Susceptibility of human leukaemias to cell-mediated cytotoxicity by interferon-treated allogeneic lymphocytes

Summary Twenty-two human leukaemias, comprising acute phase leucocytes from 13 acute myeloid and nine lymphoid leukaemias, were tested for susceptibility to spontaneous cell-mediated cytotoxicity (CMC) by untreated lymphocytes and lymphocytes treated for 18 h with 250 IU lymphoblastoid (Namalva) interferon (IFN-). IFN-amplified killing (IAK) by lymphocytes from 24 normal lymphocyte donors was checked on the K562 erythroleukaemia cell line, for comparison with IAK on fresh leukaemias. Nine leukaemias were tested with lymphocytes from three donors, nine with lymphocytes from six donors, three with lymphocytes from nine donors, and one with lymphocytes from 11 donors. Some degree of susceptibility to IAK was found in five acute myeloid and five lymphoid leukaemias, which was markedly dependent upon the source of the effector lymphocytes and did not correlate with the degree of IAK on K562. The 12 other leukaemias were virtually resistant to IAK. The results emphasize the variability in the capacity of IFN-treated lymphocytes to lyse leukaemias that have not been adapted to tissue culture. The basis of effector recognition of cell line and fresh tumour targets is discussed.     

Requirements for the stimulation of allogeneic T lymphocytes by acute non-lymphoblastic leukaemia cells

Summary Blast cell populations from 32 patients with acute non-lymphoblastic leukaemia of various morphological types have been examined for their ability to stimulate allogeneic T lymphocytes from normal donors in one-way mixed leucocyte culture (MLC). At the same time, these leukaemic cell populations were examined for the amounts of major histocompatibility complex Class I and Class II antigens they expressed, and their ability to release interleukin 1 (IL1) in culture both with and without stimulation by lipopolysaccharide. The abilities of the leukaemic cell populations to stimulate in MLC, and to produce IL1, were found to be associated with the expression of morphological characteristics of monocytic differentiation, and correlated significantly. In contrast, no correlation was observed between the extent of Class I or Class II expression and MLC stimulatory ability. Many myeloblast populations of immature phenotype were unable to stimulate allogeneic T cells despite their strong expression of these antigens. This lack of stimulatory ability was not overcome by the addition of exogenous IL1. We therefore conclude that the correlation between the production of IL1 and MLC stimulatory ability does not necessarily imply a cause/effect relationship, and that the interaction between allo-antigen and the T cell receptor together with a supply of lymphokine co-stimulator is not sufficient to activate resting T lymphocytes. The failure of some Class I and II antigen positive leukaemic blasts to stimulate in MLC even in the presence of exogenous IL1 may be due to the lack of other differentiation-associated cell surface molecules necessary for stable cell-cell interaction.     

The role of microtubules in human natural killer cell-mediated cytotoxicity

The effect of four different microtubule (MT) inhibitors on the various stages of human natural killer (NK) cell-mediated cytotoxicity was studied. The MT-disrupting effect of the drugs was monitored by indirect immunofluorescence microscopy and transmission electron microscopy. All the drugs tested, vinblastine sulfate, demecolcine, nocodazole, and taxol, had only a slight inhibitory effect on NK activity. Cells with nonfunctional MT were capable of normal conjugate formation and polarization of actin-containing microfilaments. Natural killer cell cytotoxic factor (NKCF) activity produced by cells with nonfunctional MT was slightly diminished. MT disruption caused enlargement of Golgi cisternae, but did not, however, dissociate the overall structural organization of the Golgi complex. The results indicate that fresh human NK cells are capable of lytic activity without functional MT although MT play a small supportive role in production or secretion of NKCF and mediation of the lytic activity. Previous experiments by us and others have strongly suggested that NK cells mediate their cytolytic activity by directed secretion of toxic material. As NK cells with unfunctional microtubules are capable of close to normal secretion the results presented in this report are not inconsistent with the earlier suggested stimulus-secretion model.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Ral GTPases regulate cell-mediated cytotoxicity in NK cells

NK cells are key components of the immune response to virally infected and tumor cells. Recognition of target cells initiates a series of events in NK cells that culminates in target destruction via directed secretion of lytic granules. Ral proteins are members of the Ras superfamily of small GTPases; they regulate vesicular trafficking and polarized granule secretion in several cell types. In this study, we address the role of Ral GTPases in cell-mediated cytotoxicity. Using a human NK cell line and human primary NK cells, we show that both Ral isoforms, RalA and RalB, are activated rapidly after target cell recognition. Furthermore, silencing of RalA and RalB impaired NK cell cytotoxicity. RalA regulated granule polarization toward the immunological synapse and the subsequent process of degranulation, whereas RalB regulated degranulation but not polarization of lytic granules. Analysis of the molecular mechanism indicated that Ral activation in NK cells leads to assembly of the exocyst, a protein complex involved in polarized secretion. This assembly is required for degranulation, as interference with expression of the exocyst component Sec5 led to reduced degranulation and impaired cytotoxicity in NK cells. Our results thus identify a role for Ral in cell-mediated cytotoxicity, implicating these GTPases in lymphocyte function.     

T cell-mediated cytotoxicity against dengue-infected target cells

A cytotoxic T lymphocyte (CTL) response to dengue virus-infected target cells is described. Effector cells were generated in an in vitro secondary culture and appeared to be T cells possessing both the Lyt 1.1 and Lyt 2.1 surface antigens. A stronger CTL response was noted with the H-2k haplotype compared to H-2d, and H-2 compatibility was required between CTL and target cells. CTL generated showed some cross-reactivity with target cells infected with Japanese encephalitis virus (JEV), another flavivirus, but not with target cells infected with an alphavirus, Sindbis. The significance and importance of these findings are discussed.     

A possible role of prostaglandins in the inhibition of natural and antibody-dependent cell-mediated cytotoxicity against tumor cells

We recently observed that certain tumor cell lines in tissue culture produced prostaglandins and that increased production occurred when the tumor cells were exposed to lymphocytes. The present experiments tested the effect of prostaglandins E₁ and E₂ on natural and antibody-dependent lymphocyte cytotoxicity against the same target cells in order to determine whether the production of prostaglandins by the tumor cells might influence the efficacy of the cellular immune response. Target cell lines T₂₄ and HCV₂₉ were labeled with ⁵¹Chromium and incubated at 37 °C for various times with lymphocytes prepared from venous blood of normal donors. Antiserum to T₂₄ and varying concentrations of prostaglandin E₁ or E₂ were added to the samples prior to incubation. In some experiments, lymphocytes or labeled target cells were preincubated with prostaglandins and then washed prior to their addition to the assay tubes. Cytotoxicity was determined by measuring the release of ⁵¹Chromium from the target cells after incubation. Both prostaglandins E₁ and E₂ inhibited natural and antibody-dependent lymphocyte cytotoxicity against the target cells. The effect appeared to represent a direct one on lymphocytes, and it was amplified by the presence of theophylline in the medium. Inhibition could be effected early on in lymphocyte/target cell interaction, and only a short exposure of lymphocytes to prostaglandins was required for the effect to be manifested. It thus appears that the production of prostaglandins by tumor cells may constitute a means by which the tumor cells subvert the effect of a cellular immune response that is directed against them.     

The ability of microfilariae to evade in vitro cell-mediated cytotoxicity

When microfilariae (Mf) of Dirofilaria immitis, both uterine and systemic, were incubated in an in vitro cytotoxicity assay with neutrophils and sera from dogs with occult infections, some Mf remained free of adherent cells and consequently evaded cytotoxicity. The ability to evade cytotoxicity could not be related to the age of the Mf, and host albumin was not detected on any Mf, either uterine or systemic. However, it was shown that some Mf failed to bind IgM, IgG and C3 when incubated with occult sera. It is suggested that the ability of some Mf to evade serum-dependent, neutrophil-mediated cytotoxicity in vitro was related to differences in their antigenicity.     

Lipopolysaccharide-induced suppression of antibody-directed cell-mediated cytotoxicity to chicken red blood cells

Prior treatment with commercially prepared and acetone-extracted lipopolysaccharide (LPS) was found to suppress the expression of antibody-directed, cell-mediated cytotoxicity (ADCC) to chicken red blood cells (CRBC) by spleen cells from C57/BL10, C3H, and BALB/c mice. The in vitro incubation with commercial LPS suppressed ADCC-CRBC activity of spleen cells from both C3H/HeJ and C3H/HeN mice. Only the C3H/HeN strain was suppressed when treated with purified LPS. ADCC-CRBC activity of neonatal spleen cells could be suppressed after a 3-hr in vivo incubation with LPS while adult spleen cells required a minimum of 15 hr preincubation.     

Characterization of murine cathepsin W and its role in cell-mediated cytotoxicity

Cathepsin W is a member of the papain-like family of cysteine proteases. In this report, we have isolated the cDNA for murine CtsW (mCtsW) from a splenocyte library. The deduced 371-amino-acid sequence shares 68% identity with human CtsW and includes the conserved catalytic triad cysteine, histidine, and asparagine found in all members of this family. In addition to the fulllength form of mCtsW, we have isolated an alternatively spliced form of the mRNA that lacks a complete catalytic triad. An S1 nuclease protection assay and a Western blot analysis showed that mCtsW is mainly restricted to the CD8(+) T cell and natural killer cell compartments. In addition, we confirmed that, like its human homologue, mCtsW is localized mainly to the endoplasmic reticulum and its expression is up-regulated upon activation. We also characterized the mCtsW locus using bacterial artificial chromosome clones. The gene consists of 10 coding exons and 9 introns spanning 3.2 kb. To elucidate the physiologic role of this protease, we generated mice deficient in mCtsW. Our data establish that mCtsW is not required for cytotoxic lymphocyte-induced target cell death in vitro. In addition, mCtsW deficiency does not alter the susceptibility of cytotoxic lymphocytes to suicide or fratricide after degranulation. Thus, mCtsW does not have a unique role in target cell apoptosis or cytotoxic cell survival in vitro.     

The ability of hapten-conjugated cells to induce cell-mediated cytotoxicity is affected by the mode of hapten linkage

Spleen cells from C57 $\text{B}\text{1}\text{6}$ mice were modified with varying concentrations of several haptens coupled in different ways, then assayed for their ability to induce a cytotoxic response against hapten-modified target cells when cocultured with syngeneic spleen cells. Haptens such as TNBS, DNBS, Fl-NCS, and AB-NCS which couple via the ?-amino groups of lysine were all capable of generating high levels of cytotoxicity when tested against hapten-coupled syngeneic tumor. One hapten, DNBM, which coupled via -COOH groups was effective in generating cell-mediated cytotoxicity. By contrast, similar haptens such as ABA and DNBA which couple as diazonium salts via tyrosine and histidine groups were not found to generate cytotoxic responses over wide ranges of concentrations. Possible explanations are discussed.     

Studies on the physiological manifestations of cell-mediated cytotoxicity. I. Early metabilic changes in mouse plasmacytoma cells exposed in vitro to sensitized allogeneic splenocytes.

Early and later biochemical changes occurring in neoplastic target cells interacting with sensitized lymphoid cells were investigated, with emphasis on the detection of very early damage to the target cells. The sensitizing and target cells were a BALB/c plasmacytoma; the effector cells were obtained from the spleens of specifically sensitized and normal C57BL mice. The incorporation of labeled glycine and leucine, uridine, and thymidine into macromolecular entities by the plasmacytoma cells was followed as an indication of ability to take up the precursor and/or biosynthetic capacity. Separation of target and attacking cells was effected in some experiments by use of anti-C57BL lymphocyte serum.Amino acid and uridine incorporation were markedly altered between the second and fourth hour of interaction, and specific release of ⁵¹Cr was seen only after 2 hr. In contrast, thymidine incorporation was inhibited almost immediately after contact; this inhibition was shown to occur in PCT cells incubated at various ratios with sensitized spleen cells.     

Splenic suppressor cells and cell-mediated cytotoxicity in murine schistosomiasis.

An impairment of the capacity to generate alloantigen-specific cytotoxic T lymphocytes (CTL) was observed in mixed lymphocyte cultures (MLC) established with spleen cells from mice infected with Schistosoma mansoni. This impairment, which was observed as early as the eighth week of infection, could be abrogated by the fractionation of spleen cell suspensions by the carbonyl iron/magnet method prior to the establishment of MLC. Cocultivation of normal spleen cells with increasing numbers of splenocytes from S. mansoni-infected syngeneic mice resulted in a dosage-dependent suppression of CTL generation. This "infectious suppression" was not sensitive to antiserum against mouse thymic lymphocyte antigen (MTLA). The present studies suggest the role of a macrophage rather than a T cell as the suppressor cell in this model of cell-mediated immunity in schistosome-infected mice.     

Human lectin-dependent T cell-mediated cytotoxicity against Hep-2 cells

A sensitive method for human lectin-dependent cell-mediated cytotoxicity (LDCC) is presented using HEp-2 adherent human epipharynx carcinoma cells as targets. Cytotoxicity was evaluated by detachment from the monolayer of 3H-TdR-prelabelled HEp-2 cells. Maximal LDCC was obtained in a 24 h assay with a Con A dose of 25 micrograms/ml for 50 : 1 effector-target cell ratio requiring only 2500 target cells per well. Testing of five different lymphocyte fractions: peripheral blood mononuclear cells (PBMC), monocyte-enriched adherent cells (AC), monocyte-depleted non-adherent cells (non-AC), T and non-T lymphocytes as effector cells from 25 normal individuals, suggests that LDCC to HEp-2 targets is mediated by T lymphocytes.     

Density gradient fractionation of effector cells in human natural cell-mediated cytotoxicity

In order to separate and characterize cytotoxic effector cells in natural cell-mediated cytotoxicity (NCMC), human lymphocytes were fractionated by Percoll continuous density gradient centrifugation (Pharmacia, Piscataway, N.J.). Lymphocytes from normal donors were fractionated through a 35-ml gradient and 2- or 3-ml aliquots were collected, counted, and grouped into three or more fractions in order to obtain sufficient cells for testing. Fractions of cells were tested for cytotoxicity in a 4-hr chromium release test and/or a 40-hr [³H]proline assay. Cell markers were assessed by testing for cells forming E rosettes, EA rosettes, and for cells with surface membrane immunoglobulin (SMIg). The lightest fraction contained larger cells and also usually contained the highest concentrations of cells with receptors for the Fc portion of IgG (FcR + cells), although slight variations were seen among individual donors. Results of cytotoxicity tests showed that cells from the top portions of the Percoll gradient had consistently greater cytotoxic activity on a per cell basis than the denser cells sedimenting lower. Estimation of cytotoxic activity in lytic units showed that 54–75% of the activity was recovered in the top 26–29% of the cells. This approach to investigating cell-mediated cytotoxicity should yield useful information regarding cellular interaction in, and regulation of, cytotoxic activities.     

Vulnerability of human neurons to T cell-mediated cytotoxicity

Axonal and neuronal loss occurs in inflammatory diseases of the CNS such as multiple sclerosis. The cause of the loss remains unclear. We report that polyclonally activated T cells align along axons and soma of cultured human neurons leading to substantial neuronal death. This occurs in an allogeneic and syngeneic manner in the absence of added Ag, requires T cells to be activated, and is mediated through cell contact-dependent mechanisms involving FasL, LFA-1, and CD40 but not MHC class I. Activated CD4(+) and CD8(+) T cell subsets are equally neuronal cytotoxic. In contrast to neurons, other CNS cell types (oligodendrocytes and astrocytes) are not killed by T cells. These results demonstrate for the first time the high and selective vulnerability of human neurons to T cells, and suggest that when enough activated T cells accumulate in the CNS, neuronal cytotoxicity can result through Ag-independent non-MHC class I mechanisms.