The use of the chromium-release assay to determine cytotoxicity of effector against target cells has various limitations mostly due to the inherent properties of the radioactive substance. We have developed an improved flow cytometric method that is able to measure cytotoxicity, based on two fluorescent dyes. Calcein-AM, a non-fluorescent substance which is intracellularly converted to the green fluorescent calcein by esterase activity in viable cells, is initially used to stain target cells. After incubating targets with effectors for 2 h, ethidium homodimer-1, a red DNA stain non-permeable to viable cells, is added. Dead target cells are distinguished by their double (green-red) staining. Data analysis is performed by gating the regions of living target, dead target and living effector cells, based on appropriate controls. Non-specific events are subtracted from the dead target region and the ratio of specific dead target events to total target events is expressed as percent cytotoxicity. The method is used to quantify natural killer (NK) and lymphokine-activated killer (LAK) activities against the human K562 and Daudi cell lines and the murine YAC-1 and L1210 cell lines respectively, as well as cell-mediated lympholysis (CML) exerted by tumor-infiltrating lymphocytes (TIL) against autologous and allogeneic human breast cancer tumor cells. The method is fast, reliable and correlates well with the standard 51Cr-release assay.

We examined the role of endogenously produced interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in lectin-induced nonspecific suppressor activity in vitro. The cultures consisted of highly purified T lymphocytes, autologous monocytes and phytohemagglutinin (PHA). Kinetic studies revealed peak levels for both TNF-alpha and IL-1 beta production 4 hr after initiation of cultures which then declined and reached minimal levels on day 3. At this time point maximal levels of interleukin-2 (IL-2) were detected which declined sharply 24 hr later. The decline in cytokine levels in culture supernatants was most probably due to their consumption by the mononuclear cells which were found to express specific receptors for IL-1 beta, (IL-1 beta R), TNF-alpha (TNF-alpha R) and IL-2 (IL-2R) after 3- and 6-days of culture. After their first cycle of production and consumption both monokines were reproduced and the events followed the same patterns as for the first cycle: both monokines were first produced and at the time point of their consumption, IL-2 production reached maximal levels. The requirement for IL-1 beta and TNF-alpha in both IL-2 production and generation of suppressor activity was shown by three different approaches which included (a) blocking of HLA-DR molecules on monocytes which prevented monokine consumption during the early stages of culture, (b) blocking of HLA-A,B,C molecules on monocytes which prevented monokine consumption and IL-2 production late in culture, and (c) neutralization of monokine activity late in culture which resulted in highly reduced IL-2 production. T lymphocytes harvested from such cultures exhibited diminished suppressor activity. Our data suggest that the generation of nonspecific suppressor cell activity in vitro represents a complex system that requires cell interactions via self-major histocompatibility complex (MHC) antigen recognition and two cycles of cytokine production, where IL-1 beta and TNF-alpha production and consumption is a prerequisite for IL-2 production. Since lectin-induced nonspecific suppressor activity in vitro is deficient in certain autoimmune disorders the data presented herein might help in understanding the cellular basis for this immunodeficiency.

In vitro studies have demonstrated that exposure of tumor infiltrating lymphocytes (TIL) to human recombinant interleukin-2 (rIL-2) will generate activated T-lymphocytes with major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxicity toward a panel of tumor target cells. In melanoma and ovarian carcinoma, TIL display MHC-restricted and autologous tumor-specific cytotoxicity. Such tumor-reactive cytotoxic T-lymphocytes (CTL) represent important material for understanding cellular immunity in cancer and developing specific immunotherapeutic approaches in melanoma and ovarian cancer. In breast cancer, some TIL have been demonstrated to secrete cytokines upon interaction with autologous tumor cells, indicating that autologous tumor-reactive lymphocytes may also exist among TIL in breast cancer. Therefore, the authors conducted a study to investigate the cytotoxic profile of rIL-2-activated lymphocytes in breast cancer.|Lymphocytes were isolated from primary solid tumors (TIL) of breast carcinomas (10 patients) and from peritoneal effusions (effusion-associated mononuclear cells [EAMNC]) from 2 patients with newly diagnosed metastatic breast carcinoma. Tumor infiltrating lymphocytes or EAMNC were cultured with rIL-2 in long term cultures whereby their expansion index, phenotype, and cytotoxic potential were studied.|Both TIL and EAMNC proliferated by greater than 300-fold (370-3650; mean, 1656) after 23-82 days in cultures containing mixtures of TIL or EAMNC, autologous tumor cells, and rIL-2. By fluorescence analysis, freshly isolated TIL and EAMNC were found to consist of 77.5 plus or minus 10.7% CD3+ T-cells, 33.2 plus or minus 8.9% CD4+, and 47.2 plus or minus 16.8% CD8+ cells. Their CD4 to CD8 ratio was 0.70. After expansion of lymphocytes with rIL-2 in the majority of patients (9 of 12), CD3+CD8+ T-lymphocytes were present in greater numbers than CD3+CD4+ T-lymphocytes. Recombinant interleukin-2-activated CD3+CD8+ cells exhibited preferential cytolytic activity against autologous tumor cells. The cytolytic activity of CD3+CD8+ cells was inhibited either by anti-T-cell receptor (TCR)-alpha/-beta and anti-CD3 monoclonal antibodies (MoAb) or after pretreatment of tumor target cells with MoAb against the class I MHC antigens. Recombinant interleukin-2-activated CD3+CD4+ cells demonstrated potent cytolytic activity against both autologous and allogeneic tumor cells. CD3+CD8+ T-cell clones isolated from representative TIL exhibited preferential autologous tumor-specific cytotoxicity whereas the cytolytic activity of CD3+CD4+ T-cell clones was mostly (12 of 14 clones) nonrestricted to the autologous tumor.|To the authors' knowledge, this is the first report to demonstrate that TIL from primary tumors of breast carcinomas and EAMNC from metastatic disease can be propagated in large numbers in vitro with rIL-2 while retaining autologous tumor specific and MHC-restricted CTL activity. These findings may be of importance to ongoing clinical trials using TIL or EAMNC in the immunotherapy of patients with advanced breast cancer.

We have recently demonstrated that prothymosin alpha (ProT alpha) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor alpha (TNF alpha) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT alpha in vivo. We demonstrate that i.p. injections of ProT alpha enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT alpha-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8+ cells, NK cells and activated CD3+ cells. The ProT alpha-induced effect persisted for 30 days after the end of the ProT alpha treatment period and returned to normal levels 20 days later. Splenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT alpha in vitro. The ProT alpha-induced NK and LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF alpha and IL-2 were generated in response to ProT alpha in LAK cultures. These findings suggest that ProT alpha may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF alpha in the spleen, peritoneal cavity and probably other lymphoid organs.

Circulating T lymphocytes from 20 patients with an immediate patch test reaction were tested for proliferative responses in vitro to contact allergens during both immediate and delayed skin reactions. T lymphocytes collected during the immediate patch test reaction responded specifically in the challenge allergens in the presence of autologous monocytes. Subset analysis revealed that the proliferation pattern was dominated by the CD4+ CD29+ T-cell subpopulation, whereas CD8+ or CD4+ CD45R+ cells did not respond. A similar pattern in T-cell subset proliferation was observed when cells were collected during a positive delayed skin reaction. In contrast, in the case of a negative delayed skin reaction, proliferative responses of lymphocytes to the specific allergens were dominated by CD45+ cells. The latter T-lymphocyte subset could greatly suppress in an allergen-specific manner the proliferation of CD29+ autologous cells. The in vitro allergen-specific proliferation of CD29+ or CD45R+ cells was restricted by the major histocompatibility complex class II (HLA-DR) gene products. It is suggested that allergen-specific immune responses take place in the induction and evolution of an immediate patch test reaction into a delayed one.