Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.     

In vitro depression of cellular immunity by Friend virus leukemic spleen cells.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Human lymphocyte responses are enhanced by culture at 40 degrees C.

In vitro responses of human peripheral blood lymphocytes (PBL) were found to be markedly enhanced by culture at 40 degrees C rather than at the conventional temperature of 37 degrees C. We studied proliferative responses of lymphocytes by activation by phytohemagglutinin (PHA) concanavalin A (Con A), pokeweed mitogen (PWM), and allogeneic lymphocytes in mixed lymphocyte culture (MLC) and found enhancement of DNA synthesis at the higher temperature. Cytotoxic T cell responses to allogeneic cells were also enhanced when MLC was done at 40 degrees C. These enhanced immune responses appear to be due in part to increased numbers of participating cells. If in vitro lymphocyte responses correlate with in vivo responses, then fever associated with infection or tumor may be beneficial whereas that associated with autoimmune disorders may have a detrimental effect.     

Lymphocyte cytotoxicity to influenza virus-infected cells. II. Requirement for antibody and non-T lymphocytes.

Peripheral blood lymphocytes (PBL) obtained from humans were cytotoxic for influenza virus-infected target cells. The PBL were shown to have associated influenza virus anti-hemagglutinin antibody (AHAb) detectable only by radioimmunoassay. This antibody could be removed by incubating PBL at 37 degrees C for 30 min. The lymphocyte population that was effective in this system was nonadherent and nonphagocytic cells. PBL gave comparable levels of cytotoxicity when tested by using either a xenogeneic or allogeneic virus-infected target cell. These results indicate that lymphocyte cytotoxicity to influenza virus infected cells may be mediated by small quantities of antibody and by lymphocytes that possess characteristics of K cells. No evidence for T cell-mediated cytolysis was found with this xenogeneic system.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Mechanisms of lymphocyte-mediated cytotoxicity. I. The effects of anti-human lymphotoxin antisera on the cytolysis of allogeneic B cell lines by MLC-sensitized human lymphocytes in vitro

Goat and rabbit anti-human lymphotoxin sera, IgG and F(ab')2 reagents were investigated for their capacity to effect a specific alloimmune lymphocyte-mediated cytotoxic reaction. The cytotoxic reaction employed human peripheral blood or adenoid lymphocytes sensitized in MLC to allogeneic B lymphocyte cell lines and lysis was measured in a short-term 51Cr-release assay. A polyspecific anti-LT sera (anti-WS), made against unfractionated whole supernatants from lectin-activated lymphocytes and its IgG and F(ab')2 fragments, was found to be a potent inhibitor of this reaction when the anti-WS reagent was present throughout the assay period. Absorption studies indicated the anti-WS was inhibiting cytolysis at the level of effector cell or its products. Two broadly defined antibody specificities were involved in the cytolytic-inhibitory activity of the polyspecific anti-LT; i) antigens present on the normal lymphocyte cell surface; and ii) lymphocyte surface antigens associated with activated cells. These results correlate with the previously defined antigenic structure of the LT Cx and alpha H classes. Anti-LT sera reactive with the smaller m.w. alpha and beta classes and subclasses were not inhibitory, although the anti-beta sera showed a moderate enhancing activity. The results indicated that several anti-LT antibody specificities may be required to inhibit alloimmune cytolysis. The results suggest LT molecules may mediate lymphocyte-induced alloimmune cytolysis as a multi-component toxin system, rather than as an individual toxin.     

Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, programming, and killer cell-independent steps

Various parameters of the cytolytic reaction mechanisms of the human natural killer (NK) lymphocyte were studied to characterize the lytic cycle. NK cytolysis was determined to occur in three definable steps. 1) Binding of PBL to the NK-sensitive targets Molt-4 or K562 was rapid (less than 1 min), occurred at temperatures below 37 degrees C, was Mg++3-dependent, Ca++3-independent, and was prevented by dispersion of the cells into 10% dextran. 2) Subsequent to binding, programming for lysis as determined by a Ca++ pulse method was more protracted, requiring up to 2 hr to occur and was strictly dependent on Ca++ for cytolysis to proceed. In standard cytotoxicity assays, however, programming for lysis was more rapid occurring in 10 to 30 min. Programming was inhibited by EDTA, EGTA/Mg++ and by temperatures below 37 degrees C. Furthermore, after binding but in the absence of initiation of programming for lysis, the frequency of target binding cells did not change and the NK cell did not lose its lytic potential. 3) Killer cell-independent cytolysis (KCIL) was determined by the addition of EDTA to "programmed" targets and dispersion of these cells into dextran-containing medium, which resulted in virtually 100% dissociation of conjugated cells. KCIL was Ca++ and Mg++-independent and was blocked at reduced temperatures only if the dextran was prechilled to 4 degrees C before addition. The kinetics of 51Cr release during KCIL was rapid and complete 30 min after dispersion. Interferon-activated NK cells expressed an increased rate of cytolysis in Ca++ pulse experiments. This was due to an increased rate of the Ca++-dependent step(s) during the programming events. The rate of the Ca++-independent steps, however, were similar with control and IFN-activated cells.     

DNA strand break rejoining in human lymphocytes during the S phase

Induction and repair of DNA strand breaks in asynchronous and synchronized cultures of human lymphocytes was investigated by using the alkaline DNA-unwinding technique followed by chromatography on hydroxylapatite. Strand break rejoining in exponentially growing human PHA stimulated lymphocytes, irradiated with 20 Gy of X-rays, is temperature-dependent, being fast at 37 degrees C (half-time of a few minutes), and very slow at around 4 degrees C. In synchronized cells irradiated with the same X-ray dose, the repair capacity increases during S phase reaching its maximum when DNA is entirely duplicated.     

Preliminary lymphocyte activation by a mitogen as a condition for demonstrating the ability of the Fab-fragment of normal IgG to inhibit lymphocyte transformation

Peculiarities attending inhibition of the PHA-induced blast-cell transformation of human lymphocytes by F(ab')2 fragment of rabbit IgG were studied. It was shown that the fragment did not affect the intensity of blast-cell transformation if the lymphocytes were preliminarily incubated with the fragment for 24 h at 37 degrees or 4 degrees C and then transferred to the fresh medium containing PHA. However, if the fragment was added to the cells 24 or 48 h following PHA it produced a significant inhibition of the blast-cell transformation. These data may indicate that F(ab')2 fragment interferes with the lymphocyte transformation only when the cells are already activated with PHA.     

Lymphocyte chromatin changes in Down's disease detected by heat denaturation]

By using fluorescent microscopy and acridine orange staining it was shown in the studies on short-term culture of human cells that the melting patterns of chromatin DNA of intact lymphocytes of healthy individuals represented the curves with 6 maxima (F530) at the temperature ranges of 45 degrees C, 65 degrees C, 85 degrees C, and 92 degrees C (P less than 0.01). The melting patterns of lymphocytes from patients with Down's disease represented curves with 4 maxima at the temperature ranges of 65 degrees C, 85 degrees C, 88 degrees C, 92 degrees C (P less than 0.01). No decline in the fluorescence intensity at the temperature intervals of 78 degrees C-85 degrees C was apparently due to a greater degree of condensation of definite regions of the trisomal cell chromatin complex. Possible mechanisms accounting for structural readjustments of the interphasic human lymphocyte chromatin occurring under thermal effects are discussed.     

Subpopulations of human thymus cells differing in their capacity to form stable E-rosettes and in their immunologic reactivity.

The majority of human thymus cells from young donors form stable E-rosettes with sheep red blood cells (SRBC) that do not distintegrate after prolonged incubation at 37 degrees C. With advancing age the proportion of thymus cells forming such rosettes decreases gradually. The thymus of a patient receiving prednisone treatment was found to contain only a few cells that formed stable E-rosettes. The minor population of thymus cells that fails to form stable E-rosettes (non-rosetting or NR cells) was isolated and tested for its cell surface markers and immunologic reactivity in vitro. Most of the NR-cells were capable of forming regular E-rosettes with SRBC at room temperature. Like the majority of human thymus cells they were sensitive to the cytotoxic effect of normal constituted less than 0.2% of the original thymus cell suspensions, but about 1 to 3% of the NR-population. Thymus cells from donors over the age of 36 and from a prednisone-treated child responded in vitro to stimulation with either phytohemagglutinin (PHA) or concanavalin A (Con A). Unfractionated thymus cells from children up to the age of 14 failed to react to either PHA or Con A, but their NR-population responded vigorously to both lectins. In contrast to unfractionated thymus cell suspensions from children, the NR fraction showed a significant reactivity in mixed lymphocyte cultures with mitomycin-C treated allogeneic lymphocytes. It is concluded that like the thymus of other species, the human thymus contains a minor population of cortisone-resistant cells endowed with many of the immunologic properties characteristic for periperal T lymphocytes.     

Oxygen consumption by tumor cells during membrane vesicle shedding

The incubation of mastocytoma P815 cells at low temperature (0 degrees C/1-2 hr), with a subsequent shift to greater than or equal to 20 degrees C results in the formation and shedding of membrane vesicles from the tumor cell surfaces. This process, when occurring at physiologic temperature (37 degrees C), mimics the morphological and membrane permeability changes occurring during T-lymphocyte mediated cytolysis of tumor cells. The latter is an oxygen dependent event, but it is not known whether this requirement is at the effector T cell or at the tumor cell level. The present study investigated the oxygen consumption rates of mastocytoma P815 cells induced to shed membrane vesicles by a temperature shift (0 degrees C/1-2 hrs----greater than or equal to 20 degrees C). Results showed that cells undergoing the membrane vesicle shedding process had significantly higher oxygen requirements than control non-shedding cells. Inhibition of the shedding process with deuterium oxide and hexylene glycol, reduced the oxygen consumption rates of low temperature treated cells to the level of control cells. The oxygen consumption rates of the latter were unaffected by these microtubule stabilizing agents. These data indicate that the oxygen required for immune T-cell mediated lysis of tumor cells may be at the target tumor cell level.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. I. Resolution and characterization.

Addition of high molecular weight dextran to culture medium prevents the initiation of T lymphocyte-mediated killing by holding the cytolytic T lymphocytes (CTL) and target cells in suspension and preventing intercellular contact. Suspension in 10% dextran was used to interrupt the ongoing formation of adhesions between CTL and target cells already in contact in a centrifuged pellet. The results demonstrate that 1) firm adhesions form between CTL and target cells within 1 min at 37 degrees C; 2) once formed, these adhesions are stable at low temperature and are resistant to mechanical shearing forces; 3) these adhesions can be disrupted by EDTA; 4) immediately after the adhesions form, separation of the CTL from the target cells prevents lysis of the latter; 5) after incubation of targets adhering to CTL for an additional 6 min at 37 degrees C, removal of the CTL no longer prevents target cell lysis. Thus, target cells become "programmed" for subsequent lysis within a few minutes after contact with CTL, after which lysis occurs during the next several hours without further participation of the effector cell. At 15 degrees C, adhesions form 1/17 as fast as at 37 degrees C. Programming of target cells for lysis occurs 1/76 as fast at 15 degrees C as at 37 degrees C. Thus, the programming for lysis step is about 4-fold more temperature dependent than the adhesion step. In addition to being detected by subsequent target cell lysis in 10% dextran, the adhering cell clusters can be counted with low power microscopy. This permitted verification that EDTA separates the clusters after programming for lysis is complete. Moreover, the great majority of the clusters seen at 37 degrees C are antigen-specific. Knowledge of the cluster size distribution and the subsequent level of lysis permits the deduction that not less than 6% of the sensitized peritoneal cell populations used were CTL.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

Immunomodulation of human leukocytes by staphylococcal enterotoxin A: augmentation of natural killer cells and induction of suppressor cells

Staphylococcal enterotoxin A (SEA), a protein isolated from culture supernatants of Staphylococcus aureus, is a potent T-cell mitogen and an inducer of interferon-gamma (IFN-gamma). We report here that SEA exhibits a number of significant in vitro immunomodulatory functions. In vitro treatment of human peripheral blood monocyte-depleted lymphocytes with SEA resulted in significant augmentation of their natural killer cytotoxicity against target cells from hemopoietic (K562, Daudi) or solid (melanoma, lung, colon) human tumor cell lines. SEA was found to be more effective than interferons-alpha (natural or Escherichia coli-derived) in augmenting natural killer (NK) cytotoxicity of peripheral blood lymphocytes. Studies on the kinetics of the augmentation revealed a significant increase of NK within 3 hr of in vitro treatment with SEA at 37 degrees C. A neutralizing monoclonal antibody specific for human IFN-gamma did not affect the augmentation of natural killer cytotoxicity by SEA, suggesting that SEA augmented natural killer cytotoxicity primarily by a mechanism not involving induction of interferon-gamma. Furthermore, in vitro treatment with SEA resulted in significant augmentation of antibody-dependent cell-mediated cytotoxicity and of natural killer-like cytotoxicity, generated in mixed lymphocyte culture, against the K562 targets. Induction of suppressor cells to proliferative responses of autologous or allogeneic mononuclear cells to phytohemagglutinin (PHA) or to allogeneic cells in mixed lymphocyte culture was observed after in vitro treatment of peripheral blood mononuclear leukocytes with SEA for 24 or 48 hr at 37 degrees C. In addition, the presence of SEA in mixed lymphocyte cultures (MLC) resulted in significant inhibition of the generation of specific T-cell-mediated cytotoxicity in MLC. These results suggest that SEA, which may be involved in S. aureus infections and in treatment with extracorporeal perfusion systems over S. aureus columns, can regulate a number of significant lymphoid functions.     

The human LT system. VII. Release of soluble forms and delivery to target L-929 cells by lectin-preactivated human lymphocytes in the absence of protein synthesis and secretory processes.

We have investigated the effects of inhibitors of cellular protein synthesis (emetine, cycloheximide) and secretion (colchicine, cytochalasin B) on the capacity of primary or secondary lectin-activated human lymphocytes to release LT molecules or to cause lectin-induced destruction (LICC) of murine L-929 cells in vitro. Our findings reveal: (a) agents which inhibit protein synthesis or secretion block the release of LT activity into the supernatant and LICC when primary lectin-stimulated human adenoid lymphocytes are employed as effector cells; (b) these same agents are ineffective at blocking LT release or LICC when 3- or 5-day lectin-prestimulated lymphocytes are employed; and (c) anti-human α-LT serum blocks LICC of L-929 cells mediated by primary or secondary lectin-activated human lymphocytes. The difference in participation of effector cellular processes in LICC between primary and secondary lectin-stimulated cells correlates with the findings that preactivated lymphoid cells possess high levels of preformed intracellular, as well as membrane associated, LT molecules, and that release of these materials into the supernatant or delivery to the target cell can occur independently of active protein biosynthesis or classical secretory systems.     

Studies on the mechanism of the human natural killer cell lethal hit: a new method for rapid physical isolation of lethally hit K562 targets and inhibition of cytolysis by reduced temperature or trypsin

A new method was developed which allows for rapid (2 min) physical isolation of viable K562 target cells after being programmed to lyse (lethally hit) by purified human natural killer (NK) cells (LGL). To achieve this K562 cells which were obtained from the 34-36% interface of discontinuous Percoll gradients and purified human NK cells (LGL) which were obtained from the (43-45% Percoll) interface were employed. Using a Ca2+ pulse method and the separation of NK-K562 conjugates with EDTA and rapid centrifugation on Percoll gradients at 4 degrees C we could physically isolate the lethally hit K562 cells from the LGL allowing the study of the events leading to their subsequent lysis. Lysis of "purified" lethally hit K562 cells occurred in the absence of Ca2+ or Mg2+ and was blocked by reduced temperature (4 degrees C), or by the protease enzyme trypsin. When lethally hit targets were held at 4 degrees C (to block lysis) then rewarmed to 37 degrees C lysis ensued but with a rate slower than that of control cells not held at 4 degrees C. These data support the concept that transfer of protease-sensitive and possibly temperature-dependent structures from the NK cell to the target is a requisite step in NK cytolysis.     

In vitro lymphocyte proliferation induced by radio-frequency electromagnetic radiation under isothermal conditions

Whole human blood was exposed or sham-exposed in vitro for 2 h to 27 or 2,450 MHz radio-frequency electromagnetic (RF) radiation under isothermal conditions (i.e., 37 +/- 0.2 degrees C). Immediately after exposure, mononuclear cells were separated from blood by Ficoll density-gradient centrifugation and cultured for 3 days at 37 degrees C with or without mitogenic stimulation by phytohemagglutinin (PHA). Lymphocyte proliferation was assayed at the end of the culture period by 6 h of pulse labeling with 3H-thymidine (3H-TdR). Exposure to radiation at either frequency at specific absorption rates (SARs) below 50 W/kg resulted in a dose-dependent, statistically significant increase of 3H-TdR uptake in PHA-activated or unstimulated lymphocytes. Exposure at 50 W/kg or higher suppressed 3H-TdR uptake relative to that of sham-exposed cells. There were no detectable effects of RF radiation on lymphocyte morphology or viability. Notwithstanding the characteristic temperature dependence of lymphocyte activation in vitro, the isothermal exposure conditions of this study warrant the conclusion that the biphasic, dose-dependent effects of the radiation on lymphocyte proliferation were not dependent on heating.