Rm. Zinkernagel

The Role of Antibodies in Natural and Acquired Resistance of Mice to Vesicular Stomatitis Virus

Mice infected with live vesicular stomatitis virus (VSV) produced primary antibody responses more efficiently (with doses greater than 10(2) pfu) than those injected with UV-inactivated VSV (greater than 10(6) pfu) or purified VSV glycoprotein G (equivalent to 10(7) pfu) by producing neutralizing antibodies. Very low doses of live VSV (less than 10(2) pfu) failed to prime mice. Normal mouse serum had the capacity to inactivate VSV by heat-labile and immunoglobulin-mediated mechanisms in vitro independently of specifically induced antibodies. Studies using B cell-depleted agammaglobulinaemic mice showed that their serum lacked VSV-neutralizing capacity in vitro and that naturally resistant mice became susceptible to VSV-induced paralytic disease which could be prevented by adoptive transfer of immune but also of normal serum.

Impaired generation of anti-viral cytotoxicity against lymphocytic choriomeningitis and vaccinia virus in mice treated with CD4-specific monoclonal antibody

The role of CD4+ helper T cells in induction of anti‐viral cytotoxic T‐cell response was investigated by treating normal and thymectomized C57B1/6 mice with CD4‐speciflc monoclonal antibodies (MoAb). In CD4‐specific MoAb‐treated mice infected with Vaccinia or lymphocytic choriomeningitis virus (LCMV), cytotoxic T‐cell activity was 5–15 times lower than in normal controls when measured in a 51Cr release assay and computed as lytic units 6 and 8 days respectively after virus inoculation. This difference in the levels of effector T‐cell activities did not reflect slower kinetics of cytotoxic T‐cell induction in antibody‐treated versus control mice, since it was also obvious at 8 days after infection for Vaccinia virus and 10 and 12 days after inoculation with LCMV, CD4‐specific MoAb‐induced inhibition of cytotoxic T‐cell responses in vivo was seen up to 150 days after treatment in thymectomized mice. However, no significant suppressive effect of the same antibody treatment on T‐cell cytotoxicity could be observed in animals treated on day 3 or later after infection with Vaccinia virus. Injection of CD4‐depleted mice with recombinant interleukin 2 (rIL‐2) partially corrected the impaired virus‐specific cytotoxic T‐cell response, suggesting that IL‐2 supply may be limiting in mice licking T helper cells.

Role of tumor necrosis factor in Listeria resistance of nude mice

The effects of sheep anti-murine recombinant tumor necrosis factor-alpha (TNF-α) on resistance to Listeria monocytogenes infection were studied in T cell-deficient nu/nu mice. The sheep anti-TNF-α antibody preparation was specific for TNF since it neutralized 300 U of recombinant murine TNF-α in vitro at a dilution of up to 1/1,000 but did not neutralize 32 U of interferon (IFN)-α, -β or 32 U of IFN-γ in vitro at a 1/20 dilution. When tested in vivo in sublethally Listeria-infected nu/nu or T cell-competent C57BL/6 or ICR mice, a single treatment of 0.2 ml anti-TNF-α given intraperitoneally on either day -1,0 or +1 resulted in the death of mice by day 5–7 due to the uncontrolled growth of Listeria; bacterial counts in spleen and liver were increased on days 3–5 by a factor of 10–1,000 in these organs. When examined histologically, organs from mice with the anti-TNF-α treatment contained more, and considerably bigger, lesions that exhibited central necrosis. The enhancing effect of anti-TNF-α on Listeria infection seemed greater early during Listeria infection on days 1–6 when compared to later phases of the infection around days 6–10. From the data presented we conclude that in addition to other lymphokines, such as IFN-γ, TNF-α is of importance during the entire course of a Listeria infection in nu/nu mice.

Generation of agammaglobulinaemic mice by prenatal and postnatal exposure to polyclonal or monoclonal anti-IgM antibodies.

Improved experimental conditions are described for the treatment of mice with anti‐IgM antibody, which subsequently lead to B‐cell deficiency and agammaglobulinaemia. Antibody transmitted via maternal milk alone was found to be more efficient in inducing suppression of serum immunoglobulin isotypes than prenatal transmission or postnatal intraperitoneal injections alone. However, the combined treatment by all three routes of exposure to anti‐IgM resulted in total B‐cell suppression associated with undetectable levels of all serum immunoglobulin isotypes. Furthermore, suppression of B‐cell generation was also achieved with a rat monoclonal μ‐specific antibody. The possibility of generating agammaglobulinaemic mice may be useful for investigating the influence of B cells on the generation of T‐cell reactivities, and for analysing the effects of monoclonal antibodies in the absence of interfering serum immunoglobulin.

Anti-Listeria monocytogenes immunity in mu-suppressed mice: a comparison of treatment with conventional hyperimmune rabbit anti-mouse IgM and affinity-purified, monoclonal rat anti-mouse IgM.

The capacity of anti-IgM treated, B-cell-depleted mice to control infection by Listeria monocytogenes was evaluated. Suppression was achieved with a hyperimmune rabbit anti-mouse-IgM antiserum (IRS), with affinity-purified IRS (IRP), or with an affinity-purified, monoclonal, rat anti-mouse-IgM antibody (LO-MM-9). B-cell depletion in specifically treated mice was judged to be complete by the following criteria: absence of significant response to a B-cell mitogen lipopolysaccharide, absence of B-cells with detectable IgM or kappa light chain on their surface, absence of detectable IgM, and presence of free anti-IgM antibodies in serum. BALB/c mice, conventionally treated from birth with IRS, had an increased capacity to clear L. monocytogenes from the blood during the first 5 min after intravenous infection. Furthermore, control of infection seemed to be enhanced during the first 24 h but was found to be impaired when assessed 3 and 4 days after initiation of infection. These effects were, however, not IRS specific, because control mice treated with normal rabbit serum behaved comparably. Mortality caused by 2 × 103L. monocytogenes injected intraperitoneally into BALB/c mice susceptible to L. monocytogenes was increased more in NRSthan in IRS-treated mice when both were compared with untreated control mice. Therefore, chronic injection of IRS or NRS seemed to disturb anti-L. monocytogenes immunity, rendering an evaluation of the role of antibodies impossible. Chronic treatment of mice, born from and raised by mothers similarly treated with monoclonal, rat anti-mouse IgM (LO-MM-9), showed that initial blood clearance (at 5 min), early (1–24 h) and late (3–5 day) bactericidal anti-Listeria activity in spleens and livers and also susceptibility as monitored by mortality were comparable to those observed in untreated BALB/c. These results demonstrate formally that antibodies, whether naturally pre-existant or specifically induced, do not play a role in either non-immunological early or subsequent T-cell-dependent control of primary Listeria monocytogenes infection in mice.

Distinct patterns of virus-specific T cell-mediated cytolysis of transformed versus primary target cells

Ideas about the mechanism(s) by which cytotoxic T lymphocytes(CTL) lyse appropriate target cells are still controversial. We studied the action of established murine CTL clones as well as of freshly prepared primarily induced CTL against two types of target cell. Transformed tumour cell lines (MC57G and L929) and untransformed cells such as peritoneal exudate cells (PEC) or fibroblasts were examined as target cells by scanning and transmission electron microscopy and with the 51Cr‐release assay. We found independent of which CTL used, that in transformed cells the cell membrane seemed to be the first target of CTL attack, whereas in untransformed cells the first noticeable events appeared to take place in the nucleus of the target cells; the membrane of attacked untransformed PEC or fibroblasts was found to be intact at a time when the cellular organelles already were disintegrated. The morphological observations were paralleled by differences in the kinetics of 51Cr‐release; untransformed target cells released their label only after a 2 h long period, whereas transformed cells released 51Cr considerably earlier.

Responses to Cohn's Discussion on the Zinkernagel–Hengartner ‘Credo 2004’

We have spent some 30 years to look for experimental falsification of immunological rules, e.g. falsification of the obligatory need for a linked second signal for induction or falsification of the exclusive role of thymic epithelial cells in positive selection. On the other hand, results of coevolutionary balances are difficult to falsify (e.g. serotypic definitions by protective antibodies of infectious agents are not crossreactive); therefore, we should carefully approach realities.

Cloned cytotoxic T cells specific for lymphocytic choriomeningitis virus induce acute disease and primary footpad swelling in infected mice

Lymphocytic choriomeningitis virus (LCMV) infection in mice causes a wide spectrum of diseases with various symptoms, dependent upon virus isolate, virus dose, route of infection, and the immune status of the infected host [4]. When given intravenously (i.v.) or intraperitoneally to immunocompetent mice, it causes acute disease and the virus is usually efficiently eliminated. Major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) play a key role in this virus elimination [ 10]. This has been shown by adoptive transfer experiments, by evaluating the consequences of anti-Thy 1 treatment, by analysing MHC restriction specificity of effector T ceils, and, more recently, by experiments using cloned LCMV-specific class I-restricted CTL [1]. LCMV injected intracerebrally (i.c.) to immunocompetent C57BL/6J mice causes a severe disease of the central nervous system and death within 6-9 days. This disease has been shown to result from T-cell-dependent [2] damage of infected leptomeningeal cells. The proof for T cell dependence of LCM-disease is, first, T cell-deficient or immunosuppressed mice do not develop LCM-disease [reviewed in 4] and, second, these mice do develop LCM-disease upon adoptive transfer of LCM-immune T lymphocytes [2]. The mechanism by which T cells cause LCM-disease has been under discussion for some time, but the importance of T cell-mediated inflammatory reactions versus direct cytotoxic T cell action is not clearly understood [3, 9]. LCMV-specific CTL clones derived from spleens of C57BL/10J (H-2 b) mice immunized 4-5 weeks earlier with LCMV-Armstrong were prepared by limiting dilution and recloning [7]. The growth of the clones obtained was strictly dependent on the presence of interleukin-2 (IL-2) and required repeated stimulation every 7-10 days with LCMV-infected macrophages. Clone 3.3 (as other clones), when tested in a 51 Cr-release assay, showed high LCMVspecific, H-2Db-restricted lysis of LCMV-infected target cells. When tested by immunofluorescence, these clones were found to be positive for the Thy 1.2 and Lyt 2 but negative for the L3/T4 surface markers. Clone 3.3 was tested for its ability to mediate lethal LCM-disease or to induce a delayed-type hypersensitivity (DTH)-like footpad swelling upon local transfer to appropriate recipients. Groups of 5--10 C57BL/6J mice, immunosuppressed by irradiation (750 r), were infected with 6 x 102 plaque-forming units (PFU) of LCMV-Arm