis further revealed that surface expression of CD69 on immature thymocytes inversely correlated with RAG-1 expression during positive and negative selection processes.

To address the mechanisms of tolerance to extrathymic proteins, we have generated transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) in the beta islet cells of the pancreas. The fate of LCMV-GP-specific T cells was followed by breeding the GP transgenic mice with T cell receptor transgenic mice, specific for LCMV-GP and H-2Db. These studies suggest that "Peripheral tolerance" of self-reactive T cells does not involve clonal deletion, clonal anergy, or a decrease in the density of T cell receptors or accessory molecules. Instead, this model indicates that potentially self-reactive cytotoxic T cells may remain functionally unresponsive, owing to a lack of appropriate T cell activation. Infection of transgenic mice with LCMV readily abolishes peripheral unresponsiveness to the self LCMV-GP antigen, resulting in a CD8+ T cell-mediated diabetes. These data suggest that similar mechanisms may operate in several so called "T cell-mediated autoimmune diseases". A synthetic peptide corresponding to an immunodominant epitope of lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) was used to prime or to tolerize CD8+ T cells in vivo, dependent on the mode of immunization. Peptide-specific tolerance was then examined in transgenic mice expressing LCMV-GP in the beta islet cells of the pancreas; these mice develop CD8+ T cell-mediated diabetes within 8-14 days after LCMV infection. Specific peptide-induced tolerance prevented autoimmune destruction of beta islet cells and diabetes in this transgenic mouse model.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is known to contain the biologically relevant sites for virus-neutralizing antibodies as well as T helper (Th) cell epitopes. The capacity of other VSV proteins to elicit potent Th cell responses has not yet been investigated. Additionally, a short-lived cross-reactivity between the two serologically distinct VSV serotypes Indiana (IND) and New Jersey (NJ) on the T helper cell level has been reported. Here we address the question of whether the VSV nucleoprotein (N) or matrix protein (M) can elicit T help to VSV-G-specific B cells and which of the VSV proteins contains the elements responsible for the IND/NJ cross-reactivity. The N, G, and M of the VSV Indiana serotype produced in a recombinant baculovirus system were assayed for the ability to activate VSV-specific Th cells to induce immunoglobulin class switch of neutralizing antibody responses in vivo in C57BL/6 (H-2b) mice. All three VSV-IND proteins helped in the production of neutralizing IgG antibodies to the homologous VSV-Indiana serotype but only VSV-IND N was able to trigger an IgG response to the heterologous VSV-New Jersey serotype. This data suggest that Th epitope(s) in the VSV-IND N are responsible for the observed cross-reactivity of T helper cells.

The effects of neutralizing antibodies on the disease course in mice infected with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were evaluated. Whereas non-neutralizing antisera exhibiting high enzyme-linked immunosorbent assay titers had no effect on T cell responses and their consequences, neutralizing antisera modulated them variably. Neutralizing antibodies were able to prevent lethal choriomeningitis after intracerebral infection with a neurotropic LCMV-isolate (ARMSTRONG) although they could not control local virus replication. The same antibodies exhibited little or no protective effect on choriomeningitis induced by LCMV-WE, a viscerotrope isolate. Surprisingly, these antibodies rendered mice much more susceptible to choriomeningitis after intracerebral infection with LCMV DOCILE, a very rapidly spreading lymphocyto-viscerotrope virus; in this situation antibodies prevented overwhelming infection which causes deletion of immunopathogenic cytotoxic T cell responses. Thus preexisting neutralizing antiviral antibodies had little influence on local virus spread in peripheral tissues but they reduced hematogenic spread and infection of antigen-presenting cells; thereby they influenced the primary cytotoxic T cell (CTL) response and indirectly modulated the extent of T cell-mediated immunopathology in peripheral organs. These results may explain why vaccines inducing neutralizing antibodies but no CTL may enhance an immunopathological disease caused by challenge infection with a noncytopathic virus.

The influence of antigen epitope density and order on B cell induction and antibody production was assessed with the glycoprotein of vesicular stomatitis virus serotype Indiana [VSV-G (IND)]. VSV-G (IND) can be found in a highly repetitive form the envelope of VSV-IND and in a poorly organized form on the surface of infected cells. In VSV-G (IND) transgenic mice, B cells were unresponsive to the poorly organized VSV-G (IND) present as self antigen but responded promptly to the same antigen presented in the highly organized form. Thus, antigen organization influences B cell tolerance.

After infection with some viruses that tend to persist, neutralizing antibodies are generated rather late, i.e., after 1 to 3 mo. This has been observed for HIV, Hepatitis B infection in man, or after infection with lymphocytic choriomeningitis virus (LCMV) in mice. In contrast, nonneutralizing antibodies to LCMV are generated by day 7 and reach high titers by day 10. This study attempts to evaluate reasons for late and low titered neutralizing antibody responses. After a primary infection with low doses (10(2) plaque forming units) of the poorly cytopathic LCMV-WE, neutralizing antibodies were rarely produced at detectable levels before days 60 to 120. In vivo depletion of CD8+ CTL led to a marked enhancement and acceleration of kinetics of the neutralizing antibody response. In contrast, nonneutralizing antibodies, including those specific for LCMV-GP carrying the neutralizing determinant, were detectable very early, i.e., 4 to 7 days after infection, with maximum titers usually by day 10 irrespective of the presence or absence of CTL. Mice completely lacking CD8+ T cells because of deletion by homologous recombination (CD8-/-) also exhibited neutralizing antibodies early by day 10 to 20, and by day 120 reached very high titers. The neurotropic isolate LCMV-ARMSTRONG induced low but significant titers of neutralizing antibodies relatively early (i.e., by days 7 to 10), whereas the lympho-viscerotrope mutant virus LCMV-ARMSTRONG Cl-13 did not. Early and effective CTL responses causing immunopathology (and immunosuppression) correlated with the absence of neutralizing antibodies. The discrepancy between the CTL-dependent inhibition of neutralizing versus unimpaired anti-GP ELISA responses cannot be explained by different Ag doses alone. Additionally, it may reflect different kinetics of the responses, whereby the later neutralizing responses possibly requiring IgG affinity maturation may be more susceptible to general immunosuppression; also B cells expressing neutralizing receptors, but not those with antibodies binding GP (or NP), may be actively infected, therefore they present viral peptides on class I MHC Ag and may become targets for anti-LCMV-specific CTL.

The role of costimulatory signals in T cell induction was evaluated in mice lacking the interleukin-2 (IL-2) gene. In vitro secondary antiviral T cell responses were absent unless IL-2 was added, confirming the crucial role of IL-2 in vitro. In vivo, primary and secondary cytotoxic T cell responses against vaccinia and lymphocytic choriomeningitis virus were within normal ranges. B cell reactivity to vesicular stomatitis virus was not impaired. T helper cell responses were delayed but biologically functional. Natural killer cell activity was markedly reduced but inducible. These normal in vivo immune responses in IL-2-deficient mice question the importance of IL-2 as defined by in vitro studies.

Sensitivities of in vitro and in vivo assays for the detection of lymphocytic choriomeningitis virus (LCMV)-specific CTL were compared. Measurement of primary cytotoxicity was the least sensitive of all the tested assays. However, when the same 51Cr release assays were performed after in vitro restimulation, this in vitro method was found to be more sensitive than all of the five in vivo assays tested. Assessment of CTL-mediated protection against LCMV replication in spleens was most sensitive among the in vivo tests, followed by CTL-mediated resistance to intracerebral or intraperitoneal challenge infections with vaccinia-LCMV-recombinant virus. Inhibition of LCMV-induced choriomeningitis and of the footpad swelling reaction were least sensitive in detecting LCMV-specific CTL. As discussed, the presented sensitivity gradient may most probably be generalized.

The fate of in vivo activated CD8+ cytotoxic T cells was studied in transgenic mice expressing a T cell receptor (TCR) specific for the lymphocytic choriomeningitis virus (LCMV) glycoprotein peptide 33-41 presented by major histocompatibility complex (MHC) class I molecules. LCMV infection of TCR transgenic mice induced LCMV-specific effector and memory T cells whereas injection of soluble LCMV glycoprotein peptide 33-41 resulted in tolerance by peripheral deletion and anergy of LCMV-specific T cells after an initial expansion phase. Similarly, LCMV peptide 33-41-specific tolerance could be achieved in normal C57BL/6 mice and was not abrogated by an LCMV infection. These results obtained with a classically MHC-restricted peptide antigen parallel previous findings with retroviral or bacterial superantigens and indicate a possibility to modulate specifically mature peripheral cytotoxic T lymphocytes in vivo.

The effects of formalin on the infectivity and immunogenicity of vesicular stomatitis virus (VSV) serotype Indiana were investigated. We found that formalin inactivation of VSV prevents infection of Vero cells in a concentration- and time-dependent manner, as shown by fluorometric cell analysis and inhibition of plaque formation. Inactivated VSV failed to induce significant cytotoxic T-lymphocyte responses in vivo or after restimulation in vitro. In contrast, the early immunoglobulin M (IgM) response, which is T help independent in the VSV system, was unaltered, suggesting normal antigenicity for and induction of B cells. However, no switch to IgG occurred, demonstrating failure of induction of T help. If cross-reactive T help was provided by previous infection with a second serotype of VSV (New Jersey), the IgG response was almost completely restored, confirming that the absence of IgG was due to lack of T help. A formalin-treated preparation of glycoprotein of VSV led to a delayed but otherwise normal IgG response, whereas treatment of VSV with UV light or beta-propiolactone reduced IgG titers to the same extent as did formalin. These results suggest that loss of infectivity and the ensuing lack of amplification of viral antigens of formaldehyde-inactivated VSV is the major factor impairing induction of specific T-helper cell responses.