Robert V. Blanden

T Cell Response to Viral and Bacterial Infection

TTie adaptive immune response to antigens of infectious agents is an. important component of vertebrate defence mechanisms. In many itistances, a humoral antibody response plays a key protective role. However, in certain infectious diseases, notably those caused by ititracellular parasites, recovery from primary infection, and resistance to subsequent infection with the same agent, it not mediated by free antibody, and there are now a number of clear examples of the need for a cell-mediated immune (CMI) response. Over the past few years it has become evident that activated thymusderived (T) cells are specific effector cells* in a wide variety of CMI reactions. In infectious disease models in mice, extensive evidence on this point has been obtained, particularly from studies of ectromelia virus infection (mousepox) and Listeria monocytogenes infection (a bacterial infection). This article will be devoted largely though not entirely, to recent findings in these two model systems: it is not intended to be a comprehensive review. Detailed mechanistic analysis of T cell effector functions will not be included. T cell mediated cytotoxicity in allograft rejection and tumour immunity has been comprehensively reviewed recently (Cerottini & Brunner 1973, Henney 1973). T cell-B cell interaction in humoral responses will be mentioned only in passing. Cooperation between T cells and macrophages, outlined briefly below, has been discussed in detail in other reviews in this volume and elsewhere (W.H.O. 1973).

Cell‐Mediated Immunity to Bacterial Infection in the Mouse. Thymus‐Derived Cells as Effectors of Acquired Resistance to Listeria monocytogenes

Four lines of evidence indicated that thymus‐derived (T) cells play an essential role in the expression of cell‐mediated immunity (CMI) to Listeria. a) T cell depleted (ATx‐BM) CBA mice were unable to generate antibacterial immunity. b) Responsiveness was restored to ATx‐BM CBA mice by injection of CBA X C57BL F1 thymocytes and essential CMI effector cells were derived from the F1 thymocytes (identified by anti‐H‐2 sera). c) The activity of immune cells from intact CBA mice was abolished by anti‐theta treatment but d) enriched by treatment with anti‐B cell, anti‐macrophage serum. Evidence from adult thymectomized mice and that described in b) above, indicated that T cells which had left the thymus more than 6 weeks or less than 3 weeks prior to immunization could act as progenitors of effector T cells, and that no cooperaiion between these 2 cell classes was necessary for an optimal response.

Lack of both Fas Ligand and Perforin Protects from Flavivirus-Mediated Encephalitis in Mice

ABSTRACT The mechanism by which encephalitic flaviviruses enter the brain to inflict a life-threatening encephalomyelitis in a small percentage of infected individuals is obscure. We investigated this issue in a mouse model for flavivirus encephalitis in which the virus was administered to 6-week-old animals by the intravenous route, analogous to the portal of entry in natural infections, using a virus dose in the range experienced following the bite of an infectious mosquito. In this model, infection with 0.1 to 105 PFU of virus gave mortality in ∼50% of animals despite low or undetectable virus growth in extraneural tissues. We show that the cytolytic effector functions play a crucial role in invasion of the encephalitic flavivirus into the brain. Mice deficient in either the granule exocytosis- or Fas-mediated pathway of cytotoxicity showed delayed and reduced mortality. Mice deficient in both cytotoxic effector functions were resistant to a low-dose peripheral infection with the neurotropic virus.

Effects of fourH-2K mutations on virus-induced antigens recognized by cytotoxic T cells

Lysis of ectromelia- or LCM virus-infected macrophage target cells by virus-specific cytotoxic T cells from mice immunized with the homologous virus occurred only where donors of T cells and target cells shared eitherH-2K orH-2D genes. With both viruses, use of T cell or target cell donors bearing mutations (B6.C-H-2ba, B6-H-2bh, B6-H-2bg1, and B6-H-2bg2), all of which apparently occurred in the same single genetic element in theH-2Kb region, abolished (H-2ba) or impaired (H-2bh,H-2bg1 andH-2bg2) lysis in T cell-target cell combinations that shared (apart from the mutations) all other genes in theK, I-A, orI-B regions of theH-2 complex. The data suggest that virus-induced antigenic patterns on infected B6.C-H- 2ba (mutant) cells are more different antigenically from those on C57BL/6 (wild type) cells than are those on infected cells from the other mutants -B6-H-2bh, B6-H-2bg1, and B6-H-2bg2. (B6.C-H-2ba× B6 -H-2bh)F1 mice behaved like B6-H-2bh, indicating no complementation, and confirming that theH-2K gene(s) involved in recognition of virus-infected cells by virus-specific T cells behave as a single element. These findings are discussed in relation to the nature of virus-induced antigenic patterns that are recognized by virus-specific cytotoxic T cells.

PCR AMPLIFICATION OF MURINE IMMUNOGLOBULIN GERMLINE V GENES : STRATEGIES FOR MINIMIZATION OF RECOMBINATION ARTEFACTS

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the ‘plateau phase’ of the reaction was reached. Recombination events were located throughout the ∼ 1‐kb product, with no preferred sites of cross‐over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.

Flavivirus infection up-regulates the expression of class I and class II major histocompatibility antigens on and enhances T cell recognition of astrocytes in vitro

Abstract West Nile virus (WNV) infection of astrocytes can up-regulate their expression of both class I and class II major histocompatibility complex (MHC) antigens as determined by flow cytometry with monoclonal antibodies specific for class I and class II MHC antigens. The up-regulation of class I MHC antigen expression could be partly caused by interferon secreted after WNV infection because the synthetic interferon inducer polyinosinic-polycytidylic acid (poly I : C) has similar effects. In contrast the up-regulation of class II MHC antigen expression was not induced by poly I : C. The increased MHC antigen expression by WNV infection had significant effects on T cell recognition. Thus, WNV and influenza virus A/WSN double-infected astrocytes but not astrocytes infected by A/WSN alone were lysed by influenza virus-immune cytotoxic T cells. Similarly, WNV-infected astrocytes were better stimulators than normal astrocytes for a class II MHC-reactive T cell line, both in terms of T cell proliferation and interleukin release.

The use of H-2 mutants as models for the study of T cell activation.

Studies of the role of the H-2 histocompatibility complex in the mouse have progressed rapidly over the last few years for several reasons. Firstly, there has been the ready availability of H-2 congenic lines and more recently of intra-H-2 recombinant strains. These latter have served, by using various parameters, to divide the H-2 complex into various regions and subregions, which, currently are K, I A, IB, U, IE, IC, S, G and D. Each region or subregion is identified by a marker locus, which effects transplantation phenomena (H-2K and H-2D), Ir genes or Ia specificities (IA, IB, IJ, IE, IC) serum lipoprotein levels (S) or a specificity (H-2.7) detected predominantly by hemagglutination (G). Secondly, the H-2 complex has been shown to effect not only transplantation (graft rejection) and the related phenomena of MLR, CML and the GVHR, but also many other phenomena, which are apparently not related to transplantation: the Ir genes; the susceptibility to various leukemic agents; serum lipoprotein and complement levels; specificities present selectively on B cells, and on some T cell subpopulations; size and weight of seminal vesicles and other sexual characteristics; cAMP levels in the liver and so on. This list, incomplete as it is, illustrates the

The cell-mediated immune response to ectromelia virus infection: I. Kinetics and characteristics of the primary effector T cell response in vivo

Abstract The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 × 10 2 up to 5 × 10 4 PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a 51 Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4–22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5–6 days post-infection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes . These activities were significantly sensitive to anti-δ and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days 1–4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.

Identification of cytolytic lymphocytes in West Nile virus-infected murine central nervous system.

Inflammatory cells were isolated from West Nile virus (WNV)-infected CBA/H (H-2k) mouse brains, and their function and cell surface markers were studied. Two populations of cytolytic lymphocytes were detected. One population, which lysed WNV-infected and uninfected L929 (H-2k), YAC-1 (H-2a), P815 (H-2d), OH (H-2KdDk) and 2R (H-2KkDb) target cells, had a phenotype of L3T4- Lyt2- Thy1 +/- asialo GM1+ and hence were natural killer (NK) cells. The other, which lysed WNV-infected cells to a greater extent than uninfected L929 cells, had a phenotype of L3T4- Lyt2+ Thy1+ asialo GM1- and were cytotoxic T cells. In addition, the presence of the latter population was demonstrated by the specific lysis of syngeneic WNV-infected astrocytes, a cell type which is resistant to NK cell lysis. The cell surface markers of isolated inflammatory cells were determined by two colour flow cytometry. This showed that 15 to 40% of the cells were Thy1+, among which about 3% were Lyt2+. No L3T4+ cells were detected.

The Primary in vivo Murine Cytotoxic T Cell Response to the Flavivirus, West Nile

A protocol for obtaining cytotoxic T cell responses to the flavivirus West Nile (WNV) has been developed in vivo. CBA/H (H-2k) mice were immunized with 10(6) p.f.u. WNV intravenously and their spleen cells used directly in cytotoxic assays. This method reliably produced WNV-immune Tc cells which showed WNV-specific cytotoxic activity on infected L929 (H-2k) target cells. There was inadequate lysis of infected targets by WNV-immune spleen cells when the m.o.i. was less than 100 p.f.u. WNV, or when tertiary mouse embryo fibroblasts, resident peritoneal macrophages or thioglycollate-induced peritoneal macrophages were used as targets. Only L929 cells infected for 16 h with WNV at a m.o.i. of 100 were suitable targets. Cytotoxic activity against WNV-infected target cells was first detected 4 days after immunization, peaked on day 5 and declined rapidly after day 7. An immunizing dose of 10(3) p.f.u. of WNV was adequate for significant cytotoxicity to be detected; however, the cytotoxic response increased with increasing immunizing doses to plateau levels when 10(6) p.f.u. WNV were used. The cells responsible for lytic activity were H-2-restricted, Thy-1+, Lyt-2+, L3T4- and virus-specific with respect to WNV and influenza virus.