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.     

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 killing and antibody-dependent cellular cytotoxicity: characterization of effector cells by E-rosetting and monoclonal antibodies

Recent investigations have indicated that the OKM1 hybridoma monoclonal antibody reactive with cells of the myelomonocytic series identifies a subpopulation of human peripheral blood mononuclear cells (PBMNC) which mediate natural and antibody-dependent cellular cytotoxicity (ADCC). However, it was not clear whether this OKM1+ group was heterogeneous with regard to cytotoxic function or the presence of receptors for sheep erythrocytes. Thus, the purpose of the present study was to further define the phenotype of the ADCC effector cell and natural killer (NK) cell using a combination of reactivity with hybridoma antibodies and separation of subsets by sheep erythrocyte rosette (E+) formation. Furthermore, the phenotypes of the NK population were assessed directly by performing two-color immunofluorescent staining on tumor cell conjugates. These studies led to the following conclusions: (1) that NK activity is mediated by both E+ OKM1+ and E- OKM1+ cells; the E+ OKT3+ cell possessed essentially no ADCC or NK activity; (2) that E+ OKM1+ cells mediated more NK activity on a per cell basis than E- OKM1+ cells; this was verified by separating OKM1+ cells on a cell sorter into E+ and E- with the OKT11 monoclonal antibody (anti-E-receptor antibody); (3) that E+ OKM1+ cells mediated both ADCC and NK activity; (4) that the phenotypes of PBMNC forming tumor cell conjugates were (a) OKM1+ (both E-receptor positive and negative) and (b) OKM1- E-receptor positive.     

Selective inhibition of natural killer activity by the monoclonal antibodies OKT10 and VEP10 at the single cell level

The monoclonal antibodies, VEP10 and OKT10, which have been shown to recognize determinants on human natural killer (NK) cells, inhibit large granular lymphocyte (LGL) NK activity against K562, MOLT4, and CEM tumor target cells in the single cell conjugate agarose assay. Inhibition of NK activity by monoclonal antibodies was expressed independently of effector-target cell binding, as inhibitory activity could be demonstrated when the monoclonal antibodies VEP10 and OKT10 were added to preformed conjugates or to the LGLs and targets prior to the binding event. In addition, this inhibition was exerted on the effector cell and not the target cell since VEP10 and OKT10 did not react with determinants on K562 target cells. Furthermore, the 4F2 monoclonal antibody, which reacted with determinants on the LGL and all of the targets used, effected no inhibition of NK activity. Inhibition of killing by OKT10 and VEP10 was specific to endogenous NK activity since the same antibodies did not inhibit antibody-dependent cellular cytotoxicity (ADCC), mixed lymphocyte-generated NK, or cytotoxic T lymphocyte (CTL) activities.     

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.     

Phytohemagglutinin-induced proliferation and cytolytic activity in T3+ but not in T3- cloned T lymphocytes requires the involvement of the T3 antigen for signal transmission

Proliferation and the cloning efficiency of T3+ but not T3- T cells are increased by the addition of lectins (phytohemagglutinin; PHA) to the culture medium. In contrast to that of T3+ cloned cell lines, the cytolytic activity of T3- clones is not enhanced by PHA, as we report here. We have investigated the effects of anti-T3 monoclonal antibody (MAb) and PHA on the proliferative capacity and cytolytic activity of various T3+ and T3- clones and cells to determine the possible involvement of the T3 receptor in these processes. We found that, in addition to inhibition of allospecific cytotoxicity, anti-T3 MAb can induce and/or enhance nonspecific cytolytic activity against particular target cells in cloned allospecific cytotoxic T cells (CTL) following preincubation of the effector cells with PHA or anti-T3. This enhancement of cytolytic activity is seen in T3+ but not T3- activated killer (AK) clones or fresh T3- natural killer (NK) cells and depends on the concentrations of anti-T3 MAb or PHA used. We conclude that the T3-Ti antigen-receptor complex is involved in the transmission of the activation signals by anti-T3 and PHA.     

Human natural cytotoxic lymphocytes: definition by a monoclonal antibody of a subset which kills an anchorage-dependent target cell line but not the K-562 cell line

A monoclonal mouse antibody (HNC-1A3) which defines a subset of human lymphocytes with natural cytotoxic activity was produced and studied. HNC-1A3+ cells represent 12 +/- 3% of peripheral blood mononuclear leukocytes. When sorted out using a fluorescence-activated cell sorter, they consist of 60% small lymphocytes, 35% large (predominantly agranular) lymphocytes, and 5% monocytes. They contain 30 +/- 6% E-rosette-forming cells, 6 +/- 1% OKT4+ cells, 17 +/- 6% OKT8+ cells and less than 2% OKT10+ or Leu-7 (HNK-1)+ cells. They are responsible for most of the natural cytotoxic activity against the MA-160 prostatic adenoma cell line but mediate an insignificant amount of cytotoxicity against the NK prototype target K562 cell line. Conversely, Leu-7+ cells which mediate most NK activity against K562 are weakly active against MA-160. Our data suggest a heterogeneity among leukocytes mediating natural cytotoxicity, with restricted specificities for the recognition sites on target cells.     

Human cytotoxic T lymphocytes (CTL) induced by phytohemagglutinin: conditions for CTL generation and effect of interferon

The present study demonstrates that human peripheral blood mononuclear cells (PMC) can be stimulated in vitro to become cytotoxic T lymphocytes (CTL) by PHA. A significant cytotoxic activity of PMC was detected 48 hr after the culture initiation in the presence of 5 micrograms/ml of PHA and the peak level of the activity was obtained by culturing PMC for 72 hr. The cytotoxic cells require the presence of PHA as a cell agglutinin for the expression of their cytotoxic activity. The effector cells mediating the activity were identified as T lymphocytes by E-rosette fractionation of PMC. In this system, removal of carbonyl iron phagocytosed or attached cells from PMC did not abrogate CTL generation of PMC. In addition, human alpha-interferon did not augment CTL generation or expression of their activity. Although the target cells employed were sensitive to natural killer (NK) cells, the effector cells induced by PHA did not seem to have any relation to the NK cells. The present study may provide a useful tool to analyze for precursors of killer T cells.     

Characterization of the natural killer-like lymphocytes in rheumatoid synovial fluid

IgG Fc- cytotoxic cells found in the synovial fluid of patients with rheumatoid arthritis have natural killer (NK)-like characteristics but can kill NK-resistant cell lines as well. The phenotype of these cells was defined by complement-mediated lysis with monoclonal antibodies. The synovial fluid killer cell activity was significantly reduced by treatment with complement and OKT11 and 4F2, but the cytotoxic T cells did not express the NK-related antigens OKM1 and Leu-7, nor the cytotoxic T lymphocyte-specific antigen, OKT8. These results demonstrate that the synovial fluid killer cells resemble the activated T cells generated in an autologous mixed leukocyte reaction or in the treatment of peripheral blood mononuclear cells with interleukin 2, and they are distinct from the conventional NK cells found in blood.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

Role of HLA-DR bearing Langerhans and epidermal indeterminate cells in the in vitro generation of alloreactive cytotoxic T cells in man

Human epidermal cells (EC) act as stimulator cells in the mixed-skin cell lymphocyte culture reaction (MSLR). To analyze the role of human epidermal Langerhans cells (LC) and indeterminate cells (IC), which are the only cells expressing the DR-Ia-like antigens in normal epidermis, in the generation of alloreactive cytotoxic T lymphocytes (CTL) in cell-mediated cytolysis, 18-hr 51Cr-release assays against PBL targets (targets autologous to stimulator EC) were conducted after allogeneic human MSLR. MSLR and CTL assays were conducted with, as stimulator cells, suspensions of normal human EC as controls, and EC after: (1) preincubation with anti-HLA-DR or OKT6 (specific for LC in EC suspension) monoclonal antibodies; (2) panning, a monolayer technique used to deplete EC suspensions in OKT6 or DR-positive cells. The generation of alloreactive CTL was found to occur only after allogeneic MSLR and when targets and stimulator cells were from the same donor; it was reduced by EC incubation: cytotoxic activity 26.66 +/- 3.84 (controls); 8.8 +/- 3.6 and 7.7 +/- 3.7 (EC incubated with OKT6 or anti-DR, respectively); it was reduced or abolished when the EC used in MSLR were depleted in OKT6 or DR-positive cells by panning. These findings demonstrate that human LC and IC are necessary for an optimal in vitro sensitization in MSLR and the subsequent in vitro generation of alloreactive CTL in man.     

Human cytotoxic T lymphocytes (CTL) against Epstein-Barr virus (EBV) infected cells: EBV specificity and involvement of major histocompatibility complex determinants in the lysis exerted by anti-EBV CTL toward HLA-compatible and allogeneic target cells

Human peripheral blood lymphocytes (PBL), from anti-Epstein-Barr virus (EBV)-seropositive donors, were stimulated by EBV and were shown to be cytotoxic toward autologous, HLA-compatible, and fully allogeneic EBV-transformed target cells. The lysis was not due to natural killer (NK) cells since the target cells used were resistant to lysis by fresh PBL and by virus-stimulated PBL-depleted of AET-SRBC-rosetting T cells (the latter being still fully cytotoxic on K562 NK-susceptible target cells). Conversely only E-rosette-purified (T) lymphocytes killed EBV-transformed HLA-compatible and allogeneic target cells. Moreover, anti-MHC antibodies inhibited the cytotoxicity exerted by EBV-induced cytotoxic T lymphocytes (CTL) on both autologous and allogeneic target cells. Finally the lysis was EBV specific since PHA blasts were not killed and since only EBV-transformed cells could compete for lysis with the EBV-positive target cells. Efficient competition was achieved by EBV-transformed cells autologous or allogeneic to the targets, even when effector and target cells were fully allogeneic. All together, the data suggest that human anti-EBV CTL may recognize nonpolymorphic HLA determinants on the target cells in association with the virus-induced antigens.     

A human NK and K cell subset shares with cytotoxic T cells expression of the antigen recognized by antibody OKT8

The antigen recognized by monoclonal antibody OKT8 is expressed on the cell membrane of 30 to 50% of human NK/K cells. The reactivity of OKT8 with NK/K cells was determined by indirect methods (treatment of the effector cells with OKT8 antibody and complement (C) and separation of OKT8(+) and (-) effector cell populations by fluorescence-activated cell sorting or by rosetting techniques) and, at single cell level, by C-dependent lysis of effector NK cells that bind and kill K562 targets. Analysis by indirect immunofluorescence (flow cytofluorometry) of lymphocyte subpopulations mediating NK/K cytotoxic activity and deprived of OKT8(+) T cells reveals that the NK/K cell subset bears OKT8 antigen at a density lower than that present on cytotoxic T cells. The OKT8 antigen on NK/K cells is trypsin- and pronase-sensitive, but it is resynthesized by the same effector cells during 24 hr of culture at 37 degrees C. OKT8 antibody does not inhibit NK killing, and, on a per cell basis, OKT8(+) cells within the NK/K subset mediate the same level of cytotoxic activity as OKT8(-) NK/K cells. Analogous results were obtained by using anti-Leu-2a, an antibody with the same specificity as OKT8 on cytotoxic/suppressor T cells, but not when OKT5 was used, which might identify a distinct epitope on the same antigenic molecule. The possible significance of these findings in understanding the cell lineage of NK/K cells is discussed.     

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.     

Characteristics of human NK clones: target specificity and phenotype

Clones derived from purified human large granular lymphocytes (LGL) of three different donors were expanded in culture medium supplemented with interleukin 2 (IL 2). Their cytotoxic activity was tested in a 51Cr-release cytotoxicity assay against a panel of three to five NK-susceptible tumor cell lines. Of 196 LGL clones tested, only 44 (22.4%) displayed significant cytotoxic activity. A heterogeneous pattern of reactivity was seen; 26 clones (59%) killed all the targets within the panel tested, whereas 18 clones (41%) had a more restricted specificity. Among these 18 clones, 12 lysed only one target (K562, six clones; ADCC, three clones; Daudi, two clones; MOLT-4, one clone), whereas the other six killed two different targets (ADCC and A1ab, one clone; K562 and MOLT-4, five clones). Clones derived from LGL preselected for positive reactivity with the monoclonal antibodies (MoAb) alpha OKM1, alpha OKT10 and alpha B73.1 also demonstrated either broad or restricted patterns of cytotoxicity. The LGL reactive with MoAb alpha B73.1 gave a high percentage of cytotoxic clones. Phenotype analysis showed that clones could express both antigens associated with T cells (i.e., OKT3, OKT4, and OKT8) and antigens shared by LGL (i.e., OKM1, OKT10, and B73.1). The pattern of surface markers varied considerably among the clones; however, no clear correlation between the pattern of antigenic phenotype and cytotoxic activity was seen. These data show that clones derives from purified preparations of LGL present different functional and antigenic characteristics, and support the hypothesis that the heterogeneity of the entire NK population is attributable, at least in part, to a mixture of clones that vary substantially in their target specificities and phenotypes.     

Anti-CD3 and phorbol myristate acetate regulation of MHC unrestricted T cell cytotoxicity. Lack of a requirement for CD3/T cell receptor complex expression during tumor cell lysis

The effects of anti-CD3 mAb on MHC-unrestricted cytotoxic activity of NK depleted PHA-activated human T cells were examined. Anti-CD3 mAb had variable effects on killing of K562 or Daudi targets. Whereas lower concentrations of OKT3 often inhibited lysis of either target, higher concentrations (greater than 1 micrograms/ml) frequently increased K562 killing and always augmented Daudi lysis. However, lysis of the renal cell carcinoma, Cur, was consistently inhibited by OKT3 over a broad concentration range. Such variable effects were not related to differential regulation of heterogeneous subsets of effector cells, as similar patterns of OKT3-mediated modulation of tumor cell lysis by T cell clones was also observed. Another IgG2a anti-CD3 mAb, 64.1, and either F(ab')2 fragments of OKT3 or intact OKT3 in the presence of aggregated human Ig were found to inhibit lysis of Cur, K562, and Daudi targets consistently. Additional experiments were carried out to determine whether modulation of CD3 accounted for the inhibitory effects of the anti-CD3 mAb. PMA was noted to cause modulation of CD3 from the surface of PHA or alloantigen-activated T cells, and the combination of anti-CD3 and PMA caused even more marked modulation of CD3. Whereas preincubation with PMA and/or anti-CD3 decreased alloantigen-specific cytotoxic T cell function in relative proportion to the loss of CD3 expression, no consistent relationship between CD3 expression and the capacity of PHA-activated T cells to kill Cur targets was noted. PMA alone caused no consistent alteration of Cur lysis. Moreover, in the presence of PMA, anti-CD3 mAb caused no significant inhibitory effect on Cur lysis, in spite of increased modulation and in some cases virtual total loss of surface CD3 expression. These findings indicate that when FcR interactions are prevented, anti-CD3 mAb consistently inhibit MHC-unrestricted cytotoxicity by PHA-activated T cells. Despite this, the data support the conclusion that CD3/TCR complex interactions with target cells are not required for either target cell recognition or triggering of lysis by MHC-unrestricted cytotoxic T cells.     

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.     

Cellular origin and interactions involved in gamma-interferon production induced by OKt3 monoclonal antibody

OKT3 monoclonal antibody, a human T cell mitogen, induced interferon production by cultured mononuclear cells at 10(-11) M concentrations. Interferon was secreted only under conditions wherein OKT3 was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, interferon secretion reached a peak 3 days after OKT3 stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT3 for induction. The interferon was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-alpha or -beta interferon antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT3 stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of interferon. Studies with selective pretreatments with mitomycin C suggested that gamma-interferon was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.     

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.     

Subsets of human large granular lymphocytes (LGL) exhibit accessory cell functions

The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.