Dual-parameter Flow Cytometric Analysis Research Articles

Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells.

Most HPV-positive cervical cancer cells possess wild type p53 gene, but its normal p53 functions are disrupted by expression of HPVs E6. Treatment with 0-20 microM cisplatin for 24 h in HPV16 E6 containing SiHa cells suppressed E6 mRNA, reduced E6 protein, and restored p53 expression in dose-dependent manners. Dual-parameter flow cytometric analysis indicated that sub-G(1) apoptotic cells, but not necrotic cells were the major species for cisplatin-induced cytotoxicity in SiHa cells. After 0-10 microM cisplatin treatment, slightly more apoptotic cells appeared from SiHa cells than those from dominant negative p53-transfected SiHa cells. There was no different ionizing radiation (IR)-induced apoptosis in these two different cells. On the other hand, cisplatin enhanced more IR-induced sub-G(1) apoptosis in SiHa than mp53-SiHa cells. These accompanied with prolonged p53 restoration in irradiated-SiHa cells after 24 h cisplatin treatment and thereafter. In contrast, it was not found in cells after irradiation alone. Similar results were also shown in Mdm2 expression in SiHa cells after combined treatment. Therefore, cisplatin restored p53 expression and prolonged IR-induced p53 restoration would be possible candidates to response more sub-G(1) apoptosis in irradiated SiHa cells. These results provided another new explanation on cisplatin sensitizing radiotherapy for HPV16 E6 containing cancer cells.

Assessment of quantitative dual-parameter flow cytometric analysis for the evaluation of testicular toxicity using cyclophosphamide- and ethinylestradiol-treated rats.

In the drug discovery process, effects to the human spermatogenesis must be fully evaluated before the first human trial. To estimate testicular toxicity, histopathological evaluation has been recommended in addition to the traditional mating procedure. However, it is laborious and time-consuming. Flow cytometric analysis (FCM) has also been applied to estimate testicular toxicity because of its speed, simplicity, and the objectivity of the data. Using cyclophosphamide (CP)- and ethinylestradiol (EE)-treated rat testis, we attempted to validate our dual-parameter, DNA ploidy and cell-size FCM, in a high-throughput toxicity study. Our results showed that CP damaged some spermatogonia and some early meiotic spermatocytes and EE caused severe decrease of spermatogenic cells except for spermatogonia as well as marked decrease of somatic cells, most probably Leydig cells. This is the first report discriminating between the changes of spermatogonia and that of somatic cells with FCM analysis. These results demonstrate that this method is a very useful and powerful tool to assess testicular toxicity, especially in high-throughput toxicological studies.

Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma

Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34(+)-selected PBMC, have shown no advantage for CD34(+) cell selection, in terms of the expansion achieved. Our goal was to develop procedures for consistent generation of large numbers of hematopoietic progenitor and post-progenitor cells from unselected PBMC. Unselected PBMC, collected from cancer patients undergoing apheresis prior to high-dose chemotherapy and autologous stem cell rescue, were expanded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days. The addition of 2% autologous plasma to this cytokine combination enhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8.1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold versus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given specimen was inversely related to the frequency of CD34(+) cells in the sample. CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivalent to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cultures were supplemented with autologous plasma. In plasma-free cultures, BFU-E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than with Flt3L, PIXY321, G-CSF and GM-CSF. In the presence of autologous plasma, however BFU-E expansion was higher in the Flt3L-containing media. In comparison studies, autologous plasma suppressed BFU-E expansion in SCF-containing cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplementation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures. Unselected PBMC from cancer patients can be effectively expanded ex vivo in Flt3L, PIXY321, G-CSF and GM-CSF, supplemented with autologous plasma, yielding high numbers of myeloid and erythroid progenitors.

FLOW CYTOMETRIC DETERMINATION OF METALLOTHIONEIN LEVELS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES: UTILITY IN ENVIRONMENTAL EXPOSURE ASSESSMENT

Metallothioneins (MT) are ubiquitous, low-molecular-weight proteins that exhibit high binding affinities for heavy metal ions. The expression of these cysteine-rich proteins is induced in response to various types of chemical and physical stresses and therefore can be used to assess human exposure to cytotoxic environmental agents. In the current study, MT levels of human peripheral blood lymphocytes were determined using an MT-specific antibody and flow cytometry. Treatment of human whole blood ex vivo with CdCl2 was found to induce a concentration- and time-dependent increase in lymphocyte MT levels at concentrations as low as 0.3 mu M and within a 12-h period. Interestingly, differences were observed in the magnitude of cadmium-induced MT levels in the lymphocytes of six human test subjects. Two members of the study population exhibited CdCl2-induced cellular MT levels that were up to twofold greater than the lymphocytes of other human subjects. While the lymphocytes of most test subjects exhibited a symmetric (unimodal) distribution of cadmium-induced MT-specific fluorescence, the cells of two individuals displayed a heterogeneous (nonuniform) distribution of MT levels. Dual-parameter flow cytometric analysis using phenotype-specific antibodies indicated that variations in the responsiveness of subpopulations of lymphocytes to CdCl2 were responsible for the heterogeneous distribution of MT-specific cellular fluorescence. Thelper (CD4-positive) and T-suppressor/cytotoxic (CD8-positive) lymphocytes expressed higher cellular levels of MT than other lymphocyte subpopulations (i.e., B lymphocytes, natural killer cells). Our results suggest that MT protein levels of peripheral blood lymphocytes, as determined by this flow cytometric method, may be used to assess human exposure to toxic metals and to characterize various quantitative/qualitative aspects of the response of individuals to cadmium and possibly to other types of environmental stresses.

Reduced Temperature (22°C) Results in Enhancement of Cell Killing and Neoplastic Transformation in Noncycling HeLa × Skin Fibroblast Human Hybrid Cells Irradiated with Low-Dose-Rate Gamma Radiation

The effect of reduced temperature (22 degrees C) or serum deprivation during low-dose-rate (0.66 cGy/min) gamma irradiation on cell killing and neoplastic transformation has been examined using the HeLa x skin fibroblast human hybrid cell system. The reduced temperature stops progression of these cells through the cell cycle while serum deprivation slows down cell turnover markedly. The data demonstrate an enhancement in both of the end points when cells are held at 22 degrees C compared to parallel experiments done at 37 degrees C. In operational terms, the decreased survival and increased neoplastic transformation are consistent with our earlier hypothesis of a higher probability of misrepair at reduced temperature (Redpath et al., Radiat. Res. 137, 323-329, 1994). The interpretation that this damage enhancement was associated with the reduced temperature, and not the fact that the cells were noncycling, was supported by the results of experiments performed with cells cultured at 37 degrees C in serum-free medium for 35 h prior to and then during the 12.24 h low-dose-rate radiation exposure. Under these conditions, cell cycle progression, as shown by reduction in growth rate and dual-parameter flow cytometric analysis, was considerably inhibited (cell cycle time increased from 20 h to 40 h), and there was no significant enhancement of cell killing or neoplastic transformation.

Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry

Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a heterogeneous sample.

FLOW-CYTOMETRY DETECTION OF MINIMAL DNA ANEUPLOIDY IN MATURE LYMPHOID LEUKEMIAS - COMPARISON WITH METAPHASE AND INTERPHASE CYTOGENETICS

The relative DNA content of peripheral blood cells from 79 cases with lymphoid leukemias was analyzed by a dual-parameter flow cytometric analysis. The leukemia samples corresponded to: chronic lymphocytic leukemia (CLL) 40, CLL with more than 10% prolymphocytes (CLL/PL) 12, CLL mixed 9, prolymphocytic leukemia (PLL) 5, and B-cell lymphoma in leukemic phase 13. DNA aneuploidy was found overall in 26 (32.9%) of the cases and these corresponded to: 7 (17.5%) with CLL, 7 (58.3%) with CLL/PL, 4 (44.4%) with CLL mixed, 2 (40%) with PLL and 6 (46.2%) with B-cell lymphoma. There was a good correlation between DNA content and cytogenetics/fluorescent in situ hybridization in all but 2 cases as follows: 6 of 7 cases with diploid DNA had normal karyotype and only one had trisomy 12: 4 of 6 cases with hyperdiploid DNA had trisomy 12, one had tetraploidy and only one had a normal karyotype. Two cases were hypodiploid both by DNA and cytogenetic analysis. Our findings demonstrate a higher incidence of DNA aneuploidy in B-cell lymphoma in leukemic phase, PLL, and atypical CLL in comparison with typical CLL and a good correlation with cytogenetics. We conclude that flow cytometric DNA analysis represents a useful, sensitive, and rapid method to detect and monitor minimal changes of DNA content in leukemic lymphocytes without the need of short-term cultures.

Cell cycle effects of the anti-rheumatic agent CPH82.

The benzylidated podophyllotoxin glycoside CPH82, a potentially useful drug for treatment of RA, was tested in vitro on nine human haematopoietic cell lines for cell kinetic effects. Previous studies have shown CPH82 to behave like a colchine-type 'metaphase' blocker. The distribution of cells within different cell cycle compartments (G1, S, G2 and M) was analysed by a novel method using dual parameter flow cytometric analysis of stage specific antigens (proliferating cell nuclear antigen and Ki-67). With CPH82 concentrations chosen to mimic clinical conditions, eight out of nine lines showed an accumulation of cells in the G2 phase of the cell cycle. In many lines a delayed progress through S seemed to occur. Three lines were blocked in both G1 and G2, whereas the major effect on one line (HL-60) was an accumulation of cells in the G1 phase. Progression of M cells seemed only slightly delayed for some cell lines. In comparison with two related 'metaphase' blocking agents (podophyllotoxin and taxol), CPH82 had a different and dose-dependent pattern of cell cycle retardation. It is speculated that the cell kinetic action of CPH82 might give insight into the question why it, unlike other 'metaphase' blockers, has proved valuable in the treatment of RA.

Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured asa function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction (∼ 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.

Recombinant Human Granulocyte Colony-Stimulating Factor in Combination with Continuous Infusion of Cytosine Arabinoside for the Treatment of Refractory Acute Myelogenous Leukemia

Because recombinant human granulocyte colony-stimulating factor (G-CSF) has been reported to increase the sensitivity of acute myelogenous leukemia (AML) blast cells to cytosine arabinoside (Ara-C) in vitro, we treated a patient with refractory AML with an Ara-C-based regimen in combination with G-CSF (G-CSF/Ara-C therapy). G-CSF (50 micrograms/m2/day, subcutaneous injection) was administered simultaneously with a continuous intravenous infusion of Ara-C (70 mg/m2/day). Complete remission was achieved, however, severe neutropenia, documented infection, stomatitis, and diarrhea were observed. In vitro studies using 3H-thymidine uptake and dual parameter flow-cytometric analysis of DNA and proliferating cell nuclear antigen showed that leukemic blast cells in S phase were increased after incubation with G-CSF. G-CSF also enhanced expression of the transferrin receptor (CD71) on blast cells in vitro. These observations suggest that G-CSF/Ara-C therapy may be useful in the treatment of high-risk AML.

Herpes simplex virus induces unscheduled DNA synthesis in virus-infected cervical cancer cell lines

We evaluated herpes-simplex-virus-type-2(HSV2)-induced unscheduled DNA synthesis in virus-infected cervical cancer (HeLa, CaSki, C-33A, and SiHa) cells. HSV2 replication was approximately 100-fold more efficient in the HeLa cells than in less susceptible C-33A and SiHa cells. In dual parameter flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation, HSV2-infected HeLa cells showed a rapid increase in the proportions of DNA-synthesizing G1- and S-phase cells, whereas in C-33A and SiHa cells, the proportions of DNA-synthesizing G1- and early S-phase cells were increased late in the infection. Blocking of HSV2 replication by phosphonoformate inhibited virus-induced changes in HeLa cells, but not in C-33A and SiHa cells. Anti-BrdU antibodies exhibited a coarse globular nuclear staining pattern in the C-33A cells, while the other cells showed speckled and/or fine globular nuclear fluorescence. Anti-ICP8 (HSV-specified major DNA-binding protein) antibodies revealed that, in C-33A cells, ICP8 remained in the cytoplasm, whereas in the other cells, speckled or globular nuclear fluorescence was found. Our results showed that HSV2 induced the unscheduled synthesis of cellular DNA, which was host-cell-dependent, and in virus infected C-33A cells, it may be attributable to both viral and cellular proteins.

Human immunodeficiency virus-related lymphomas: a possible association between tumor proliferation, lack of ploidy anomalies, and immune deficiency.

In an attempt to identify a biologic basis for the aggressive clinical behavior of human immunodeficiency virus (HIV)-associated lymphomas (HAL), dual-parameter flow-cytometric analysis was performed on 22 paraffin-embedded biopsy specimens. Cases were analyzed for DNA ploidy, the percentage of cells in S-phase (proliferative activity), and content of a recently identified proliferation-associated nuclear antigen, p105. The DNA-content analysis of 22 HALs was compared with that of 109 cases of intermediate-grade non-Hodgkin's lymphoma (NHL) unrelated to the acquired immune deficiency syndrome (AIDS) studied previously in our laboratory and 125 cases of high-grade NHL reported in the literature. The proliferative activity was higher in intermediate-grade HAL relative to non-AIDS NHL (24.0% v 10.4%; P = .03), and in high-grade HAL in comparison with NHLs of similar histology unassociated with HIV infection (24.8% v 19%), although the latter did not reach statistical significance. The number of mitoses per 10 high-power fields was found to correlate with the percentage of cells in S-phase (r = .68; P = .0004). Although p105 content tended to be higher in HAL than in an AIDS-related complex (ARC)-associated hyperplastic lymph node control, no statistically significant associations were found between p105 content and proliferative activity or the number of mitoses per 10 high-power fields. When compared with non-AIDS NHLs of comparable grade, there was a trend toward a lower incidence of DNA aneuploidy in both intermediate- (25% v 56%) and high-grade (38.5% v 60%) HALs. The higher proliferative activity and lower incidence of DNA aneuploidy found in HAL relative to non-AIDS NHL of comparable histologic grade may represent differences in pathogenesis and may underlie the poor prognosis of HIV-associated NHL.

B‐cell surface phenotypes of proliferating myeloma cells: Target antigens for immunotherapy

Dual-parameter flow cytometric analysis of B-cell antigens and DNA content was used to determine the phenotypes of proliferating tumor cells (S-phase cells) from 30 patients with multiple myeloma. B4 (CD19), J5 (CALLA, CD10), B1 (CD20), and monotypic surface immunoglobulin (Slg) were expressed heterogeneously in 24 patients. J5 and monotypic Slg were found most frequently but were always expressed on a significantly lower percentage of cells than the antigens typically associated with plasma cells, cytoplasmic immunoglobulin (Clg) and T10 (CD38). S-phase cells were found in each antigen(+) subset. B antigen(+) cycling cells were demonstrated in 16 patients whose marrow or blood cells expressed B antigens exclusively in the hyperdiploid fraction and therefore were certainly part of the myeloma clone. Similar to the low level of proliferative activity of the T10(+), Clg(+), and PCA1(+) subsets, the percentages of cycling cells of the preplasma cell B-antigen-bearing myeloma subsets ranged from less than 1% to 12%. The tumor cells of four patients were also studied with dual-color surface antigen analysis and demonstrated independent expression of B antigens, with only rare coexpression of T10 and monotypic Slg, J5, or B4. These findings are consistent with the presence of distinct myeloma subsets bearing differing B phenotypes in the same tumor and provide evidence that the proliferation in myeloma is occurring at various developmental stages in the malignant B lineage. These antigens may be important targets for immunologic therapy aimed at eliminating the entire proliferating compartment of this B-cell tumor.

Dual-parameter flow cytometric analysis of an early lymphocyte activation antigen (CK226) and DNA content.

This study provides a direct correlation, via dual-parameter flow cytometric analysis (simultaneous assessment of surface immunofluorescence and DNA content), between activated T-cell entry into the S/G2/M phases of the cell cycle and the kinetics of expression of a novel T-cell activation antigen, termed CK226. This molecule was identified by the specific monoclonal antibody on the leukaemic T-cell line CEM/K, and it was expressed by 8-30% of resting peripheral blood lymphocytes and the majority of monocytes and granulocytes. A large fraction of activated lymphocytes acquired the CK226 antigen before DNA synthesis. Moreover, this molecule was expressed on virtually all G0/G1 and S/G2/M phase cells by day 2 after phytohaemagglutinin (PHA) activation and at day 6 after stimulation in a mixed lymphocyte culture. The time course of expression of other known activation antigens, such as Tac and transferrin receptor, was comparable to that of CK226. Based on the relationships between CK226 expression on cycling cells and the stimulatory effects of the specific monoclonal antibody, we conclude that CK226 should be considered an early activation antigen, which defines a new pathway of T-cell activation.

Prediction of thioguanine-induced cytotoxicity by dual-parameter flow cytometric analysis.

A method is presented for the quantitative analysis of delayed cytokinetic effects resulting from the treatment of L1210 cells with 6-thioguanine (TG). By using dual-parameter (DNA/protein) flow cytometry, we could observe the accumulation of late S/G2/M cells with abnormally high green fluorescence (i.e., protein content), indicative of unbalanced growth. The use of mitotic cells from a pseudotetraploid line (HT29) as external markers for both red and green fluorescence facilitated highly reproducible measurement of the mean green fluorescence (GFLmean) of the arrested late S/G2/M population. We found that the dose dependence of the observed GFLmean values followed the same unusual biphasic pattern as did cytotoxicity in this cell line, indicating that this parameter might be a suitable means of predicting TG-induced toxicity in vivo. We propose that the low background expected for this kind of measurement would make it particularly appropriate for the analysis of clinical specimens (e.g., mononuclear bone marrow cells) from leukemic patients receiving thiopurines, to monitor (and, hopefully, predict) their response to treatment.

Dual parameter analysis of myeloma cells by flow cytometry. DNA content of cells containing monotypic cytoplasmic immunoglobulin.

Dual-parameter flow cytometric analysis of monotypic cytoplasmic immunoglobulin (CIg) and DNA content on 15 myeloma marrows allowed S-phase determination of the CIg(+) tumor separately from the CIg(-) hematopoietic cell pool. The median percentage of the CIg(+) cells in S-phase was 2% compared with 5% for the CIg(-) cells. The median survival of patients with more than 2%, and those with 2% or less CIg(+) S-phase cells was 2 months and more than 13 months, respectively, from the time of study. Ploidy analysis identified four patterns of plasma cell DNA content: entirely hyperdiploid, entirely diploid, combined diploid and tetraploid stemlines, and tumors containing diploid and aneuploid CIg(+) cells. A monotypic CIg(+) double stemline myeloma was distinguished from two aneuploid tumors containing admixed normal, diploid polyclonal plasma cells. This technique provides an improved and expedient means for determining the proliferating fractions of myeloma cells and enhances recognition of double stemline tumors and clonal evolution in myeloma.

Nutrient modification of proliferation and radiation response in EMT6/RO spheroids

The effects of external nutrients on the growth and radiation response of EMT6/Ro spheroids were studied by maintaining spheroids in media with different concentrations of glucose, amino acids, and vitamins. Compared to spheroids grown in normal glucose concentration (5.5 mM), spheroids grown in higher glucose media (24.8 mM), demonstrated no difference in initial volume doubling time, clonogenicity, number of proliferating cells, or cell cycle distributions. However, histology sections revealed that, spheroids grown in higher glucose concentration had a thicker viable rim than spheroids grown in normal glucose media. Two-step acridine orange staining and dual parameter flow cytometric analysis, in addition to continuous [3H]-thymidine labeling techniques, showed that spheroids grown in higher glucose had 2 to 3 times the fraction of quiescent cells, when compared to normal glucose spheroids. When irradiated in ice to reoxygenate, the Do's were similar in the normal and the higher glucose spheroids, but the Dq's were reduced in the higher glucose spheroids in the presence of increased amino acids and vitamins. When irradiated in air at 37 degrees C, spheroids grown in the higher glucose media were more sensitive (decreased Do), and had a smaller hypoxic fraction than when grown in normal glucose media. For spheroids grown in the same glucose concentrations but increased concentrations of amino acids and vitamins, there was generally an increased Do under all irradiation conditions. Some of these differences in radiation sensitivity could be correlated to differences in cellular glutathione levels of these spheroid cells.

Dual parameter flow cytometric analysis of DNA content, activation antigen expression, and T cell subset proliferation in the human mixed lymphocyte reaction.

This study provides direct correlation via dual parameter flow cytometry (simultaneous assessment of immunofluorescence and DNA content) between mixed lymphocyte reaction (MLR) responder cell entry into the S/G2/M phases of the cell cycle with the kinetics of expression of two activation-associated cell surface proteins, Tac (IL 2 receptor) and 4F2 (unknown metabolic function). A small population of activated cells was identifiable by expression of both Tac and 4F2 antigens before peak DNA synthesis in the MLR. This population of activation antigen-positive cells expanded linearly in size from days 3 to 7 of culture. Treatment of immature MLR cultures with anti-4F2 Mab and complement (C) before DNA synthesis (treatment on day 3, peak DNA synthesis on days 5 to 6) resulted in blunted proliferation and activation antigen expression when the same culture was analyzed after maturation on day 6, indicating that the activated population had been previously detected and removed by anti-4F2 Mab + C. The 4F2 antigen was expressed on a greater percentage of cells in the MLR at all times (days 3 to 9) than was Tac, was present on virtually all S/G2/M phase responder cells, and a large fraction of cells remained intensely 4F2+ subsequent to peak DNA synthesis. In contrast, after initially preceding responder cell entry into the S phase of the cell cycle, the kinetics of Tac antigen expression closely paralleled the kinetics of responder cell proliferation. A subpopulation of cycling responder cells was noted in all MLR cultures studied that expressed Tac antigen weakly or not at all. Cells within both T4 and T8 cell subsets proliferate with similar kinetics in response to alloantigen. The possibility that activation antigens can be utilized to study effector cell generation in the MLR and that this flow cytometric technique may be utilized to analyze the response to various alloantigens is discussed.