Characterization of the phorbol 12, 13-dibutyrate (P(Bu)2) induced binding between human lymphocytes

The mechanisms, cell surface structures, and cell types involved in the phorbol 12,13-dibutyrate (P(Bu)₂)-induced binding between human lymphocytes were studied. Induction of cell aggregation by 20 min treatment with P(Bu)₂ required Ca²⁺, an intact membrane, functional microfilaments, and the possible participation of an esterase or, less likely, a protease. Trypsin-sensitive cell surface structures were needed and neuraminidase (NANase) treatment slightly increased the intercellular binding. Retinoic acid, an anti-tumor promoting agent, was inhibitory. Calmodulin-dependent processes, microtubules, phospholipid methylation, intracellular levels of cyclic adenosine monophosphate, and cellular secretion did not seem to be involved. Cell conjugation between 24 hr P(Bu)₂-treated and untreated cells required participation of trypsin-sensitive cell surface structures in each of the interacting cells and NANase treatment of one partner slightly increased the intercellular binding. Thymocytes, T cells, mature B and Epstein-Barr virus-transformed B cells aggregated while pre-B, early B, and intermediate B lymphocytes derived from representative malignancies did not. The lack of aggregation was not due to the absence of phorbol ester receptors. It is concluded that the P(Bu₂)-induced intercellular binding is mediated by cell surface proteins, depends on certain enzymatic activities and metabolic events and involves certain cell types.     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

The polysaccharide K (PSK) potentiates in vitro activation of the cytotoxic function in human blood lymphocytes by autologous tumour cells

Summary We have studied the effect of polysaccharide K (PSK) in the in vitro recognition of ex vivo carcinoma, sarcoma and lymphoma cells by the autologous blood lymphocytes. In 4/25 experiments PSK treatment activated the lymphocytes for auto-tumour lysis. Tumour cells alone generated lytic activity both in short- (16 h) and in longterm (6 days) mixed lymphocyte/tumour cell cultures (MLTC), in 2/12 and 3/13 cases respectively. The tumours that activated the lymphocytes expressed high levels of major histocompatibility complex (MHC) class I molecules. In vitro cytokine (interferon and tumour necrosis factor ) treatment of the tumour cells elevated the amounts of class I antigens and the treated cells acquired stimulatory potential. When PSK was added to the MLTC, in which untreated tumour cells were used, lytic potential was induced in 9/13 short-term and in 11/12 long-term cultures. It is noteworthy that in the presence of PSK the untreated, negative or low-class-I-expressor tumours also activated the cytotoxic function of the lymphocytes in 4/5 long-term and in 6/7 short-term cultures. Even in the case of those lymphocytes that could be activated by PSK or tumour cells alone, the simultaneous exposure was more efficient. The effect of PSK was dose-dependent, being optimal at 1 µg/ml and 10 µg/ml. The presence of EDTA and/or cytochalasin B in the cytotoxic test performed with the activated effectors abrogated the lysis, indicating the requirement of contacts with the effector cells.     

Phorbol esters inhibit the functional activation of cytotoxic precursors in mixed lymphocyte cultures

We have investigated the effect of phorbol esters on T cell activation and generation of suppressor and cytotoxic activity in mixed lymphocyte cultures (MLC). The presence of 30 nM P(Bu)2 during the sensitization phase inhibited the generation of allospecific cytotoxicity and also decreased the killing potential against NK-sensitive targets. The inhibition was not mediated by direct blocking of the lytic capacity nor by suppression of clonal expansion of cytotoxic cells through modulation of lymphokine production. The presence of P(Bu)2 enhanced cell proliferation, but inhibited the functional activation of lymphocytes and consequent generation of Dr antigen-positive T cells. Because the presence of the compound did not affect the MLC-induced generation of suppressor activity, it is likely that P(Bu)2 selectively blocks the maturation of cytotoxic precursors. Surface-marker analysis with OKT monoclonal antibodies revealed that the effects on lymphocyte activation were associated with a decrease in OKT3 and OKT4 reactivity and an increase in the percentage of OKT8-positive cells. The decrease in OKT4 reactivity was not due to selective loss of this lymphocyte subpopulation, because P(Bu)2 was equally mitogenic for the purified OKT4- and OKT8-positive subsets. The results suggest that the effect of P(Bu)2 on cell differentiation and its ability to modulate the expression of functional markers in lymphocyte subsets may interfere with T-T cell cooperation that controls the functional maturation of cytotoxic precursors.     

Serum levels of the low-affinity interleukin-2 receptor molecule (TAC) during IL-2 therapy reflect systemic lymphoid mass activation

Summary Expression of the low-affinity interleukin-2 (IL-2) receptor molecule (TAC) has been associated with lymphocyte activation, in vitro and in vivo [Greene WC (1987) Clin Res 35:439]. We have used an enzyme-linked immunosorbent assay (ELISA) to quantify the role of released and cell-bound IL-2 receptor following in vitro or in vivo activation of human lymphocytes with IL-2. In vitro experiments, culturing fresh peripheral blood lymphocytes in 30 U/ml IL-2 (corresponding to the steady-state IL-2 concentration achieved in patients receiving IL-2 in our clinical trials), showed that the levels of IL-2 receptor released into the culture media exceeded the levels of cell-associated receptor, with both rising in parallel to the cytotoxic activity of the peripheral blood lymphocytes (PBL) against cultured tumor cells. In 12 patients receiving high-dose IL-2 for the treatment of various malignant neoplasms, the levels of IL-2 receptor released into the serum rose dramatically during the IL-2 infusion, and then fell following cessation of the IL-2 infusion. This heightened release of IL-2 receptor into the serum occurred during the episodes of profound lymphopenia that developed within hours after patients began an IL-2 infusion. Following each 4-day infusion of IL-2, a rebound lymphocytosis was observed, as has been previously reported. Serum IL-2 receptor levels do not rebound in parallel; rather, they reach a plateau near the end of the 4-day infusion and then decrease upon cessation of IL-2. These changes in serum IL-2 receptor levels accompany changes in lytic activity of circulating PBL on Daudi target cells. These results suggest that lymphocyte populations exposed to IL-2 in vivo are activated to become cytotoxic, release TAC, and relocate in non-peripheral blood compartments. Following cessation of the IL-2 infusion these activated lymphocytes return to the peripheral circulation and do not secrete TAC as vigorously as while influenced directly by the IL-2 infusion.This research was supported by NIH contract CM-47669, NIH grants CA-32685, and RR-03186, American Cancer Society CH-237B, and a research fellowship of the Leukemia Society of America (P. Fisch), a Ewing Foundation Fellowship (S. Voss) and Lutheran Brotherhood/Life and Health Insurance Medical Research Fund M. D./Ph. D. Training Fellowship (S. Voss)     

Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons

Summary Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic acitvity against the YAC-1 lymphoma cell line and to a lesser extent against P815 mastocytoma cells. The association of IL-2 and LPS had an additive effect on induction of cytotoxicity. The IL-2-induced cytotoxic activity lasted for the whole of the culture; however, the addition of LPS at the initiation of the culture increased the cytotoxic activity during its the early phase, the increment being followed by a fall of lytic activity after 72 h of culture. Assessment of interferon (IFN) in the culture supernatants showed (a) a production of IFN by IL-2-supplemented cultures, (b) a more potent IFN production by cultures treated with IL-2 plus LPS (including 20% IFN/), (c) and that indomethacin (1µm) potentiated the effect of either IL-2 or LPS used alone but did not significantly increase the cytotoxic activity of cultures treated with IL-2 plus LPS (the one that produced a high level of IFN). When cultures were treated by an anti-IFN antibody we observed no change in the cytotoxic activity; however, in the presence of anti-IFN/ serum the cytotoxic activity of cultures treated with IL-2 plus LPS was inhibited after 24 h but stimulated after 72 h. When cultures treated with IL-2 plus LPS were supplemented with both indomethacin and anti-IFN/ the cytotoxic activity assessed after 72 h of culture was maintained at the same level as that of IL-2-treated cultures, hence the fall after 72 h of the cytotoxicity of cultures initiated in the presence of LPS alone was affected by both the immune serum and the cyclooxygenase inhibitor. Altogether these data show that when splenocytes are cultured for more than 72 h in the presence of IL-2 and LPS their cytotoxic activity decreases, and it is likely that this diminution is linked to the endogenous prodution of prostaglandin E₂ and IFN/.     

Inhibition of cytotoxic responses by prostaglandin E2 in the presence of interleukin 2

Experiments are described which provide direct evidence for a strong suppressive effect of prostaglandin E₂ (PGE₂) on the activation of cytotoxic T lymphocytes (CTL). The experiments also tested the hypothesis that PGE₂ inhibits cytotoxic responses exclusively by preventing the helper T cells from producing interleukin 2 (IL-2). In support of this hypothesis we found that indomethacin enhanced cytotoxic responses in the absence but not in the presence of concanavalin A-activated spleen cell supernatant (CSCS) indicating that the endogenously produced prostaglandin(s) inhibited primarily the production of IL-2. However, the addition of PGE₂ to microcultures was found to inhibit in a dose-dependent manner the activation of cytotoxic responses against I-region compatible stimulator cells even in the presence of CSCS. This inhibition was not observed in the presence of I-region incompatible stimulator cells, indicating that the inhibitory effect of PGE₂ did not result from a general toxic effect on the culture. The results argue against the notion that PGE₂ inhibits cytotoxic responses exclusively by preventing the production of IL-2.     

Functional characteristics of the intercellular adhesion molecule-1 (CD54) expressed on cytotoxic human blood lymphocytes

We have shown that intercellular adhesion molecule-1 (ICAM-1) (CD54) positive cells are mainly responsible for the natural cytotoxic function of human blood lymphocytes. The evidences were the inhibition of cytotoxicity by anti-ICAM-1 (LB-2) monoclonal antibodies (mAb) and the loss of lytic activity after removal of the ICAM-1+ cells. In addition, the cytotoxic potential of the separated ICAM-1- lymphocyte population after activation appeared in parallel with the expression of this molecule. The ICAM-1+ lymphocytes lysed both LFA-1 (CD11a/CD18 or Leu-CAMa) positive and negative cell lines, and pretreatment of the effectors with the LB-2 mAb also inhibited the lysis of LFA-1- targets. The results point to a yet unrecognized role of ICAM-1 on the lymphocytes. Kinetics experiments suggested that pretreatment of lymphocytes with alpha-ICAM-1 (LB-2) mAb did not inhibit the promptly established lytic interactions but influenced later events, recycling and/or recruitment of effectors. It is possible that the cytotoxic potential is regulated by contacts between the members of the lymphocyte population and that these events occur via their ICAM-1 and LFA-1. Exposure of lymphocytes to NK-sensitive targets for 16 hr elevated their cytotoxic potential. The function of activated lymphocytes was not inhibited by the LB-2 mAb.     

Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retarets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8⁺ and CD4⁺ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V11, in particular CD8⁺ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.     

Activation of human blood lymphocyte subsets for cytotoxic potential

Blood lymphocytes of individuals differ in the spontaneous cytotoxic potential exerted in vitro against certain cell lines (natural killing, NK). In the low NK donors, the activity can be enhanced by short-term IFN pretreatment of the effectors (interferon activated killing, IAK) and by addition of PHA to the short-term assay (lectin-dependent cellular cytotoxicity, LDCC). Lymphocyte subpopulations fractionated on the basis of nylon adherence, SRBC, and EA rosette formation differ in their response to these measures. The results obtained with IFN-treated lymphocytes of low NK donors were similar in strength to the spontaneous activity of the high NK donors. Therefore, the distinction between NK and IAK is only operational. The nylon passed E receptor-negative and low-avidity E receptor-positive cells had the strongest NK activity. These subsets can be triggered for enhanced activity by IFN. In the majority of the cases the high-activity E-receptor-positive subset which did not sediment with EA indicators had low NK effect and was not triggered by IFN. Addition of PHA to the lytic assay, however, induced activity in the subset. Realization of DNA synthesis was not necessary for the lytic performance. The PHA-imposed triggering event was not dependent on IFN production nor on induction of the competence for IFN response. The results showed that all non-B lymphocyte subsets separated on the basis of nylon wool adherence, SRBC, and EA rosetting contain cells with lytic potential if the appropriate stimulus is used. The relative activities of the subsets against K562 and Daudi differed. Cells which rosetted readily with EA indicators had weak effect against Daudi.     

B-Lymphocyte Proliferation during Bovine Leukemia VirusInduced Persistent Lymphocytosis Is Enhanced by T-Lymphocyte-Derived Interleukin-2

Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5⁺ B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-γ) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Comparison between crude Interleukin-2 and recombinant Interleukin-2 in maintaining killing activity of cultured lymphocytes

Peripheral blood lymphocytes, regional lymph node lymphocytes or malignant effusion lymphocytes from cancer patients were incubated with crude IL-2 (cIL-2) for 13 days. These effectors, which frequently expressed IL-2 receptor (IL-2R), proliferated well and possessed augmented killing activity against fresh autologous tumor cells and K562. However, when recombinant IL-2 (rIL-2) was added for the last 4 days of culture instead of cIL-2, IL-2R expression and killing activity against fresh autologous tumor cells decreased significantly (P<0.05). Phenotypic analysis indicated that cIL-2 significantly promoted the expansion of the cytotoxic population (CD8⁺ .11b^–)(P<0.05). The decreases in killing activity and IL-2R expression were restored by 0.004% PHA plus rIL-2, but not in the presence of rIFN-, rIL-1, rIL-l, rIL-4 or rIL-6. PHA-free cIL-2 maintained killing activity, but not IL-2R expression.We conclude that some factors in cIL-2 and a low dose of PHA-P are necessary for the maintenance of killing activity and IL-2R expression of cultured lymphocytes in the late phase of culture.     

Differential Regulation of HLA-DR Expression and Antigen Presentation in Toxoplasma gondii-Infected Melanoma Cells by Interleukin 6 and Interferon γ

CD4⁺ cytotoxic T lymphocytes (CTL) clones, YT-4 and YT-9, specific for Toxoplasma gondii (T. gondii)-infected melanoma SK-MEL 28 (P36), were generated from the peripheral blood lymphocytes (PBL) of a patient with chronic toxoplasmosis. These CTL clones were shown to secrete significant amounts of interleukin 6 (IL-6) and interferon γ (IFN-γ) upon antigen (Ag)-specific stimulation. Downregulation of human leukocyte antigen (HLA)-DR surface expression and HLA-DR mRNA levels in P36 cells were observed when P36 cells were infected with T. gondii. Such downregulated HLA-DR expressions of 71 gondii-infected P36 cells were upregulated by treatment with both recombinant IL-6 (rIL-6) and recombinant IFN-γ (rIFN-γ). The antigen-presenting ability of T. gondii-infected P36 cells to T. gondii-infected cell-specific CTL was enhanced by rIFN-γ but not by rIL-6. The present study reveals the existence of differential regulation of HLA-DR expression and Ag presentation in T. gondii-infected melanoma cells by IL-6 and IFN-γ.     

Interleukin 2 enhances natural killing of normal lymphocytes

Purified Interleukin 2 (IL-2), free of interferon (IFN), significantly enhanced NK activity of normal human peripheral blood mononuclear cells (PBMC). This enhancing activity was absorbed by IL-2 receptor-bearing cells but was not blocked by antibody to α-IFN. IFN in the culture supernatants was greatly increased after stimulation with poly(I:C) plus IL-2. There was less IFN produced by either modulator acting alone. Stimulation of PBMC with IL-2 and/or poly(I:C) increased the proportion of OKM1⁺ cells and anti-Leu-7⁺ cells. When cells expressing either surface antigen were specifically lysed to deplete NK, cytotoxic activity could be restored by overnight incubation in IL-2. This result suggests that IL-2 stimulates the development of NK cells from precursors that lack cell surface OKM1 or Leu-7. IL-2 acted directly on large granular lymphocytes and did not require the presence of adherent cells. These results suggest that IL-2 may act synergistically with other IFN inducers and may play an important role in the regulation of NK cells.     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Differential effect of cyclosporin-A on the mixed-lymphocyte culture-induced proliferative and cytotoxic responses of T lymphocytes with different cell densities

We have analyzed the effect of cyclosporin-A (CsA) on the proliferative and cytotoxic responses induced by mixed-lymphocyte cultures (MLC-s) on low and high density T lymphocytes. Allogeneic stimulation had a different impact on the two subsets. Proliferative and cytotoxic responses were inversely correlated; i.e., high density cells proliferated but exerted low levels of cytotoxicity while the lytic activity of the low density subset was stronger and the proliferation was weak. CsA impaired the proliferative and cytotoxic responses of the high density T lymphocytes but influenced less markedly the response of the low density cells. In both subsets CsA inhibited the MLC-induced interleukin 2 (IL-2) production. The generation of specific cytotoxicity was markedly suppressed by CsA, whereas the generation of anomalous activity was less affected. Addition of exogenous IL-2 to the CsA-containing cultures fully restored the proliferation and the generation of nonspecific cytotoxicity. In contrast, addition of interferon gamma (IFN-gamma) restored neither of these responses. However, for complete restoration of the stimulation-specific cytotoxicity, addition of both lymphokines was required. Taken together these results suggest that the CsA-induced suppression of the lymphokine production has different consequences in the low and high density subsets; the expression of anomalous and specific cytotoxicities require different signals; CsA interferes with several steps in the T-cell activation.     

IL-2- and IFN gamma-enhanced natural cytotoxic activity: analysis of the role of different lymphoid subsets and implications for activation routes

To elucidate the immunoregulatory mechanisms by which human recombinant interleukin 2 (r.IL-2) and human recombinant interferon (IFN)-gamma influence natural cytotoxic activity of human peripheral blood lymphocytes (PBL), experiments were performed in which we studied: the kinetics of augmentation of natural cytotoxic activity by IL-2 and IFN-gamma; the phenotypes of the natural cytotoxic precursor cells acted upon by IL-2 and IFN-gamma; the role of IL-2-induced IFN-gamma in the natural cytotoxic activity enhancing effects of IL-2 and (d) the surface phenotypes of the natural cytotoxic cells activated by IL-2 or IFN-gamma. Three phenotypical distinct precursor cells can be identified. The phenotypes of the precursor cells sensitive to r.IFN-gamma are T3-, Leu 7+or-, FcR-gamma+. The phenotypes of the precursor cells sensitive to r.IL-2 are T3-, Leu 7-, FcR-gamma+or-. It appears that during prolonged culture relatively more of the FcR-gamma- precursor cells acquire natural cytotoxic activity upon stimulation with r.IL-2. Antibodies to IFN-gamma, known to neutralize both recombinant and natural IFN-gamma, completely inhibited the IFN-gamma-induced augmentation of natural cytotoxic activity but only slightly affected the IL-2-mediated augmentation. This indicates that only part of the augmenting effect of r.IL-2 is mediated through IL-2-induced IFN-gamma. This implies that r.IL-2 augments natural cytotoxic activity predominantly through an IFN-gamma-independent pathway. The phenotype of the effector cells expressing natural cytotoxic activity after a 4-day culture period with IL-2 or IFN-gamma was analyzed by depletion experiments. Virtually all the cells that exerted the IFN-gamma-enhanced natural cytotoxic activity are FcR-gamma+, whereas a portion of the cells that exerted the IFN-gamma-enhanced natural cytotoxic activity are FCR-gamma-. Both natural killer cells and lymphokine-activated killer cells contribute to natural cytotoxic activity. The relative contributions of both activities to the natural cytotoxic activity augmented by IL-2, respectively, IFN-gamma, is discussed.     

Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta.

The effect of transforming growth factor-beta 1 (TGF-beta) on activation-induced CD8+ T cell cytotoxicity and gene expression was investigated. TGF-beta was demonstrated to inhibit pore-forming protein (PFP) mRNA expression and total benzoyloxycarbonyl-L-lysine thiobenzyl ester esterase activity in CD8+ T cells cultured with IL-2 and OKT3 mAb for 6 to 18 days. Consistently, in the absence or presence of TGF-beta, the PFP mRNA expression and lymphokine-activated killer (LAK) activity of CD8+ T cells were closely correlated. The inhibitory effects of TGF-beta on both CD8+ T cell PFP mRNA expression and LAK activity were reversible by removal of TGF-beta from the culture. Expression of lymphokines, adhesion/recognition molecules, and activated p55 IL-2R, previously implicated in the lytic mechanism of cytotoxic lymphocytes, either was not detectable or did not correlate with TGF-beta inhibition of LAK activity. In addition, independently of effector/target cell binding, the lectin- or heteroconjugated antibody-dependent cellular cytotoxicity of IL-2/OKT3 mAb-activated CD8+ T cells was inhibited by preculture with TGF-beta. TGF-beta also inhibited the rapid activation-induced expression of PFP mRNA and cytotoxic potential in resting T cells, thereby indicating that the effect of TGF-beta was independent of T cell proliferation. TGF-beta inhibition of CD8+ T cell PFP mRNA expression and cytotoxic potential was TGF-beta dose dependent; however, a variety of activation stimuli (including IL-2, IL-6, and OKT3 mAb) were all similarly inhibited by TGF-beta. Therefore, TGF-beta may be an important general regulator of CD8+ T cell cytotoxic function, in particular by suppressing expression of PFP, a major cytolytic protein implicated in the lytic function of cytotoxic lymphocytes.