Stimulation with autologous lymphoblastoid cell lines: lysis of Epstein-Barr virus-positive and -negative cell lines by two phenotypically distinguishable effector cell populations

Human lymphocytes, stimulated in vitro for 6 days with x-irradiated or glutaraldehyde-treated autologous Epstein-Barr (EB) virus-transformed lymphoblastoid cell lines (LCL), are cytotoxic for autologous and allogeneic EB+ LCLs as well as for several EB- cell lines that are also susceptible to lysis by interferon-activated natural killer (NK) cells. To determine whether the apparent nonspecific lysis mediated by LCL-stimulated cells is due to a mixture of effector cells directed against different target cells, advantage was taken of our recent finding that monoclonal antibody OKT8 reacts with human cytotoxic T lymphocytes but not with NK cells or NK-like cells generated in mixed leukocyte cultures. The depletion of OKT8+ cells from LCL-stimulated cultures by treatment with OKT8 and complement abolished or markedly depleted cytotoxicity against all EB+ target cells tested, whereas cytotoxicity against EB-, NK-sensitive cell lines including K562, MOLT-4 and HSB-2 was not or only minimally reduced. These results indicate that stimulation with autologous LCL results in the generation of OKT8+ cytotoxic T lymphocytes that lyse EB virus-transformed LCL and OKT8- NK-like cells that lyse EB-, NK-sensitive cells.     

Depletion of human NK cells with monoclonal antibodies allows the generation of cytotoxic T lymphocytes without NK-like cells in mixed cultures

In vitro stimulation of human mononuclear cells with x-irradiated autologous lymphoblastoid cell line (LCL) or allogeneic normal cells in mixed leukocyte cultures (MLC) was previously shown to result in the generation of OKT3+ OKT8+ cytotoxic T lymphocytes (CTL) lytic for allogeneic and autologous LCLs and also of natural killer- (NK) like cells that are OKT3- and primarily OKT8- and are lytic for HLA- NK-sensitive K562 cells. The origin of the NK-like cells was not previously known because, although the majority of fresh human NK cells react with monoclonal antibodies OKM1 and B73.1, lymphocytes bearing these markers are not detected several days after the onset of MLC, when NK-like cells are present. In this study, experiments were undertaken to determine whether NK-like cells generated after stimulation with x-irradiated pooled allogeneic normal cells (poolx) or with autologous LCL are derived from cells expressing antigens reactive with monoclonal antibodies OKM1 or B73.1, which react with fresh NK cells. Mononuclear cells, depleted of monocytes, were stained with OKM1 or B73.1 and fluorescein-labeled goat anti-mouse IgG. Lymphocytes depleted of OKM1+ or B73.1+ cells, by fluorescence-activated cell sorting, and lymphocytes that were stained but not sorted were stimulated for 7 days with either poolx or autologous LCL. The generation of NK-like activity was decreased at least 90% after depletion of cells reactive with OKM1 or B73.1, whereas the generation of CTL against autologous and allogeneic LCL was minimally affected. These findings show that NK-like cells generated in MLC are derived from cells that express the phenotype of fresh NK cells (OKM1+ or B73.1+) and that CTL can be generated in cultures in which relatively little NK-like activity is concomitantly detected, by depleting NK cells with monoclonal antibodies before stimulation.     

Sensitization of lymphocytes against pooled allogeneic cells. II. Characterization of effector cells cytotoxic for autologous lymphoblastoid cell lines

Stimulation of human lymphocytes in mixed leukocyte culture (MLC) with x-irradiated pooled allogeneic normal cells (poolx) was previously shown to result in generation of effector cells cytotoxic for autologous Epstein-Barr virus- (EBV) transformed lymphoblastoid cell lines (LCL). This study was undertaken to determine whether lysis of the autologous EBV- transformed LCL cells by pool-stimulated cells is mediated by cytotoxic Tc lymphocytes (Tc) or natural killer- (NK) like cells, both of which are generated in MLC. In the first series of experiments, proliferating cells were eliminated by treatment of pool-stimulated cells with 5 X 10(-5) M 5-bromodeoxyuridine (BUdR) and light. The remaining cells failed to lyse allogeneic normal lymphocytes and autologous LCL cells, whereas cytotoxicity against NK-sensitive K562 leukemia cells was retained. In the second series of experiments, pool-stimulated effector cells were treated with monoclonal anti-human Tc cell antibodies, OKT3 or OKT8, and complement (C). The cells recovered after antibody and C treatment were diminished in their ability to lyse allogeneic normal lymphocytes as well as autologous LCL cells, whereas their cytotoxicity against K562 leukemia cells was unaffected. These combined results provide strong evidence that lysis of autologous LCL cells by lymphocytes stimulated with pooled allogeneic normal cells is mediated by Tc rather than NK-like cells.     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Differentiation of NK-like cells from OKT3-, OKT11+, and OKM1+ small resting lymphocytes by culture with autologous T cell blasts and lymphokine

NK-like cells have been generated in vitro from a resting lymphocyte population of PBMC by 8 days culture with mitomycin C-treated autologous T cell blasts and lymphokine. The responder lymphocyte population was purified to the extent that it lacked classical NK cells, and lacked the precursors of MLC-derived NK-like cells and of lymphokine-activated killer cells. The NK-like cells were not generated when the responder lymphocytes were cultured with either T cell blasts or lymphokine alone. Thus, at least two signals are required for their activation. Metabolically inactive T cell blasts plus lymphokine were effective in stimulating the generation of NK-like cells, suggesting that a membrane determinant on the T cell blasts was involved in activation. The phenotype of the NK-like cells and their precursors was analyzed by monoclonal antibody and complement treatment. The phenotype of both precursor and effector cells was OKT3-, OKT11+, and OKM1+, with a distinct pattern of reactivity with OKT8 and Leu-7 for each individual donor tested. The NK-like cells were morphologically large granular lymphocytes, and they killed a variety of target cells. These studies show that signals provided by autologous T cell blasts and lymphokine are essential in triggering the differentiation of NK-like cells from appropriately purified resting lymphocytes. This mechanism of activation could occur in vivo, leading to the generation of NK cells subsequent to an antigen-specific T cell response.     

Lysis of fresh human tumour cells by autologous tumour-associated lymphocytes: Two distinct types of autologous tumour killer cells induced by co-culture with autologous tumour

Summary The specific and natural killer (NK)-restricted nature of auto-tumour cytotoxicity of tumour-associated lymphocytes was studied in cancer patients with malignant pleural effusions. Large granular lymphocytes (LGL) and small T lymphocytes were isolated from carcinomatous pleural effusions by centrifugation on discontinuous Percoll gradients. Tumour cells freshly isolated from pleural effusions were classified according to their susceptibility to lysis by Percoll-purified LGL from the blood of normal donors in a 4-h ⁵¹Cr release assay. Of 12 NK-sensitive tumour samples, 11 were killed by autologous fresh effusion LGL, whereas only 2 were lysed by autologous T cells. Neither LGL nor T cells were cytotoxic to NK-resistant autologous tumour cells. T cells and LGL were each cultured in vitro with autologous tumour cells for 6 days. Effusion LGL maintained their auto-tumour killing activity in 10 of 12 autologous mixed lymphocyte-tumour cultures (MLTC) with NK-sensitive tumour, while LGL lost the activity when cultured alone. Removal of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL enriched effector cells. Autologous MLTC-derived LGL could also kill NK-sensitive allogeneic effusion tumour cells and K562 cells, as did fresh LGL. In autologous MLTC LGL failed to acquire lytic function to NK-resistant autologous tumour cells. In contrast, in vitro activation of effusion T cells with autologous tumour cells induced auto-tumour killer cells in 9 of 12 NK-sensitive tumour samples and 3 of 6 NK-resistant tumour cases. However, cultured T cells were incapable of killing allogeneic tumour cells and K562 cells. In the autologous MLTC effusion T cells proliferated vigorously in response to autologous tumour cells, whereas LGL showed no proliferation. The enrichment of blasts from cultured T cells on discontinuous Percoll gradients resulted in an enhancement of auto-tumour cytotoxicity, with no reactions recorded in blast-depleted, small, resting T cells. These results indicate that two distinct types of auto-tumour-recognising lymphocytes, LGL and T cells, are present in carcinomatous pleural effusions of cancer patients and that each effector type recognises different membrane moieties of autologous effusion tumour cells.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.     

NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]

We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Characterization of human large granular lymphocyte subpopulations: comparison of the phenotype of NK cells and of interleukin 2-dependent progenitors of cytolytic effector cells

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.     

Natural killer (NK)-like cytotoxic activity of allospecific T cell receptor-gamma,delta+ T cell clones. Distinct receptor-ligand interactions mediate NK-like and allospecific cytotoxicity

We recently generated a series of human alloantigen-specific, CD3+,TCR-gamma,delta+ clones by stimulating CD3+,CD4-,CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). As reported previously, these clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Further studies of these and other TCR-gamma,delta+ clones, described in this report, indicate that most but not all of these clones express the NK cell associated marker, NKH-1 or Leu-19, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not an irrelevant LCL or NK-resistant line, Raji. TCR-gamma,delta+ clones which lacked expression of Leu-19 lysed their allospecific targets but had no detectable NK activity. The allospecific cytotoxicity of Leu-19+ and Leu-19- clones was inhibited by mAb to CD3 or the TCR delta-chain. In contrast, the NK-like activity of the Leu-19+ clones was enhanced by these antibodies over a wide range of antibody concentration. Although mAb to LFA-1 markedly inhibited both the allospecific and NK-like activity of these clones, an HLA class I framework specific mAb (W6/32) had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the TCR-gamma,delta+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+,TCR-gamma,delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Activation of autoreactive cytolytic T lymphocyte clone against human melanoma by anti-T3 monoclonal antibody and autologous accessory cells

A cytotoxic T-lymphocyte (CTL) clone Tc1.8 was derived in a limiting dilution culture from a single cell that was derived from melanoma-involved lymph node lymphocytes activated in in vitro coculture against the autologous melanoma cells (VIP). The clone Tc1.8 (T3+, T8+, T4-, and Leu7-) expressed restricted cytolytic activity against only the autologous target VIP. As it aged in continuous culture containing interleukin 2, Tc1.8 lost cytolytic activity. The cytolytic function could be restored, however, with monoclonal antibody (MoAb) against T3 (OKT3) or with F(ab')2 fractions of OKT3, and upon restimulation with irradiated accessory cells. OKT3-mediated reinduction of cytotoxicity by the aged Tc1.8 could not be achieved if the T3 molecules were modulated from the effector cell surface following overnight incubation of Tc1.8 with saturating concentrations of OKT3 MoAb. Following reactivation with OKT3 Tc1.8 gained cytolytic function against NK targets in addition to VIP. Reactivation with F(ab')2 fractions of OKT3 and with autologous accessory cells, however, maintained its restricted antigen fidelity. The NK-like activity of Tc1.8 upon reactivation with OKT3 resulted from conjugate formation between the activated Tc1.8 and NK targets via the activating ligand itself. Thus, upon stimulation with anti-T3 MoAb and with autologous accessory cells, independently, the autoreactivity could be restored in an aged and inactive CTL clone.     

Human T cell clones exerting multiple cytolytic activities show heterogeneity in susceptibility to inhibition by monoclonal antibodies

Human cytotoxic T cell clones (CTL) were obtained by limiting dilution after in vitro priming against an allogeneic Epstein Barr virus (EBV)-transformed B cell line (B-LCL) BSM. Three OKT3+, OKT8+ E rosette-forming (RFC) but EA gamma-RFC- clones with cytotoxic activity against the stimulator cell and one "non-cytolytic" clone were expanded for over 50 generations and further characterized. Clone G9 showed allospecific lysis of Cw3+ lymphocytes and B cell lines. Three cytolytic clones (G9, D11, and A3) showed cytotoxicity to the stimulator B-LCL, to the human plasma cell leukemia-derived line LICR-LON-HMY2 and to short-term cultured melanoma cells (O-mel). Four other EBV-transformed B-LCL unrelated to the stimulator B-LCL were not lysed. These clones also exerted cytotoxic activity against NK-sensitive target cells (TC), e.g., the erythroleukemia cell line K562. Other NK-sensitive TC, e.g., lymphoma-derived Daudi cells, were killed provided they were pretreated with phytohemagglutinin (PHA). Cytolytic activity against the B-LCL cell LICR-LON and O-mel, but not against K562 or PHA-treated target cells, was inhibited by monoclonal anti-HLA ABC antibodies (MCA). The cytolytic activities of OKT3+,8+ clones G9 and A3 but not that of OKT3+,8+ clone D11 were inhibited by OKT8. Another MCA, 13.3, directed against the murine glycoprotein T-200, inhibited the cytolytic activity of clone D11 against K562 but not against the stimulator cells. Clone G9 was not inhibited by MCA 13.3. The four clones, including the OKT4+ "non-cytotoxic" clone K12, exerted lytic activity against TC that are normally resistant to lysis provided these TC were pretreated with PHA. The TC specificity range of the clones was confirmed by cold target inhibition experiments. A correlation between blocking of lytic activity by cold TC and the percentage of conjugate formation with the particular cold TC was observed. Because these clones also show differential susceptibility to inhibition of lysis by various MCA, it is concluded that human cytotoxic T cell clones can exert multiple lytic activities, i.e., the operationally defined lytic mechanisms differ at least at certain stages of the lytic cycle.     

Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts.

This study was undertaken to characterize the phenotype and function of lymphocytes derived from endomyocardial biopsies in heart transplant patients. To this aim, tissue infiltrating lymphocytes were derived from seven heart transplant patients and were analyzed for the expression of a panel of markers, including CD3, CD4, CD8, CD16, CD56, CD45RA, CD45RO, alpha/beta and gamma/delta T cell receptor, and for their ability to lyse a series of targets, including NK-sensitive K-562 targets, NK-resistant Raji targets, donor related, and unrelated normal splenocytes. Our data show that the majority of cultured lymphocytes expressed the CD3+ phenotype and the alpha/beta T cell receptor. The CD4 and CD8 molecules were heterogeneously expressed among T cell lines tested. Concerning cytotoxic related markers, a significant percentage of cells were CD56+. The evaluation of CD45 isoforms showed that both "naive" and "memory" cells were present among heart TIL. Cytotoxic in vitro studies demonstrated that all our T cell lines showed an efficient cytotoxic machinery when tested against NK-sensitive targets. A marked lysis of donor-related splenocytes was demonstrated in all patients tested. To investigate the role of CD3 and HLA class I molecules in the cytotoxic mechanisms taking place in human heart allograft rejection mechanisms, TIL were assessed for their lytic activity against different targets in the presence of anti-CD3 and anti-HLA class I monoclonal antibodies (mAbs). Although donor-specific cytotoxicity was considerably inhibited by the anti-CD3 mAb, no inhibitory effect was displayed by this antibody on TIL-mediated cytotoxicity against donor-unrelated splenocytes. Anti-HLA class I mAb was able to inhibit both allospecific and nonallospecific cytotoxicity. These data suggest that different types of cytotoxic cells may be propagated from biopsy specimens of heart transplant patients.     

Characterization of the T cell-mediated cellular cytotoxicity during acute infectious mononucleosis

Primary infection with EBV during acute infectious mononucleosis (IM) is associated with a cytotoxic response against allogeneic target cells. C depletion with anti-CD3 (OKT3) and anti-CD8 (OKT8) mAb decreased the allogeneic cytolysis of two EBV-infected lymphoblastoid cell lines (LCL) by 96% and 89%, respectively. Complement depletion with the NK cell-specific mAb Leu-11b and NKH-1a resulted in only a slight decrease (less than 35%) in the lysis of these LCL. mAb inhibition studies with OKT3 and OKT8 inhibited the allogeneic lysis of two LCL by 87% and 82%, respectively. The alloreactive cytotoxic response was strongly inhibited by mAb specific for MHC class I determinants (W6/32, 65% inhibition and BBM.1, 58% inhibition). Acute IM lymphocytes lysed the allogeneic EBV-negative cell lines HSB2 (45%) and HTLV-1 T cell lines (16%). NK cell-depleted lymphocytes from an acute IM patient demonstrated preferential lysis of K562 transfected with human HLA-A2 (73%) compared with the K562 transfected control (20%). Cold target competition studies with allogeneic and autologous target and competitor LCL demonstrated no significant competitive inhibition between allogeneic and autologous cells. We interpret these results as evidence that 1) the acute IM-alloreactive cytotoxic response is mediated primarily by CTL; 2) these alloreactive CTL lyse allogeneic target cells irrespective of EBV antigenic expression; 3) MHC class I expression is sufficient for allogeneic recognition and lysis of target cells; 4) distinct effector CTL populations mediate lysis of autologous and allogeneic target cells; and 5) during acute IM, EBV infection results in the induction of both virus-specific and alloreactive CTL populations.     

Stimulation of human lymphocytes with irradiated cells of the autologous Epstein-Barr virus-transformed cell line. I. Virus-specific and nonspecific components of the cytotoxic response

Peripheral blood lymphocytes were cocultivated with irradiated cells of the autologous EB virus-transformed cell line at different responder:stimulator (R:S) ratios and the cytotoxic response was assayed up to 12 days later. In cocultures set up at a R:S ratio of 4:1, the response from both EB virus antibody-positive (seropositive) and negative donors was dominated by a broad-ranging NK-like cytotoxicity which did not segregate within the E-rosette-forming subpopulation of effector cells. In contrast, cocultures set up at a R:S ratio of 40:1 and harvested after 10 to 12 days gave rise, in the case of seropositive donors only, to effector T-cell preparations which appeared to be both EB virus specific and HLA-A and B antigen restricted. Strong lysis of the autologous virus-transformed cell line and demonstrable activity against certain allogeneic HLA-A and/or B antigen-related virus-transformed lines occurred in the absence of any significant killing either of the corresponding lines from HLA-unrelated donors or of a variety of EB virus genome-negative target cells (K562, HSB2, BJAB) particularly sensitive to NK-like cytotoxicity; furthermore, lysis of the autologous cell line by these effector T cells was specifically inhibited by monoclonal antibodies binding to HLA-A, B, and C antigens on the target cell surface. This work demonstrates that an HLA-restricted EB virus-specific cytotoxic T-cell response can indeed be induced in vitro by stimulation of fresh lymphocytes with autologous EB virus-transformed cells providing cocultures are set up at the correct R:S ratio.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

Comparison of recombinant-interleukin-2-activated peripheral blood and tumor-infiltrating lymphocytes of patients with epithelial ovarian carcinoma: Cytotoxicity,growth kinetics and phenotype

Summary We have compared the growth and tumordirected cytotoxic efficacy of recombinant-interleukin-2-(rIL-2)-activated peripheral blood (PBL) and tumor-infiltrating lymphocytes (TIL) from patients with epithelial ovarian carcinoma. These studies demonstrated that TIL and PBL displayed similar levels of cytotoxicity and a broad range of target cell killing, as exemplified by their reactivity against autologous and allogeneic ovarian tumors as well as against tumor cell lines. No specificity of autologous tumor cell killing was manifested by TIL. Even though TIL of some patients showed higher proliferative activity (especially at the later times in rIL-2 culture) this was not a general phenomenon. In fact, in one case TIL did not proliferate at all, and in the other case the PBL proliferated more actively. While the cultures were composed primarily of CD3⁺ lymphocytes, the major cytotoxic cells displayed the CD56⁺ and CD16⁺ phenotype. Addition of OKT3 mAb to rIL-2 cultures resulted in an increased proliferative index, but showed only a minor effect on the cytotoxic potential of cultured lymphocytes. The therapeutic potential of rIL-2-activated TIL and PBL is discussed.Recipient of the Florence Maude Thomas Cancer Research Professorship