Clustered CD3/TCR complexes do not transduce activation signals after bispecific monoclonal antibody-triggered lysis by cytotoxic T lymphocytes via CD3.

Bispecfic mAb (bsmAb) directed against the CD3/TCR complex and a tumor-associated Ag (TAA) induces CTL-mediated lysis of TAA+ target cells. We have investigated whether bsmAb-pretargeted CTL can enter multiple lytic cycles. BsmAb-pretargeted CTL retained bsmAb-targeted lytic capacity for at least 24 h when exposed to medium without TAA+ target cells. Exposure of bsmAb-pretargeted CTL to TAA+ target cells resulted in a rapid loss of bsmAb-targeted cytotoxicity of TAA+ or Fc gamma R+ target cells, although the CTL retained surface bsmAb. Moreover, addition of rabbit anti-mouse lg to these CTL did not induce calcium mobilization. These CTL still showed Ag-specific cytotoxicity and cytolysis of anti-CD3 mAb-expressing hybridoma cells. Readdition of bsmAb to CTL that had lost bsmAb-targeted cytotoxicity instantly restored the bsmAb-targeted lytic activity of the CTL. Hence, as in Ag-specific cytolysis, bsmAb-pretargeted CTL can enter multiple bsmAb-targeted cytolytic cycles. Surprisingly, exposure of bsmAb-pretargeted CTL to Fc gamma R+ cells did not result in loss of bsmAb-targeted cytolysis of TAA+ cells. Fluorescence microscopic analysis revealed that bsmAb-mediated interaction with TAA+ cells, but not with Fc gamma R+ cells, resulted in clustering of bsmAb-pretargeted CD3/TCR complexes on the CTL surface. On the basis of the observed correlation between clustered bsmAb-pretargeted CD3/TCR complexes and loss of bsmAb-targeted cytotoxicity, we hypothesize that clustered CD3/TCR complexes can no longer transduce signals.

Optimization of culture conditions for activation and large-scale expansion of human T lymphocytes for bispecific antibody-directed cellular immunotherapy.

We investigated the optimal culture conditions (i.e., activation procedure, medium composition and type of culture vessel) for rapid in vitro expansion of large numbers (greater than 5 x 10(9) of blood T lymphocytes. These expanded lymphocytes can be targeted to be cytotoxic to ovarian carcinoma cells with a bispecific monoclonal antibody (BsAb) specific for CD3 and for the ovarian carcinoma-associated antigen MOv18. Both phytohemagglutinin (PHA) and monoclonal antibody (MAb) CD3 induced rapid T-cell proliferation, although the growth kinetics after PHA activation were slightly faster. A 50-fold increase in cell number was obtained after 14 and 16 days for PHA and CD3 MAb, respectively. The induction of BsAb-directed cytolysis was faster after CD3 MAb than after PHA activation of lymphocytes, but became similar around day 20. A mixture of media consisting of 78% RPMI 1640, 20% AIM-V and 2% human plasma (Mix-med) yielded better results than 100% AIM-V medium. Culture of lymphocytes in polyolefin bags, compared with tissue culture flasks, or cryopreservation did not affect lymphocyte yield and function. In most cultures the proportion of CD8+ lymphocytes increased, suggesting a growth advantage of CD8+ over CD4+ lymphocytes in this culture system. A protocol employing PHA activation, Mix-med and polyolefin bags has been used successfully to activate and expand blood lymphocytes for the first 5 patients entered into a phase-I/II clinical trial for the intraperitoneal treatment of ovarian carcinoma using CD3 x anti-MOv18 BsAb-directed T lymphocytes.

Leucoagglutinin induces differential proliferation of lymphocyte subsets.

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1.

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.

Cellular cytotoxicity assessed by the 51Cr release assay. Biological interpretation of mathematical parameters.

This study deals with the interpretation of primary data of the 51Cr release assay for cellular cytotoxicity. In particular the dose-effect relationship between increasing numbers of lymphoid cells and the percentage of target cells killed has been considered. The number of target cells killed depends on the frequency and the activity of cytotoxic cells. These two parameters are often not distinguished from each other, which causes confusion and frequently results in vague descriptions of cytotoxicity data. Because in many cases not all cells in the target cell population can be lysed, we recommend the introduction of the plateau value for target cell kill. This maximum of target cell kill is a measure of the frequency of lysable target cells, but also depends on the cytotoxic cell activity. Description of the dose-effect curve by y = A(1 - e-kx) allows the simultaneous calculation of the maximum kill (A) and the frequency of cytotoxic cells (k) in the lymphoid cell population (x). Results are presented which indicate that A and k represent totally independent biological parameters the use of which permits a more objective description of cytotoxicity data.

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones.

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.

Rapidly expanded activated human killer cell clones have strong antitumor cell activity and have the surface phenotype of either T gamma, T-non-gamma, or null cells.

Cloned lymphoid cell lines showing cytolytic activity were derived from natural killer (NK) cell-enriched cell fractions obtained by fluorescence-activated cell sorting of cells that reacted with B73 .1, an NK cell-specific monoclonal antibody (MCA). The clones were cultured for more than 30 generations (i.e., more than 10(9) descendants from a single cell). The rapid expansion was achieved by using a special culture system developed for this purpose and based on the use of two types of allogeneic feeder cells. Three phenotypically different types of cytotoxic clones were obtained. These clones showed a broad spectrum of cytolytic activity against several NK-susceptible and NK-nonsusceptible tumor target cells. One of these clones had the following binding pattern to MCA: B73 .1+, T3-, T4-, T8-, HNK1 -, and Lyt-3-. These cells formed rosettes with IgG-coated erythrocytes but not with sheep erythrocytes, and therefore might be null cell-derived. Most of the cytotoxic clones showed the following phenotype: B73 .1+, T3-, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma +. These clones were probably derived from T-gamma cells. In addition, one clone with cytolytic activity was derived from B73 .1- cells. This had the phenotype B73 .1-, T3+, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma-, and may be of T-non-gamma cell origin. About 10 noncytolytic clones showed the phenotype B73 .1-, T3+, T4, or T8+, HNK1 -, Lyt-3+, Ia+, E+, and EA-gamma -. An absolute correlation was found between the presence of the B73 .1 antigen, the absence of the T3 marker, and the capacity of the cells to form EA rosettes. Furthermore, all clones except one (Lyt-3-) formed E rosettes. Although the in vitro life span varied from clone to clone, B73 .1- clones generally grew faster and for longer times (greater than or equal to 50 generations) than did B73 .1+ ones (less than or equal to 40 generations). The cytolytic activity, cell surface phenotype as determined with MCA, rosette formation, and target cell specificity spectrum remained stable over the entire culture period. We conclude that the majority of the activated MHC-nonrestricted cytolytic clones obtained in this culture system show a particular phenotype. These cells can be expanded to large numbers. Whether or not these clones might be derived from B73 .1+, HNK1 + NK cells with the morphologic appearance of large granular lymphocytes will be discussed.

Clonal expansion of human B73.1-positive natural killer cells or large granular lymphocytes exerting strong antibody-dependent and -independent cytotoxicity and occasionally lectin-dependent cytotoxicity.

B73.1-positive natural killer (NK) cells were separated from B73.1- cells by fluorescent-activated cell sorting and used for the generation of cytotoxic clones. The B73.1+ and B73.1- cells were cultured at one responder cell per well in medium with feeder cells and interleukin 2 but without lectins. The plating efficiency of B73.1- cells (about 4%) but not of B73.1+ cells was increased 3- to 7-fold by addition of lectin. All B73.1+ clones showed a high cytolytic activity against a wide variety of human NK-susceptible target cells, such as K562, MOLT-4 and HSB, but not against murine cell lines (P815). They also lysed several NK-insensitive Epstein-Barr virus-transformed B cell lines (B-LCLs), Daudi cells, the bladder tumor cell line T24 and melanoma tumor cells. B73.1+ clones also exerted antibody-dependent cellular cytotoxicity. Only 1 of 20 clones derived from the B73.1- fraction showed a similar pattern of cytolytic activity. In addition, some clones derived from both B73.1+ and B73.1- fractions were able to exert lectin-dependent cellular cytotoxicity.