Jane E. Allan

Dissection of an inflammatory process induced by CD8+ T cells

A massive delayed type hypersensitivity (DTH) reaction occurs in the cerebrospinal fluid (CSF) of mice with lymphocytic choriomeningitis (LCM). In this article, Peter Doherty and colleagues analyze this reaction together with the population dynamics of the regional lymph node to give a comprehensive picture of the events underlying this CD8+ T-cell-mediated immunopathological disease. Their findings are of general relevance to the understanding of inflammation.

Virus-specific memory T cells are Pgp-1+ and can be selectively activated with phorbol ester and calcium lonophore

Memory lymphocytic choriomeningitis virus (LCMV)-immune cytotoxic T-lymphocyte precursors (CTLp) can be stimulated to proliferate and to mediate specific cytotoxic activity following incubation with phorbol myristate acetate (PMA), calcium ionophore (CaI), and interleukin 2 (IL-2). This protocol can be used to selectively induced virus-specific CTL activity under both bulk culture and limiting dilution conditions, in the absence of added antigen. There is no concurrent stimulation of alloreactive CTLp. Proliferation of the effector Lyt-2+ population in medium containing PMA and CaI requires L3T4+ cells, which can be replaced by adding IL-2, and the development of cytotoxicity is totally IL-2 dependent. The LCMV-specific memory T cells are also characterized by the expression of the Pgp-1 (Ly24) glycoprotein. The availability of this marker, together with the capacity to selectively stimulate primed CTLp in the absence of antigen, should greatly facilitate the analysis of T-cell memory in virus infections.

Nature of the Inflammatory Process in the Central Nervous System of Mice Infected with Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis (LCM) is an inflammatory disease that occurs in immunologically competent adult mice after infection of the brain with LCM virus (LCMV). Tremors develop in the legs, head, and tail, and characteristic extensor spasms of the hind legs are found during the terminal stage of disease, usually within 1 week of infection.

Consequences of a single Ir-gene defect for the pathogenesis of lymphocytic choriomeningitis.

The H-2Ld allele has been identified by others as the sole Ir gene in the H-2d haplotype for the cytotoxic T lymphocyte (CTL) response to mouse lymphocytic choriomeningitis virus (LCMV). The BALB/c-H-2dm2 (C-H-2dm2) mutant lacks H-2Ld, and thus should be ideal for assessing the contribution of virus-immune CTL to LCM immunopathology. Comparison of the C-H-2 dm2 mice with congenic BALB/c mice revealed that there is a delay of about 24 h in the onset of severe inflammatory process and symptoms in the mutant strain, but the absence of H-2Ld did not prevent the later development of fatal disease in mice injected intracerebrally (i.e.) with neurotropic LCMV. This could indicate that virus-immune CTL are not the major mediators of clinical LCM. Spleen cells from LCMV-primed BALB/c mice did not show CTL activity for LCMV-infected C3H.OH, C-H-2dm2, or (CBA × C-H-2dm2)F1 target cells. However, immune lymphocytes from both the mutant and the F1 strains lyse virus-infected BALB/c cells. Furthermore, BtO.HTG and, in some experiments, B10.A(5R) mice generated CTL lytic for LCMV-infected BALB/c, C-H-2dm2, and (CBA × CH-2dm2)F1 macrophages. Apparently H-2Ld is immunodominant in the H-2d restricted response to LCMV. However, in the absence of H-2Ld, it seems that H-2Kd and, to a lesser extent, H-2Dd also serve as Ir genes for the CTL response in this infection. Even so, the absence of the H-2Ld-restricting element results in a disease process which is either delayed in onset or less severe.

Immune T cells can protect or induce fatal neurological disease in murine lymphocytic choriomeningitis

Adoptively transferred immune spleen cells induce fatal neurological disease in cyclophosphamide-suppressed recipients injected intracerebrally (ic) with a large, but not small, dose of neurotropic lymphocytic choriomeningitis (LCM) virus. The elimination of virus from brain in the latter group, which survives without developing symptoms, depends upon the presence of Lyt 2+ lymphocytes. However removal of Lyt 2+ subset which is cytotoxic in vitro does not diminish the severity of the inflammatory process in vivo, though the onset of clinical disease is delayed in mice given Lyt 2-depleted populations and a larger ic dose of virus. The present findings are consistent with the idea that fatal LCM results from acute, synchronous damage to key functional cells in the central nervous system by virus-immune Lyt 2+, lymphocytes. Even so, if the number of virus-infected CNS cells is still relatively small at the time of T cell invasion, neurological symptoms are not recognized and the mice survive.

The acute inflammatory process in murine lymphocytic choriomeningitis is dependent on Lyt-2+ immune T cells

Virus-immune spleen cells induce fatal immunopathology following adoptive transfer into adult C57B1/6J mice that have been infected with lymphocytic choriomeningitis virus (LCMV) and immunosuppressed with cyclophosphamide. This is accompanied by the development of potent cytotoxic T-lymphocyte (CTL) activity of donor origin in the recipient spleen. Both the capacity to trigger the acute meningitis observed at 72 hr and to generate CTL effectors in lymphoid tissue are completely abrogated by the removal of Lyt-2+ cells from the donor population. However a lower level of inflammatory process in the central nervous system may emerge, in the absence of significant CTL function in recipient spleen, by 5 days after transfer of the Lyt-2-depleted cell population. Treatment of the transferred cells with antibody to the L3T4 marker does not reduce either the severity of inflammation or the level of CTL effector function in the recipient. Thus Lyt-2+ cells are required for the acute, fatal immunopathology characteristic of LCM, but it is not clear that in a more chronic situation, they are the sole effectors capable of triggering inflammatory process in this disease.

Characterization of Interferon Induction in Mice of Resistant and Susceptible Strains during Murine Cytomegalovirus Infection

Comparison of the induction of plasma interferon (IFN) by murine cytomegalovirus (MCMV) in BALB/c and CBA mice demonstrated similar kinetics of induction with maximum titres at 6 and 48 h after infection. Although neutralized by antibody to type I (alpha and beta) IFN, the IFN was predominantly acid-labile. The 6 h titre was markedly dependent upon the dose and method of preparation of MCMV and also the strain of mouse used. Higher plasma IFN titres were found in CBA than in BALB/c mice following both intraperitoneal or intravenous infection but this strain variation was not apparent in IFN levels in the spleen and liver. IFN induced by MCMV in CBA and BALB/c mice was equally effective at inhibiting MCMV replication in vitro. Cultured embryo fibroblasts derived from CBA and BALB/c mice produced similar levels of IFN, but it was not detected until 24 h after infection. Use of irradiation chimeras prepared from H-2-compatible high IFN producers (CBA) and low producers (B10.BR) showed that the level of IFN produced at 6 h was dependent upon bone marrow-derived cells.

Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition

The amino acid sequence YPHFMPTNL of pp89, the ie1-encoded product of murine cytomegalovirus (MCMV; Smith strain), constitutes an immunodominant T cell epitope recognized in association with H-2Ld. Nucleotide sequencing of MCMV isolates derived from wild mice identified variation between amino acids 147-192 of pp89 in 19 of 27 isolates, including the region encompassing the CTL epitope (amino acid residues 168-176). Four groups of isolates with naturally occurring variant sequences for the CTL epitope were defined: (1) YPHFMPPNL; (2) YPHFMPPSL; (3) YPHFIPPSL; and (4) YLDFMPPNL. The remaining isolates, and the laboratory strains K181 and Vancouver, showed complete identity with the Smith strain. Polyclonal pp89 (Smith strain)-specific CTL only weakly recognized target cells infected with MCMV from most variant groups. No lysis of cells infected with isolate N1 from group 4 was detected. Analyses of cross-reactive recognition of YPHFMPTNL peptide-coated targets by CTL primed with variant MCMV isolates showed that the group 2 and 3 isolates, G4 and K6, respectively, but not the group 4 isolate N1, elicited CTL that exhibited a cross-reactive response. Furthermore, while the group 2 and 3 isolates G4 and K6 were able to prime CTL responses that displayed reactivity to homologous pp89 variant nonapeptides, the group 4 isolate N1 failed to do so. Finally, while immunization of mice with the nonapeptide YPHFMPTNL conferred significant protection against the laboratory strain K181 [correction of Kl81], no evidence of protection was observed for the group 2 and 4 variants G4 and N1, respectively. These observations raise the possibility that clinical isolates of HCMV may also differ in sequence from potential vaccine strains at immunodominant epitopes for CD8+ T cells thus reducing the efficacy of vaccination.

Consequences of Cyclophosphamide Treatment in Murine Lymphocytic Choriomeningitis: Evidence for Cytotoxic T Cell Replication in Vivo

Administration of a large dose (400 mg/kg) of Cyclophosphamide (Cy) prior to infection with lymphocytic choriomeningitis virus (LCMV) suppresses the virus‐immune cytotoxic T lymphocyte (CTL) response. Treatment with 150 mg/kg of Cy before, at the time of, or on the day after LCMV infection has little effect on CTL function. In contrast, when given on 2‐7 days after LCMV, this dose profoundly depresses the day‐8 CTL response and delays the onset of neurological disease. Administration of as little as 50 mg/kg of Cy has a marked effect on CTL activity when given 5 days after virus. Therefore it seems likely that CTL are replicating for 5 or 6 days during this primary response. Multiplication of these T cells is apparently completed by day 8, since inoculation of 150 mg/kg of Cy at this time has little effect on the level of CTL activity measured on day 9. The CTL activity is not reconstituted by in vitro culture with added helper factors. The interpretation that CTL are replicating in vivo following inoculation of LCMV is in accord with other analyses of virus‐specific precursor frequency in primed and naive mice.

Contributions of host and donor T cells to the inflammatory process in murine lymphocytic choriomeningitis

The severe inflammation characteristic of the infection of adult mice with murine lymphocytic choriomeningitis virus (LCMV) is induced earlier in unsuppressed, virus-infected recipients by the adoptive transfer of class I MHC-compatible, CD4- CD8+ LCMV-immune spleen cell populations. The time to onset of fatal LCM may also be slightly diminished, though not to the extent that would be expected from the enhanced kinetics of the extravasation of cells into cerebrospinal fluid. The development of symptoms is thus not solely related to the magnitude of the inflammatory process. The majority of the T lymphocytes in the inflammatory exudate are of host origin and have the size characteristics of resting cells, while the minority population of donor T cells show more of a lymphoblast morphology. The findings are consistent with the idea that relatively few CD8+ virus-immune effectors trigger an inflammatory process which consists largely of secondarily recruited host T cells and monocyte/macrophages.