Review

Multiple mechanisms of lymphocyte-mediated killing

In this paper, we study the competition between tumour progression due to cancer cell replication and tumour regression due to immune cell cytotoxicity. We show that the increase of specificity alone during the development of the immune response is not sufficient in general to reject the tumour. Moreover, we describe three distinct and independent mechanisms that can, in the absence of external treatment, produce regular temporally alternating phases of progression and regression: (i) a deviation from homeostasis where the population of cytotoxic cells varies; (ii) a competition for space between replicating and dead cancer cells; (iii) a formation of multi-conjugate cellular complexes where one cytotoxic cell may bind and kill several cancer cells. In the latter case, the coexistence of a low cancer cell population (interpreted as tumour dormancy) and a high population state is observed.

Norepinephrine (NE) has been shown to inhibit human peripheral blood-derived natural-killer (NK) cell cytotoxicity (NKCC) in vitro. We demonstrate in this study that NE not only inhibits IL-2-activated NKCC but antibody-dependent cellular cytotoxicity (ADCC) as well. NK cytotoxicity by purified NK cells against K562 (NKCC) and against Raji cells (ADCC) were inhibited by NE (1–100 μM) by more than 50% in a 4-h ⁵¹Cr release assay. The mechanism underlying the inhibition has been examined. NK cytotoxicity is dependent on target recognition and formation of NK–target conjugates, and activation by IL-2 is dependent on the secretion of cytokines (such as TNF-α) by NK cells. We hypothesized that the inhibition of NK functions by NE may be due to disruption of NK–target conjugation, blocking programming for lysis, and/or inhibition of cytokine secretion. Pretreatment of human peripheral blood mononuclear cells (PBMC) with NE for 15 min significantly reduced the binding to K562 cells by CD16⁺ NK lymphocytes. In the presence of K562 cells, NE down-regulated the expression of CD16 (FcγRIII) by human PBMC, an NK cell receptor responsible and necessary for ADCC and cytokine secretion. We also demonstrate that NE inhibited the IL-2-mediated up-regulation of the activation marker CD69. At concentrations of 10⁻⁶ to 10⁻⁵ M, NE inhibited TNF-α, IFN-γ, and GM-CSF secretion by NK cells, which are essential for IL-2-driven NK maturation and functions. In addition, using single-cell analysis, NE pretreatment of lymphocytes reduced the frequency of killer cells in the NK–K562 conjugate population in a concentration-dependent manner, indicating an inhibition of the programming for lysis by NK cells. In summary, these data demonstrate that NE-induced inhibition of NK cytotoxicity is manifested at multiple levels, including a modification of NK cell receptor ligation to target cells, blockade of NK cytokine secretion necessary for NK maturation and differentiation, and inhibition of the target-induced activation of the cytotoxic mechanism(s) in NK cells. Thus, sympathetic activation, as often induced experimentally, may profoundly impair natural cellular immunity through varied measurable pathways.

The factor(s) responsible for the reduced B cell number and increased T cell infiltrate in T-cell-rich large-B-cell lymphomas (TCRBCLs) have not been well characterized. We studied 18 TCRBCLs and 12 diffuse large-B-cell lymphomas (DLBCLs) to compare the 1) predominant T cell subpopulation(s), 2) expression of cytotoxic granule proteins (TIA-1 and granzyme B), 3) level of tumor cell apoptosis (Apoptag system, Oncor, Gaithersburg, MD), and 4) expression of Ki-67 (Mib-1) and apoptosis-related proteins (fas (CD95), bcl-2, and p53). T cells in TCRBCLs and DLBCLs were predominantly CD8⁺ T cells expressing αβ T-cell receptors and TIA-1 (16 of 18 TCRBCLs with >50% TIA-1⁺ small lymphocytes) but lacking granzyme B (16 of 18 TCRBCLs with <25% granzyme B⁺ small lymphocytes). Scattered apoptotic tumor cells (confirmed with CD20 co-labeling) were present in 15 of 18 TCRBCLs, with 14 of 15 cases having <10% apoptotic cells. No apoptotic cells were seen in 12 of 12 DLBCLs. In 16 of 16 immunoreactive TCRBCLs, <25% tumor cells were bcl-2⁺, whereas 6 of 12 DLBCLs had >50% bcl-2⁺ tumor cells. CD95 (fas) expression was also lower, with 3 of 18 (16.7%) TCRBCLs versus 4 of 12 (33%) DLBCLs having >25% CD95⁺ tumor cells. TCRBCLs and DLBCLs had similar levels of p53 and Ki-67 (Mib-1) expression. Thus, T cells in TCRBCLs are non-activated cytotoxic T lymphocytes (TIA-1⁺, granzyme B⁻). Tumor cell apoptosis (perhaps cytotoxic T cell mediated) may partly account for the decreased number of large (neoplastic) B cells in TCRBCLs, but other factors (ie, decreased bcl-2 expression) may also be needed.

We reported the characterization of three serine proteases (granzymes 1, 2, and 3) from human cytotoxic T lymphocytes. In this study, human granzymes 1,2, and 3 were purified from the cytoplasmic granules of lymphokine activated killer (LAK) cells by gel filtration and cation exchange chromatography. Human perforin was purified by phenyl superose and heparin—agarose chromatography. Each purified granzyme was used with purified perforin to study DNA fragmentation in target cells of both human and murine origin. As measured by agarose gel electrophoresis and [¹²⁵I]dUrd assay, the granzymes induced oligonucleosomal DNA fragmentation and [¹²⁵I]dUrd release respectively from various target cells. Murine target cells were generALLy more susceptible to nuclear DNA release than were human targets. Both enzyme activity and nuclear DNA breakdown were significantly inhibited by 3,4-dichloroisocoumarin (DCI) or by heat inactivation of each granzyme. Perforin alone or granzyme alone failed to fragment nuclear DNA in various target cells. We conclude that human granzymes are an important family of effector molecules that with perforin induce DNA fragmentation in susceptible target cells.

Effector-target conjugation is described quantitatively by binding isotherms which are characterized by three parameters, the maximum effector and target conjugate frequencies, α_max and β_max, and the dissociation constant of the conjugates formed, K_D. In this paper the application of non-linear data-fitting techniques, as well as linear transformations of the binding isotherms that permit us to use standard regression analysis, has been tested to calculate estimates of these parameters in the NK-MOLT4 and NK-K562 effector-target systems. Both unweighted and weighted data were used to calculate α_max, β_max and K_D for six different donors which were used as a source of NK cells. The results obtained have shown that these regression methods are useful for revealing potential disparities between binding efficiencies in effector-target systems.

Perforin is a cytolytic mediator produced by killer lymphocytes, and is stored in and released by cytoplasmic granules. The protein is partially homologous to the terminal components of the membrane attack complex of complement and produces pores of up to 20 nm in diameter on target membranes. Its genomic and protein structures have recently been unraveled, and its function elucidated through the availability of genetically engineered, perforin-deficient mice. Here Chau-Ching Liu, Craig M. Walsh and John Ding-F Young briefly outline certain biochemical and molecular features of perforin, and discuss the still-evolving issues concerning the relevance of perforin and Fas in cell killing.