Polyadnylic acid sequences in RNA able to transfer specific delayed type hypersensitivity in vitro.

Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with 10^?3M cycloheximide, (Cy)], although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

1,25-Dihydroxyvitamin D3 enhances the generation of nonspecific suppressor cells while inhibiting the induction of cytotoxic cells in a human MLR.

The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), has been shown in both in vitro and in vivo experiments to be immunoregulatory. We analyzed the effects of the hormone on the human mixed lymphocyte reaction (MLR), the in vitro model of allograft response. Suppressor-cell activity of MLR-generated effector cells was enhanced by calcitriol (10(-10) to 10(-8) M). This suppressor activity was nonspecific since calcitriol-generated effector cells could suppress a primary MLR with stimulators and/or responders heterologous to the effector-generating MLR. Calcitriol (10(-9) to 10(-7) M) was also effective in preventing the generation of cytotoxic T cells when tested in a 51Cr release assay. While no differences were observed in the phenotypic analyses of the MLR-generated effector cells between 1,25-D3-treated cells and control cells, a significant reduction of class II antigen expression was observed in the presence of the hormone. The effects of calcitriol on human MLR are similar to those observed with cyclosporine.     

Studies on the mechanism of activation of human natural killer function by interferon and inhibitors of thymidylate synthesis

Previous publications from this laboratory have demonstrated that agents such as methotrexate (MTX), 5-fluorodeoxyuridine (FUdR), trimethoprim, and D-glucosamine (D-GlcN), which are known to inhibit thymidylate synthesis, can augment human NK activity in vitro. Furthermore, this augmentation was inhibited by exogenous thymidine (TdR) at concentrations of 10(-6) to 10(-7) M. In this report, underlying mechanisms of action of FUdR, D-GlcN, and IFN are compared. Each of these agents increased the lytic activity of effector cells bound to targets but did not increase the percentage of conjugates formed. The augmentation could be induced in a population highly enriched for NK cells (Leu-1 lb positive in phenotype). FUdR and D-GlcN could not induce any augmentation in a Leu-1 lb-negative subpopulation whereas IFN could induce significant lytic activity. alpha-Amanitin, an inhibitor of RNA polymerase II, blocked the activation of NK activity by all three reagents; hence gene expression was required. Comparison of [35S]methionine-labeled proteins by two-dimensional gel electrophoresis revealed that six new proteins were induced in IFN-treated cells. Three of these were similar in pI and molecular weight to the newly synthesized proteins in the D-GlcN-treated cells. One protein was synthesized in increased amounts in the FuDR-treated cells and it was not common to either of the other treatments. Evidence to date is consistent with the hypothesis that separate mechanisms underlie the activation of NK cells by IFN and thymidylate synthesis inhibitors, although the existence of a final common pathway for all NK response modulators cannot be excluded at the present time.     

Induction of granzyme B and T cell cytotoxic capacity by IL-2 or IL-15 without antigens: multiclonal responses that are extremely lytic if triggered and short-lived after cytokine withdrawal

The purpose of these studies was to determine the minimal requirements to induce granzyme B, cytotoxic granules and perforin-dependent lytic capacity. To our surprise, both IL-2 and IL-15 induced not only proliferation, but also profound granzyme B and lytic capacity from CD8+ T cells in the absence of antigen or TCR-stimulation. Mouse splenocytes were incubated with mouse r-IL-2 or r-IL-15 for three days, tested by anti-CD3 redirected lysis and examined for intracellular granzyme B and for T cell activation markers. With 10(-8) M IL-2 or IL-15, there was excellent lytic activity at 1:1 effector to target ratios mediated by T cells from wild-type but not from perforin-gene-ablated mice, consistent with multiclonal activation. Lower interleukin concentrations induced less lytic activity. Granzyme B was undetectable on day 0, and greatly elevated on day 3 in CD44hi CD8+ T cells as detected by flow cytometry. Cytokines alone elevated the granzyme B as much as concanavalin A combined with the cytokines. Some ex vivo CD8+ T cells were CD122+, as were the cultured granzyme B+ cells, thus both populations had low-affinity receptors for the interleukins. Only some of the activated cells were proliferating as detected by CFSE labeling. When the cytokines were withdrawn, the cells lost lytic activity within 24 h and then within the next 24 h, died. Our results suggest that high concentrations of either IL-2 or IL-15 will activate the lytic capacity and granzyme B expression of many T cells and that antigen recognition is not required.     

Studies on the mechanism of lymphocyte-mediated cytolysis. IX. Relationships between antigen recognition and lytic expression in killer T cells.

The relationship between antigen recognition and lytic expression by killer T cells was studied by co-culturing two effector cell populations. When antigen recognition was bidirectional (e.g., b anti-d cells cultured with d anti-b) there was a loss of lytic activity in both populations. In contrast, when antigen recognition was unidirectional (e.g., a anti-d co-cultured with d anti-b) then the loss of lytic activity only occurred in that direction; i.e., there was a marked decline in the d anti-b activity but no change in the a anti-d population. These studies suggest: i) that mere proximity to a killer cell does not lead to target cell death; ii) that accommodation of the T cell's antigen receptor is necessary for the cell to express its lytic potential; and iii) there is direct linkage between the T cell's antigen receptor site and its killing mechanism.     

IL-4-induced lymphokine-activated killer cells. Lytic activity is mediated by phenotypically distinct natural killer-like and T cell-like large granular lymphocytes

The purpose of the current study was to characterize lymphokine-activated killer (LAK) activity induced with IL-4/B cell stimulatory factor-1 and to compare IL-4-induced LAK activity with IL-2-induced LAK activity. Culture of murine lymphocytes with high concentrations of IL-4 induced nonspecific lytic activity against a wide variety of tumors. Lytic activity induced by IL-4 increased with increasing concentrations of IL-4 over the range of 1.0 to 25 ng/ml. The kinetics of LAK induction by IL-4 and IL-2 were similar; however, IL-4 was less effective than IL-2 in maintaining lytic activity for longer culture periods and provided lower viable cell yields than did IL-2. Similar to IL-2, IL-4 induced blastogenesis and the generation of large granular lymphocytes, all LAK activity observed was exclusively associated with the large granular lymphocyte fraction, and the cytolytic effector cells were heterogeneous in regards to cell surface phenotype. The majority of IL-4-induced lytic activity was associated with mutually exclusive NK-like (i.e., NK-1.1+ Lyt-2-) and T cell-like (i.e., NK-1.1- Lyt-2+) LAK cells. The precursors for each subset were distinct and expressed the asialo-GM1+ Lyt-2- and the asialo-GM1+ Lyt-2+ phenotypes, respectively. Although IL-4-induced LAK effector cells were morphologically and phenotypically similar to IL-2-induced LAK cells, IL-2 generated equivalent numbers of T cell-like and NK-like LAK cells, whereas IL-4 generated 3.5-fold more T cell-like LAK cells than NK-like LAK cells. It might eventually be possible to exploit the preferential activation of T cell-like LAK by IL-4 for therapeutic advantage.     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in alpha-minimal essential medium containing either vehicle, genistein (l0(-7) - 10(-5) M) or daidzein (10(-7) - 10(-5) M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [3H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein ( 10(-5) M) or daidzein ( 10(-5) M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10(-7) M) or cycloheximide (10(-6) M) in the absence or presence of isoflavones. Moreover, when genistein (10(-7) 10(-5) M) or daidzein (10(-6) and 10(-5) M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10(-7) M). In addition, [3H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10(-6) and 10(-5) M) or daidzein (10(-5) M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

Accessory cells, dendritic cells, or monocytes, are required for the lymphokine-activated killer cell induction from resting T cell but not from natural killer cell precursors

In this study we have investigated the role of accessory cells in the development of lymphokine-activated killer cells (LAK) from highly purified human NK and small resting T cell progenitors. As accessory cells we used autologous, as well as allogeneic, monocytes, and dendritic cell enriched cells. Both NK and T cells were able to generate LAK activity, but their activation requirements were different. NK cells were activated merely by IL-2, and accessory cells did not enhance their lytic activity in the presence or absence of IL-2. Conversely, T cells were practically unresponsive to even high concentrations of IL-2 having a strict requirement for accessory cells for the development of lytic activity and proliferation. Accessory cells differed in their ability to activate T cells presumably depending on their ability to induce IL-2 synthesis, allogeneic dendritic cells being the most effective accessory cells and IL-2 synthesis stimulators. Allogeneic accessory cells could induce lytic activity in T cells even in the absence of exogenous IL-2. Thus, accessory cells play a central role in expanding the LAK effector cell population.     

Murine trophoblast resists cell-mediated lysis. I. Resistance to allospecific cytotoxic T lymphocytes

Research on the mechanisms of nonrejection of the fetoplacental allograft has focused on the tissues composing the fetomaternal interface, of which the placental trophoblast, the tissue directly confronting the maternal environment, is considered a prime candidate responsible for the survival of the fetus. We recently developed a method for isolating murine trophoblast, and found that a proportion of trophoblast cells from mature placentas, cultured for 2 days, express class I antigens on their surface, and this expression can be enhanced in vitro by interferon-alpha/beta (IFN-alpha/beta). In the present study, it was determined that cultured trophoblast cells from day 14 placentas were resistant to allospecific cytotoxic T lymphocytes (allo-CTL), while being susceptible to alloantibody and complement-mediated lysis. The trophoblast cells remained resistant to allo-CTL-mediated lysis despite IFN-mediated enhanced expression of class I H-2 antigens on their surface and the addition of phytohemagglutinin into the assay. Inhibition of protein synthesis also had no effect. In contrast, fetal fibroblasts, isolated from the same conceptuses, were readily susceptible to allo-CTL-mediated lysis. That the trophoblast cells do interact with the effector cells was shown by their ability to specifically inhibit the lysis of tumor target cells in a dose-dependent manner. Additionally, trophoblast culture supernatants did not inhibit the lytic activity of allo-CTL, even when concentrated 10- to 25-fold, indicating that a soluble suppressor factor was not inactivating the effector cells. These results suggest that trophoblast cells have a protein synthesis-independent mechanism of resistance to lysis by allo-CTL, which could play an important role in protecting the fetoplacental allograft from maternal immune rejection.     

Natural cytotoxic cells and tumor necrosis factor activate similar lytic mechanisms

The lytic activity of natural cytotoxic (NC) cells has several characteristics which clearly distinguish it from other cell-mediated lytic activities and from most soluble cytolytic factors. An exception is the lytic activity mediated by tumor necrosis factor (TNF). In this paper, we report a detailed comparison of NC and TNF lysis of target cells which are used as prototype NC targets or TNF targets, and show that the two cytolytic activities have very similar, if not identical, lytic mechanisms. We present data showing that target cells which are NC-sensitive are also TNF-sensitive and that target cells which are NC-resistant are also TNF-resistant. Moreover, cells selected either in vivo or in vitro for NC resistance are selected for TNF resistance, and cells selected for TNF resistance are selected for NC resistance. The analysis of the kinetics of 51Cr release mediated by NC cells or by TNF show that both activities affect similar kinetics, in that there is no cell lysis for several hours after targets and effectors first interact. However, NC and TNF lytic activities can be distinguished. By using the cell lines 10ME or B/C-N as targets, it can be shown that whereas NC-mediated lysis is dependent on protein synthesis, TNF-mediated lysis is not. We also show that targets which are resistant to NC-mediated lysis because they express a protein synthesis-dependent resistance mechanism also require protein synthesis to resist TNF-mediated lysis, suggesting that the same resistance mechanism protects cells against both NC cells and TNF. Together, these data strongly support the hypothesis that NC cells and TNF activate the same lytic mechanism within target cells and that TNF may mediate the lytic activity of NC effector cells.     

Cutting edge: CCR7+ and CCR7- memory T cells do not differ in immediate effector cell function

It has been proposed that expression of the chemokine receptor CCR7 represents a defining factor for nonpolarized central (CCR7(+)) and polarized effector memory (CCR7(-)) T cells. In this study, we have tested this hypothesis using in vivo-activated T cells from P14 and SMARTA TCR-transgenic (tg) mice specific for MHC class I- and II-restricted epitopes of the lymphocytic choriomeningitis virus (LCMV) glycoprotein. CCR7 cell surface expression on TCR-tg cells was monitored with a CC chemokine ligand 19-Ig fusion protein. CC chemokine ligand 19-Ig staining separated TCR-tg cells activated by LCMV infection into CCR7(-) and CCR7(+) effector/memory T cell populations. Nonetheless, both T cell populations isolated from spleen and liver produced identical amounts of IFN-gamma after short-term Ag stimulation. Furthermore, CCR7(+) and CCR7(-) CD8 TCR-tg cells from LCMV-infected mice exhibited similar lytic activity against LCMV peptide-coated target cells. These results question the proposed concept of differential effector cell function of CCR7(+) and CCR7(-) memory T cells.     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.     

Studies on T cell clonal expansion. II. The in vitro differentiation of pre-killer and memory T cells.

Spleen cells from C57BL/6 mice immunized with a DBA/2 mastocytoma (P815) were harvested at various stages of the immune response and cultured in vitro in the presence and absence of antigen. Killer T cell activity in immune spleens could not be demonstrated until 6 or 7 days after antigen, but spleen cells harvested as early as 3 or 4 days and cultured for 24 hr at 37 degrees C showed significant cytotoxicity. This increased activity was not augmented further by culturing with antigen. "Memory" T cells, whose in vitro differentiation into killer cells required the presence of antigen, could not be demonstrated until 9 or 10 days after alloantigenic stimulation. Once produced, however, these cells persisted for at least 6 months. Memory cells, like killer T cells bound avidly to homologous allogeneic monolayers. There were indications that the memory T cell pool was heterogeneous. On one hand, when cells harvested 10 days after stimulation were exposed to antigen in vitro, their lytic activity increased within 24 hr but showed no further increases when the culture period was extended. In contrast, 45-day-old immune cells showed increasing lytic activity throughout a 4-day exposure to antigen. Augmentation of lytic activity in both cell populations was independent of DNA synthesis through the first 24 hr of culture. Subsequent increases in the activity of 45-day cells was dependent upon cell proliferation. Both the antigen-independent augmentation of lytic activity which followed culturing of immune cells, and the antigen-induced differentiation of memory cells were reversibly inhibited by a series of drugs which raised lymphocyte cAMP levels.     

Expression of CD161 (NKR-P1A) defines subsets of human CD4 and CD8 T cells with different functional activities

A subset of T cells in human peripheral blood expresses CD161 (NKR-P1A) receptors that are primarily associated with NK cells. In the current study we isolated blood T cell subsets according to the expression of CD161 and examined their contents of naive, central memory, and effector memory cells and their capacities for proliferation, cytokine secretion, and natural cytolysis. We found that CD4+CD161- and CD8+CD161- subsets contained predominantly naive T cells that secreted high levels of IL-2 after in vitro stimulation, and CD4+CD161int and CD8+CD161int subsets contained predominantly effector and central memory T cells that secreted high levels of IFN-gamma and TNF-alpha. All of these subsets showed vigorous proliferation after stimulation in vitro, but none had NK lytic activity. Unexpectedly, the CD8+CD161+ cells contained an anergic CD8alpha+CD8betalow/-CD161high T cell subset that failed to proliferate, secrete cytokines, or mediate NK lytic activity.     

Tumor-specific responses in lymph nodes draining murine sarcomas are concentrated in cells expressing P-selectin binding sites.

Tumor-draining lymph node (TDLN) cells develop substantial antitumor activity after activation on immobilized alphaCD3 and culture in low-dose IL-2. This study found that the minor subset of TDLN T cells expressing binding sites for the adhesion receptor P-selectin (Plig(high) T cells) produced T lymphoblasts with the most tumor-specific IFN-gamma synthesis in vitro and antitumor activity following adoptive transfer in vivo. The Plig(high) T cells constituted <25% of the cells with the phenotype of recently activated cells including high levels of CD69, CD44, or CD25, and low levels of CD62L. The cultured Plig(high) TDLN were 10- to 20-fold more active against established pulmonary micrometastases than cultured unfractionated TDLN, and >30-fold more active than cultured TDLN cells depleted of the Plig(high) fraction before expansion (Plig(low) cells). Tumor-specific IFN-gamma synthesis in vitro paralleled the antitumor activities of the cultured fractions in vivo, implying that increased Tc1 and Th1 effector functions contributed to the tumor suppression. Neither nonspecific interaction with the P-selectin chimera used for sorting nor endogenous costimulatory activity in the Plig(high) fraction accounted for the marked increase in antitumor activities after culture. The cultured Plig(high) fraction contained a variety of potential effector cells; however, the CD8 and CD4 subsets of alphabeta T cells accounted for 95-97% of its antitumor activity. The authors propose that P-selectin sorting increased antitumor activities by concentrating Tc1 and Th1 pre-effector/effector cells before culture.     

Natural killer cells in rhesus monkeys: properties of effector cells which lyse Raji targets

Spontaneously occurring natural killer cell activity of rhesus monkey peripheral blood mononuclear cells was assayed against five human cell lines, three of which were Epstein-Barr virus (EBV) positive, including the human B cell line Raji. The lytic activity to Raji cells was high, significantly higher than to any other cell line tested. Raji cells are normally insensitive to spontaneous lysis by human NK cells, and the contrasting vigor of the rhesus monkey cytolytic activity to Raji prompted us to investigate the properties of this effector cell. We found the effector cell-mediating lysis of Raji to be nonadherent and phagocytic with lytic activity slightly enhanced in the E-rosette-forming cell (ERFC+) fraction and decreased in the ERFC- fraction. Further isolation of FcIgG receptor-positive and FcIgG receptor-negative subsets by rosetting resulted in significant enrichment of NK activity to Raji in the positive fraction and a loss of activity in the negative fraction. Depletion studies with various monoclonal antibodies (mAb's) confirmed that nearly all lytic activity was contained in the CD16+ (Leu 11b+) population, while subsets of effector cells expressed CD2 (9.6) and CD8 (OKT8). Depletion of CD4 (OKT4)-, HLADR (OKIa)-, or LFA1 (MAC-1)-positive populations failed to reduce NK activity. We compared the phenotypic properties of alloimmune effector cells exhibiting specificity for allogeneic donor targets with those exhibiting lysis of Raji targets. Results indicated that allospecific cytotoxic T lymphocytes expressed a CD16-, CD2+ phenotype, a pattern distinct from that of the effector cell population recognizing Raji targets. The presence of CD2 mAb's in the culture had no effect on NK lytic activity. In contrast, mAbs CD8 and Leu 11b were inhibitory. This would suggest a functional role for CD8 and FcIgG molecules in the lysis of Raji cells by rhesus effectors. In summary, these studies describe a distinct population of effector cells in the blood of rhesus monkeys which exhibit spontaneous lytic activity to Raji cells and exhibit the properties of NK cells.     

Augmentation of antileukemia lytic activity by OKT3 monoclonal antibody: synergism of OKT3 and interleukin-2

We have shown that short-term incubation (45 min) of peripheral blood lymphocytes of normal donors with OKT3 monoclonal antibody (MoAb), directed against T-cell-associated antigen CD3, resulted in an acquisition of lytic activity against fresh leukemic cells. Induction of such antileukemia activity was specific for OKT3, since Leu-1 MoAb (directed against another T cell surface molecule, CD5) did not induce a lytic effect. The OKT3-generated antileukemia effect was displayed against various types of leukemia including chronic myelogenous leukemia and acute myelogenous leukemia of various histological subtypes (M1, M2, M5). We furthermore demonstrated that OKT3 MoAb substantially enhanced leukemia killing by interleukin-2 (IL-2)-activated killer cells obtained from peripheral blood of patients with leukemia. Of most importance was the observation that the combined treatment of effector cells with IL-2 and OKT3 MoAb resulted in the highest levels of lysis of both autologous and allogeneic fresh leukemic cells that have been observed in leukemic patients to date. Of importance was to note that OKT3 treatment was effective in induction of cytotoxic activity also in patients whose effector cells were unresponsive to stimulation with IL-2 alone. All of these observations suggest that IL-2-activated and OKT3-MoAb-treated effector cells may represent the most aggressive population of antileukemia-directed killer cells and may play a significant role in the treatment of human leukemia.