Kwok-Nam Leung

An Analysis of Effector T Cell Generation and Function in Mice Exposed to Influenza A or Sendai Viruses

The activation and effector function of T cells are governed by a dual specificity of recognition system involving, on the one hand, a foreign antigen such as a hapten, a soluble protein molecule or a viral antigen; and, on the other hand, a self antigen coded for by the major histocompatibility complex (MHC). According to the H-2 region involved, mouse T lymphocytes can be subdivided into two subsets T cells which recognize antigen in association with I region MHC products and T cells which recognize antigen associated with K,D region MHC products. The H-2 restriction pattern and the Lyt phenotype of T lymphocytes are closely linked: I region restricted T cells are Lyt 1*, 2 ,3 ; K,D region restricted T cells have the Lyt T, 2*, 3* phenotype. In the latter case, it may be that the observed correlation reflects participation of Lyt 2, 3 antigens in the structure of the T cell receptor (Hollander et al. 1980, Sarmiento et al. 1980). T cells of both these subsets take part in a variety of functions. It has been proposed, and we would support the concept, that T cells evolved as part ofthe defence system to infectious diseases, and viral infections may have played a particularly important role. Thus, infection of cells by many different viruses results in the early expression of viral antigens at the surface of infected cells.

Cells Mediating Delayed‐Type Hypersensitivity in the Lungs of Mice Infected with an Influenza A Virus

Effector cells that demonstrate delayed‐type hypersensitivity (DTH) on transfer with antigen to naive mice can he recovered from the lungs of mice inoculated intranasally 6 days earlier with a lethal dose (usually 5 × 104EID50) of influenza A virus. The activity recovered was proportional to the dose of virus instilled intranasally and the extent of lung consolidation observed. Active cells could also be recovered from the draining lymph nodes and from the peripheral blood. The effector cells were identified as T lymphocytes of Ly I phenotype and required I‐region sharing between donor and recipient for activity to be elicited. They were cross‐reactive within the A group of influenza viruses. Two experiments are reported in which immune cell preparations that expressed DTH activity but had very little cytotoxic T cell activity were transferred to mice inoculated 1 or 2 days earlier with a lethal dose of virus. The mice were not protected from death, and in both experiments, the recipient mice died more rapidly than the controls. These results contrast with earlier results in which cell preparations with high cytotoxic T‐cell activity were shown to protect recipient infected mice from death.

Two T-Cell Populations Mediate Delayed-Type Hypersensitivity to Murine Influenza Virus Infection

Two classes of T lymphocytes can mediate delayed‐type hypersensitivity (DTH) to influenza virus in the mouse. If non‐infectious virus preparations are used to sensitize for or to elicit a DTH response, the effector cells are found to be Ly‐1‐positive and are I‐region‐restricted. If infectious virus is used both to sensitize for and to elicit the reaction, a second set of effector cells is also detected, which are Ly‐2,3‐positive and are D‐ or K, D‐region‐restricted. The latter cells are cross‐reactive within the A strains of influenza viruses, and pretreatment of the mice with high doses of cyclophosphamide markedly decreases their generation in the spleens of sensitized mice, suggesting that the cells that demonstrate DTH activity in vivo may also have cytotoxic activity in vitro.

Different functions of subsets of effector t cells in murine influenza virus infection.

Abstract Enriched preparations of secondary effector T cells to influenza virus were tested for their in vivo biological function by adoptive transfer to mice 24 hr after an intranasal inoculation of infectious influenza virus. One class of cells which were Lyt 1+2−3−, I region-restricted, and could mediate DTH reaction failed to reduce lung virus titers 5 days after transfer and caused a higher mortality rate in the recipient mice than in the controls. A second class of cells which were Lyt 1−2+3+, K,D region-restricted, and were cytotoxic and could mediate DTH activity substantially reduced lung virus titers 5 days after transfer. The influx of mononuclear cells to the lungs after adoptive cell transfer was measured by injection of [125I]UdR 24 hr prior to harvest of lung cells, using both infected CBA and athymic BALB/ c nude (nu/nu) mice as recipients. I region-restricted cells caused increased cellular infiltration which was very marked in athymic mice. It was concluded that this reaction significantly contributed to the observed immunopathology in infected mice. Transfer of K,D region-restricted cells reduced the cellular infiltration in infected CBA mice and caused only a slight increase in infected athymic mice. The evidence supported the concept that the second class of cells exerted their protective (antiviral) effect in vivo by direct lysis of virus-infected cells rather than by liberation of lymphokines.

Modulatory Effects and Action Mechanisms of Tryptanthrin on Murine Myeloid Leukemia Cells

Leukemia is the disorder of hematopoietic cell development and is characterized by an uncoupling of cell proliferation and differentiation. There is a pressing need for the development of novel tactics for leukemia therapy as conventional treatments often have severe adverse side effects. Tryptanthrin (6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a naturally-occurring, weakly basic alkaloid isolated from the dried roots of medicinal indigo plants (Ban-Lan-Gen). It has been reported to have various biological and pharmacological activities, including anti-microbial, anti-inflammatory, immunomodulatory and anti-tumor effects. However, its modulatory effects and action mechanisms on myeloid cells remain poorly understood. In this study, tryptanthrin was shown to suppress the proliferation of the murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. It also significantly reduced the growth of WEHI-3B JCS cells in vivo in syngeneic BALB/c mice. However, it exhibited no significant direct cytotoxicity on normal murine peritoneal macrophages. Flow cytometric analysis showed an obvious cell cycle arrest of the tryptanthrin-treated WEHI-3B JCS cells at the G0/G1 phase. The expression of cyclin D2, D3, Cdk 2, 4 and 6 genes in WEHI-3B JCS cells was found to be down-regulated at 24 h as measured by RT-PCR. Morphological and functional studies revealed that tryptanthrin could induce differentiation in WEHI-3B JCS cells, as shown by the increases in vacuolation, cellular granularity and NBT-reducing activity in tryptanthrin-treated cells. Collectively, our findings suggest that tryptanthrin might exert its anti-tumor effect on the murine myelomonocytic leukemia WEHI-3B JCS cells by causing cell cycle arrest and by triggering cell differentiation.

Production of DTH in the Mouse to Influenza Virus: Comparison with Conditions for Stimulation of Cytotoxic T Cells

The kinetics of sensitization and elicitation of delayed‐type hypersensitivity in mice to both infectious and non‐infectious preparations of influenza virus was found to be similar to that of some protein antigens and to other viruses. Sensitization was achieved without added adjuvant. Maximum DTH was elicited in the footpad 6 days after sensitization. Adoptive transfer experiments showed that the effector cells were in the Ig‐negative fraction of the spleen and were sensitive to anti‐θ and complement. A comparison was made of conditions for the generation of DTH activity with cytotoxic T cells. The route of inoculation was important. With a high dose (103 HAU) of virus, subcutaneous inoculation was the most efficient and intravenous injection the least efficient for sensitizing for DTH, whereas the reverse was found for cytotoxic T‐cell generation. Second, treatment of mice with cyclophosphamide (Cy) had differential effects. Preinjection of a large dose (200 mg/kg) into mice 2 days before sensitization with virus resulted in an increase In the DTK response and a 90% reduction in cytotoxic T‐cell activity in the spleens of the treated mice. The Cy‐injected mice had reduced (70%) anti‐haemagglutinin levels compared with the controls. This may be the explanation for the enhanced DTH response, since transfer of specific antibody to sensitized mice before injection of the eliciting virus substantially reduced the DTH response. Pretreatment with Cy did not affect the generation of DTH effector cells, since spleen cells from these and control mice had similar levels of activity.

The generation of 'cytotoxic' macrophages in mice during infection with influenza A or Sendai virus.

Injection of infectious but not of non‐infections influenza A virus or of infectious or non‐infectious Sendai virus intraperitoneally into mice induces the generation of plastic‐adherent cells that arc able to effect release of 51Cr from labelled virus‐infected target cells but not from labelled, uninfcctcd cells. Their activity is greatly diminished by exposure to silica or carrageenan but not by anti‐Thy I antibody and complement treatment. Similarly, the activity of the cell preparation cannot be explained by contamination with natural killer or ‘K’ cells. Thus, the effector cells were identified as macrophages and for convenience are called ‘cytotoxic macrophages’. The maximum cytotoxic activity was recovered from the peritoneal cavity 5 days after virus injection and declined thereafter. Although the effector cells are cross‐reactive in that cells activated by an influenza A strain virus lyse target cells infected with the same or other A strain viruses or with Sendai virus, there is preferential lysis of cells infected with the homologous virus. The action of the effector cells is not H‐2‐restrictcd. Preliminary experiments showed that similar effector cells can be recovered from the lungs of mice 5 days after intranasal inoculation of infectious influenza virus, so they may contribute to the host control of the disease.

Distinguishing between mouse IL-3 and IL-3 receptor-like (IL-5/GM-CSF receptor converter) mRNAs using the polymerase chain reaction method

A set of primers (MF43, MF44 and MF45) were designed and used in the polymerase chain reaction to distinguish between the expression of mouse IL-3 receptor and mouse IL-3 receptor-like mRNAs. Primers MF43 and MF45 were specific for IL-3 receptor mRNA while the primers MF44 and MF45 were specific for IL-3 receptor-like mRNA. Primers MF44 and MF45 could not amplify IL-3 receptor cDNA even at an annealing temperature of 46 degrees C which is 20 degrees C below the melting temperature of the primers, or at high template concentrations (up to 100 ng cDNA). The optimal range of Mg2+ concentrations for the two pairs of primers MF43, MF45 and MF44, MF45, were essentially the same and this permits comparisons of the expression level of these two mRNAs under identical PCR conditions. Both the IL-3 receptor and IL-3 receptor-like mRNAs could be detected in normal bone marrow cells and IL-3-dependent cell lines (FDC-P1 and 32D cl-23), as well as in the IL-3 independent cell lines P388D1 and WEHI-3B, the latter being a constitutive producer of IL-3. In contrast, neither species of mRNA was detected in the T lymphoma cell line (EL-4). The ratio of IL-3 receptor-like mRNA to IL-3 receptor mRNA was usually greater than 1, except in 32D cl-23 cells where it was 0.66.

Decreased expression of J11d antigen during monocytic differentiation of M1 myeloid leukemic cells

Four myeloid cell lines (M1, WEHI-3B D+, FDC-P1, and 32D) were screened for the presence of J11d antigen. One of these cell lines, the myeloid leukemia M1, was found to express a high level of J11d antigen on the cell surface. Recombinant mouse leukemic inhibitory factor (rm-LIF), recombinant human LIF (rh-LIF), and steroids (hydrocortisone and dexamethasone) could induce M1 cells to undergo monocytic differentiation. The level of J11d antigen was greatly reduced after treatment of the cells with LIF or steroids. Western blotting revealed that the apparent molecular weight of the J11d antigen on M1 cells was 45-48 kDa. Furthermore, the level of J11d mRNA was also reduced during LIF-induced differentiation of M1 cells.

In Vivo Collaboration between Precursor T Cells and Helper T Cells in the Development of Delayed‐Type Hypersensitivity Reaction to Influenza Virus in Mice

Nude, athymic mice do not mount a delayed‐type hypersensitivity (DTH) response to influenza A virus. A single injection of T helper cells (γ‐irradiated, 2‐day immune spleen cells) or three injections over 3 days of a concanavalin‐A‐activated spleen cell supernatant to virus‐sensitized nude mide resulted in a ‘normal’ DTH response when the mice were challenged with the virus. It was previously shown that the cells responsible for the reaction were T cells and required I‐region compatibility. Injection of T helper cells into normal mice did not affect the level of the subsequent DTH response. However, injection of such cells into mice pretreated with anti‐thymocyte serum (ATS) restored the ability of the mice to mount a DTH response. The results show that (1) nude mice contain precursor T cells for influenza virus antigen; (2) an I region‐restricted response can be generated in the absence of a thymus; and (3) in vivo collaboration between DTH T‐cell precursors and helper T cells can be shown to occur in congenitally nude mice and ATS‐treated mice.