Inhibitory activity of medium-dialyzed transfer factor on lymphocyte blastogenesis.

Human transfer factor was prepared from normal donors with marked skin reactivity against common microbial antigens by dialysis of lymphocyte extracts versus tissue culture medium (TFmd). Several batches of TFmd were tested for their ability to specifically increase the DNA synthesis of unsensitized lymphocytes in vitro in the presence of the corresponding antigens (PPD, SKSD, Candida, Thistoplasmin). No transfer or immunologic reactivity by TFmd was observed. There was, however, a significant unspecific inhibition by TFmd of lymphocyte cultures stimulated by antigens, PHA or allogeneic cells.     

Lymphocyte responses to syngeneic antigens. I. Enhancement of the murine autologous mixed lymphocyte response by polyethylene glycol

The normally weak murine T-cell proliferative response against autologous non-T stimulator cells (the autologous mixed lymphocyte culture (MLC) was enhanced markedly by inclusion of the hydrophilic polymer, polyethylene glycol (PEG), into the culture medium. Potentiation of the autologous MLC was indicated on the basis of increased [³H]TdR incorporation by responding cells, as well as by the numbers of viable cells recovered from mixed cell cultures. PEG is not a polyclonal activator of T and/or B lymphocytes, since nylon wool nonadherent lymphoid cells (T cell-enriched fraction), nylon wool adherent cells (B cell-enriched fraction) and T cell-deficient “nude” spleen cells were not stimulated into DNA synthesis when cultured separately with PEG. Inclusion of 4% PEG into the culture medium was found to optimally enhance autologous MLC, although concentrations between 2 and 5% also significantly elevated responsiveness. At a responder/stimulator ratio of 1:2, autologous MLC yielded peak [³H]TdR incorporation after 5 days of culture. At lower ratios (1:1 and 2:1), however, Δ cpm of autologous MLC continued to increase over a culture period of 7 days. Enhanced responsiveness in the presence of PEG was observed in strains of mice representing a variety of H-2 haplotypes, indicating that at least the potential for autoreactivity of this type is a naturally occurring and widespread characteristic of murine species. An absolute requirement for purified T responder cells was necessary in the autologous MLC, since unseparated lymphoid cell responder LN or spleen cells demonstrated marked proliferation when cultured alone in medium containing PEG. The proliferation of T cells to autologous non-T cells within the same unseparated lymphoid cell preparation appears to be responsible for this phenomenon. Ia antigens expressed by the stimulator cells are involved in the induction of T-cell response, since anti-Ia sera added directly to the cultures inhibited the autologous MLC, but did not affect other T-cell responses to alloantigens or mitogens. Despite the marked proliferation observed in the autologous MLC performed in the presence of PEG, there was no generation of cytotoxic effector cells. Thus, PEG does not appear to add, or alter determinants on stimulator cells to an extent that they are recognized as foreign by precursor cytotoxic T cells. Although the mechanism of enhancement of autologous MLC by PEG is not totally defined, it appears, at least functionally, to promote cellular interactions that occur normally between T cells, B cells, and macrophages. In this respect, PEG will be a powerful and useful probe to dissect the cellular interactions that take place in autologous responses.     

Histamine-releasing activity (HRA). I. Production by mitogen- or antigen-stimulated human mononuclear cells.

Supernatants from 1- to 2-day cultures of human mononuclear cells induced the release of histamine from basophils. Generation of this histamine-releasing activity (HRA) was stimulated by addition of concanavalin A to the cell cultures. Mononuclear cells were also cultured with SKSD and Candida albicans antigens. Stimulation of HRA production by these antigens was correlated with positive delayed skin reactions. Serial dilutions of supernatants assayed for HRA provided a semiquantitative determination of the level of HRA in mitogen- or antigen-stimulated samples. Antigen increased HRA production when added during the first or second day of culture. Generation of HRA probably requires active protein synthesis, since puromycin was inhibitory, and since preformed HRA could not be recovered from lysed cells. HRA was detected in supernatants after 4 hr, and the effects of antigen stimulation were apparent after 8 hr of culture. Replacement of supernatants with fresh culture medium allowed continued synthesis of substantial quantities of HRA during the second day of culture. A linear correlation was observed between the amount of HRA produced and the mononuclear cell concentration. Our findings provide evidence for the interaction of lymphocytes and basophils via a soluble mediator.     

Comparison of collagen gels and mammary extracellular matrix as substrata for study of terminal differentiation in rabbit mammary epithelial cells

Mammary epithelial cells were prepared by collagenase digestion of tissue from mid-pregnant rabbits and cultured for up to 6 days on either collagen gels or an extracellular matrix prepared from the same tissue. The behaviour of the cells in serum-supplemented medium containing combinations of insulin, prolactin, hydrocortisone, estradiol and progesterone were monitored by measuring rates of casein synthesis, lactose synthesis, DNA synthesis and protein degradation. After 6 days, epithelial cells on floating collagen gels showed substantial increases in casein synthesis and DNA synthesis over freshly-prepared cells, following a decline during the first 3 days when the collagen gels are contracting. The optimum hormone combination for casein synthesis was insulin + prolactin + hydrocortisone, whereas for optimum DNA synthesis the additional presence of estradiol and progesterone was required. Cells on extracellular matrix showed increased rates of both casein synthesis and DNA synthesis by day 6 in the presence of insulin + prolactin + hydrocortisone, with additional estradiol + progesterone having an inhibitory effect. Whereas on day 2 rates of intracellular protein degradation were generally lower in cells on extracellular matrix, by day 6 rates of protein degradation were lowest in cells cultured on collagen gels with insulin + prolactin + hydrocortisone. In all cases, rates of lactose synthesis fell to low levels as the culture proceeded. Pulse-chase labelling of freshly-prepared cells with [32P]orthophosphate in medium containing serum and insulin + prolactin + hydrocortisone demonstrated that newly-synthesized casein was degraded during its passage through the epithelial cell. The influences of the collagen gels and extracellular matrix and of the hormone combinations on epithelial cell differentiation and secretory activity are discussed.     

Dissociation of differentiation and proliferation in the primary induction of cytolytic T lymphocytes by alloantigens

The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.     

Lymphocyte reactivity in pregnant women and newborn infants.

The mitotic response to phytohaemagglutinin (PHA) was determined in lymphocytes of mothers and their newborn infants obtained at delivery and seven days later by measuring the rate of 125 I-idoxuridine uptake into DNA in lymphocytes cultured in their own plasma and after washing and resuspension in fetal bovine serum. There was no difference in the unstimulated counts of maternal lymphocytes taken at delivery, whether unwashed or washed, compared with those from nonpregnant controls. With PHA stimulation the mitotic response of the maternal lymphocytes cultured in their own plasma was reduced compared with that of the control lymphocytes but washed maternal cells showed a similar response to the controls. These findings suggest that the reduced lymphocyte mitotic response to PHA in pregnancy is due to a plasma inhibitory factor This inhibition was not evident in maternal blood taken seven days after delivery. DNA synthesis in unstimulated cultures from newborn infants at birth and seven days after birth was greater than that in adult control cultures. With PHA stimulation the mitotic response of cord-blood lymphocytes cultured in their own plasma paralleled that of control lymphocytes but washed newborn cells showed a greater response. Thus plasma suppression similar to that observed in the mother seems also to affect infants at birth. This inhibition was not demonstrable in blood taken from infants of 7 days.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

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

Comparative study of the proliferative activity of human lymphocytes from different pairs of donors in a mixed culture in vitro

The proliferative response of human lymphocytes was studied in one-way and two-way mixed lymphocyte cultures (MLC). The maximal proliferation was shown to be attained in a two-way MLC containing unequal quantities of the cells from two paired donors. It was also demonstrated in a one-way MLC that the cells of one of the two paired donors are more effective responders. The most powerful proliferation in the MLC of these donors was observed in the cultures containing the excess of more effective cells. Thymosine increased the response of human lymphocytes in the two-way MLC. In vitro cell preincubation reduced the response in the cultures with a high proliferative response in the control. It has been thus demonstrated that lymphocytes from paired donors possess different functional activity in the MLC.     

Effects of insulin, hydrocortisone and prolactin on cell morphology, growth and survival of mammary organoids from mid-pregnant mice cultured on collagen gels.

Mammary epithelial organoids consisting of groups of lobular-alveolar acini were prepared from mid-pregnant mice and cultured for 24, 48, 96 and 192 hr on attached collagen gels in the presence of combinations of insulin, hydrocortisone and prolactin. The organoids rapidly attached to the gels and with all the combinations of hormones used colonies of cells spread out as a monolayer from the organoids within 48 hr. Although colony formation continued for up to 192 hr in culture, the maintenance of parental organoid structure after 96 and 192 hr was strongly favoured when hydrocortisone was present in the culture medium. The presence of hydrocortisone produced a dose-dependent increase in the amount of organoid DNA associated with the collagen substratum but decreased the rate of DNA synthesis by the organoids, as measured by the incorporation of labelled thymidine into DNA, in a dose-dependent manner under these conditions. The results suggest that the presence of hydrocortisone minimised the loss of cells from the collagen matrix in these cultures.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

In vitro response of subpopulations of human lymphocytes. II. DNA synthesis induced by anti-immunoglobulin antibodies.

A significant and constant increase in DNA synthesis was observed in human lymphocytes cultured in the presence of purified anti-immunoglobulin antibodies specific for human IgG, IgA, and IgM. This has been found in cultures of lymphocytes isolated from blood, tonsils, spleen, and lymph nodes. The optimal culture conditions for blood and tonsil lymphocytes were determined. As a rule 6-day cultures containing 2 x 10(6) cells/ml and 100 mug/ml of antibody yielded the highest 3H-thymidine uptake. Purified T cell cultures could not be stimulated, whereas a low response could be observed in most of the purified B cell cultures. Optimal culture conditions were the same for the B and total tonsil lymphocytes. However, when the purified B cells were totally depleted of T cells, no response was observed. A T and B cell synergy has been demonstrated by supplementing B cell cultures with purified T cells, whether treated or not with mitomycin. These experiments indicated a permissive and potentiating effect of T cells on the B cell response. Cultures containing mitomycin-treated B cells and purified T cells (mB + T) could be stimulated by a-Ig, thus indicating a T cell proliferation. In keeping with this finding was the observation of an increased response of total lymphocytes supplemented with T cells but not with B cells. Adherent cells are necessary for an optimal response to a-Ig; they enhanced the B cell proliferation observed in (Tm + B) cultures and suppressed the response of T cells in (T + Bm) cultures.     

Cyclosporine-induced tolerance in experimental organ transplantation. Evidence of diminished donor-specific cytotoxicity relative to donor-specific proliferative response

Alloreactivity of intragraft and peripheral blood lymphocytes from tolerant canine lung allograft recipients was examined. Tolerance was induced by variable periods of treatment with cyclosporine. Analysis of effector cells from lung allografts (obtained by bronchoalveolar lavage) revealed the absence of specific cytolytic T lymphocyte (CTL) activity and the presence of a low level of cytolytic activity detected in a lectin-dependent cell-mediated cytotoxicity assay. In contrast, high levels of specific CTL activity and lectin-dependent activity were detected in cell preparations from lung allografts undergoing rejection. Tolerant recipients retained normal ability to generate specific CTL activity to third party alloantigens in mixed lymphocyte cultures (MLC) but had diminished ability to generate CTL to donor alloantigens in recipient X donor MLC. Addition of exogenous interleukin 2 to these MLC was unable to restore donor-specific CTL activity. Lymphocytes from tolerant recipients were, however, capable of generating proliferative responses and lectin-dependent cytotoxicity on exposure to donor alloantigens in MLC. Evidence presented in this report suggests that the lectin-dependent cytolytic activity generated in these MLC is mediated by lymphokine-activated killer cells. Such cells are likely to be activated by interleukin 2 released in the proliferative response. The results support the proposal that the cyclosporine-induced tolerant state is characterized by the relative inability to respond against major histocompatibility complex class I antigens in contrast to class II antigens and/or minor histocompatibility antigens since MLC-induced CTL are directed, for the most part, against class I molecules.     

Alteration of the antigenicity of human lymphocytes by plant lectins and short-term tissue culture.

Human lymphocytes studied after being placed in culture for 1–6 wk progressively lost stimulating ability, i.e., lymphocyte defined antigens, when tested in one way mixed lymphocyte culture (MLC) but retained several other identifiable membrane components as well as the capacity to respond to mitogenic stimuli. Lymphocytes placed in culture with motogenic doses of PHA and Con-A after 1 and 2 wk strongly stimulated autologous responding fresh lymphocytes, but the MLC response of allogeneic fresh lymphocytes to stimulating lectin treated cells was even lower than the response to stimulating allogeneic cultured lymphocytes. The HL-A antigens on lectin treated cells or on lymphocytes through 6 wk in culture were clearly identifiable. Assays for T cell rosettes and B cell surface immunoglobulin showed both cell types to be present in numbers equal to fresh lymphocytes for up to 5 wk after culturing. However, the Fc receptor site on B cells was lost from cultured lymphocytes at the same time that MLC stimulation was lost. It is concluded that plant lectins can unmask new mitogenic sites on the cell surface as well as mask or delete existing sites, and that culturing lymphocytes for 1–6 wk will produce somewhat similar modulations. Modulation of surface membrane components by tissue culture or lectins may, therefore, have a profound effect in altering transplantation immunogenicity.     

Effect of activated lymphocytes on the regulation of hematopoiesis: suppression of in vitro granulopoiesis by OKT8+Ia+T cells induced by alloantigen stimulation

We studied the effects of alloantigen-stimulated lymphocytes in the regulation of hematopoiesis. Alloantigen-stimulated lymphocytes were harvested on days 2 to 3, days 6 to 7, or days 9 to 10 of MLC and were tested for their effects on granulocyte/macrophage progenitor cells (CFU-C). Dose-dependent suppression of CFU-C was observed when alloantigen-stimulated lymphocytes from days 6 to 7 and days 9 to 10 MLC were added to the cultures of autologous or allogeneic bone marrow cells for CFU-C assays. Suppressive activity was detected in the T cell fraction but not in the non-T cell fraction. For further characterization of these CFU-C/suppressor cells, alloantigen-stimulated lymphocytes were treated with radiation (2000 rad) or with monoclonal antibodies against T cell subsets and complement (C) before culture. Suppressive activity was completely abolished by treatment with OKT8 or OKIa1 antibodies and C whereas suppression was retained after radiation treatment. These observations suggest that CFU-C/suppressor cells can be induced by alloantigen stimulation in MLC and that they are radioresistant OKT8+ and Ia+ T cells.     

Suppressive effects of cyclosporin A on the induction of alloreactivity in vitro and in vivo

The purpose of the present study was the investigation of the effect of cyclosporin A (CsA) on the induction of alloreactivity in vitro and in vivo. Addition of CsA to mouse mixed lymphocyte cultures (MLC) not only inhibited lymphocyte proliferation but also prevented the generation of alloreactive cytolytic lymphocytes (CL). It was necessary to add CsA within the first 3 days of a 5-day MLC in order to achieve a significant suppressive effect. Lymphocytes, after being cultured in MLC with CsA for 4 days or longer, were incapable of being activated upon re-exposure to the same alloantigens although their responses to unrelated antigens remained intact, indicating antigen specificity of the suppression induced by CsA and its long-lasting effect. Furthermore, lymphocytes from mice treated with CsA after allosensitization failed to manifest primary cytotoxicity and could not be reactivated in a secondary MLC. Finally, CsA had no effect on those CL already generated, suggesting that CsA acts upon the induction of CL rather than the effector phase.     

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

The effect of hydrocortisone on lymphocyte-mediated cytolysis

This paper reports the results of experiments designed to analyze the mechanism by which hydrocortisone suppresses the cell-mediated cytolysis produced by sensitized lymphocytes. We used an in vitro system in which rat lymph node cells were sensitized to, and caused cytolysis of mouse fibroblasts.We found that hydrocortisone probably suppresses cytolysis by preventing the primary activation of the cytolytic mechanism by target cell antigens. Suppression was most efficient when hydrocortisone was added at the beginning of the cytolytic reaction. The cytolytic mechanism itself appeared to remain intact, and could be activated by the lectin concanavalin A (con A) despite the presence of hydrocortisone.Suppression of cytolysis could not be related to any general inhibition of DNA, RNA, or protein synthesis. The influence of hydrocortisone on cytolysis was not modified by vitamin A (retinol), an agent which antagonizes the effect of hydrocortisone on lysosome membranes.Hydrocortisone was found to be less effective in suppressing the activity of lymphocytes that had been sensitized initially in the presence of hydrocortisone.     

Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.     

The human mixed-lymphocyte culture (MLC) interaction after in vivo allo-immunization. II. HL-A specificity of early proliferative response

Human volunteers were immunized with a single intradermal dose of allogeneic buffycoat cells. When donor and recipient differed for four HL-A antigens belonging to the first and second series, an increased MLC proliferation early in the cultures was observed when responding lymphocytes from the immunized individual were confronted with stimulating cells from his donor. These findings were not observed when the incompatibility between donor and recipient involved only one second series HL-A antigen. The specificity of the altered response was studied by confronting responding lymphocytes from the immunized individual with different third-party stimulating cells. Most of these combinations revealed an early increased proliferation compared to control combinations with responding lymphocytes from nonsensitized individuals. However, the early increased responsiveness was significantly more pronounced in combinations where the stimulating cells shared two or more “private” HL-A determinants with the immunizing donor. It is concluded that a major part of the early increased responsiveness is due to proliferation of lymphocytes with receptors for HL-A (SD) determinants.     

Cytotoxic activity of lymphocytes. VII. cellular origin of alpha-lymphotoxin.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.