Immune Monitoring in Xenotransplantation: The Multiparameter Flow Cytometric Mixed Lymphocyte Culture Assay

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1 Cytometry (Communications in Clinical Cytometry) 42: (2000) Immune Monitoring in Xenotransplantation: The Multiparameter Flow Cytometric Mixed Lymphocyte Culture Assay Sicco H. Popma, 1 * Alyssa M. Krasinskas, 2 Andrew D. McLean, 1 Wilson Y. Szeto, 1 Daniel Kreisel, 1 Jonni S. Moore, 2,3 and Bruce R. Rosengard 1 1 Department of Surgical Research, University of Pennsylvania, Philadelphia, Pennsylvania 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 3 Cancer Center Flow Cytometry and Cell Sorting Facility, University of Pennsylvania, Philadelphia, Pennsylvania Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ( 3 H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with 3 H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4 7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical 3 H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient. Cytometry (Comm. Clin. Cytometry) 42: , Wiley-Liss, Inc. Key terms: proliferation; carboxyfluorescein succinimidyl ester; multiparameter mixed lymphocyte cultures Donor organ shortage has triggered the search for alternative organ sources. Currently, genetically engineered pigs, expressing human complement inhibitors to prevent antibody-mediated hyperacute rejection (1,2), are thought to be the most promising source for donor organs in the near future (3). Our laboratory is interested in the human cellular immune response against xenogeneic porcine cells. Mixed lymphocyte cultures (MLC) are widely used in transplant immunology as an in vitro measure of recipient helper T-cell responses against donor stimulator cells. Mismatches between donor and recipient allogeneic or xenogeneic major histocompatibility complex antigens can induce a cell proliferative response (4,5). Traditionally, responder (recipient) cell proliferation has been quantitated by particle emission of 3 H-thymidine incorporation into newly synthesized DNA. To measure only the responder cell proliferation, the stimulators are treated with ionizing irradiation from a 137 Cs source or by mitomycin C to incapacitate stimulator (donor) cell proliferation before these cells are cultured with the responder cells, the so-called one-way MLC. However, the conventional method does not allow phenotypic analysis of proliferating cells within a heterogeneous MLC. Therefore, our goal was to develop a method, using fluorescent cell proliferation markers in flow cytometry, to measure multiple parameters such as proliferation, phenotype, and viability of the responding cells within heterogeneous xenogeneic MLC. *Correspondence to: Sicco H. Popma, Department of Surgical Research, University of Pennsylvania, 352 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA spopma@mail.med.upenn.edu Received 23 March 2000; Accepted 27 June Wiley-Liss, Inc.

2 278 POPMA ET AL. Fluorescent cell dyes, such as PKH derivatives, first described by Horan and Slezak (6,7), have been used in flow cytometric in vitro and in vivo assays to assess T-cell proliferation (8) and cell-mediated lysis (9) in murine and human models and lymphocyte migration (10,11) in murine models. Recently, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) has also been used in similar applications. New assays, using CFSE, such as measurement of in vivo cell proliferation of adoptively transferred cells, have also been developed (12,13). CFSE is a green fluorescent cytoplasmic dye that covalently binds to intracellular macromolecules with a low turnover (14). With its excitation at 491 nm and emission 518 nm, it can be easily detected using conventional tabletop flow cytometers. Because CFSE-labeled cells can be detected in vivo for up to 11 weeks in a murine model (15), it is ideal for in vivo cell tracking experiments. When cells divide, the CFSE-labeled macromolecules of the cell membrane segregate into two daughter cells such that each daughter cell contains half the CFSE content of the parent cell. The fluorescence intensity of each generation is half that of the previous generation, a property that allows tracking of proliferated cells by flow cytometry. CFSE-labeled cells, counterstained with other fluorescein-labeled antibodies and viability markers, allow multiparameter characterization of proliferating cells (16). Thus far, the use of CFSE or PKH to measure proliferation of human cells has been applied to mitogen-stimulated cultures and antigen-specific T-cell responses (17), but not to heterogeneous culture systems such as our xenogeneic MLC. Multiparameter flow cytometric analysis of mixed lymphocyte cultures (MFC-MLC) permits assessment of viability and phenotype of dividing cells within a heterogeneous culture system. We propose that the use of CFSE in the MFC-MLC can complement traditional 3 H-thymidine incorporation MLC studies. This approach will provide the basis for further understanding of the cellular interactions involved in donor recipient responses in xenotransplantation and allow real-time monitoring of the immune status of patients undergoing this type of transplant. MATERIALS AND METHODS Cell Preparation Heparinized peripheral blood was obtained from healthy volunteers and from Hanford miniswine (Charles River Laboratories Inc., Worcester, MA). Peripheral blood lymphocytes (PBLs) were isolated under sterile conditions using density centrifugation with Lymphocyte Separation Medium (ICN Biomedicals Inc., Aurora, OH). PBLs were washed twice in AIM-V medium (Gibco BRL, Gaithersburg, MD), handled under sterile conditions and kept on ice. PBLs were fractionated into two cell populations: plastic adherent cells enriched for antigen-presenting cells (APCs) and plastic-adherent cell and nylon wool adherent cell-depleted PBL, enriched for T cells. Antigen-Presenting Cell Isolation Adherent cells were isolated according to previously described procedures (5). Briefly, PBLs were adjusted to cells per milliliter in AIM-V medium, and 25 ml was transferred to a 75 cm 2 culture flask (Becton Dickinson, Franklin Lakes, NJ) and incubated flat at 37 C and 5% CO 2. After 2 h, the flask was removed from the incubator and the nonadherent cells were collected. Briefly, the flask was washed once with 10 ml warm (37 C) media followed by 10 ml of cold (4 C) AIM-V; the adherent cells were scraped from the flask bottom and the flask was washed once with 10 ml cold AIM-V. The collected cells were centrifuged (400g), resuspended in cold AIM-V, and kept on ice until use. This fraction had an increased potency to stimulate cell proliferation (data not shown) and will be referred to as APCs throughout this article. T-Cell Isolation Enriched T-cell fractions were obtained as described (5). PBLs, depleted of APCs, as described above, were centrifuged and adjusted to cells per milliliter in warm AIM-V. A maximum of 2 ml of cell suspension was loaded onto a 1-g nylon wool column (Polysciences Inc., Warrington, PA) and incubated for 45 min at 37 C in 5% CO 2 (20). Nonadherent cells were eluted from the column with warm AIM-V. The first 15-ml cell suspension was collected and washed once in cold AIM-V and stored on ice. The plastic and nylon wool adherent cell-depleted fraction routinely yields more than 90% CD3-positive T cells and will be referred to as T cells throughout this article. APCs were sufficiently removed from the isolated cells as detected by flow cytometry ( 2% CD14-positive cells) and lack of stimulatory capacity as detected by 3 H-thymidine incorporation (data not shown). CFSE Labeling The CFSE labeling method was adapted from a previous described protocol (18). Human PBL or T cells were washed twice in 10 ml phosphate buffered saline (dpbs, Gibco BRL) at room temperature. The cells were adjusted to 10 7 cells per milliliter, and CFSE (Molecular Probes, Eugene, OR) in dimethylsulfoxide was added to a final concentration of 5 M from a 1,000 stock. The cells were vortexed for 10 s and then incubated for 10 min in the dark at room temperature with gentle shaking. After incubation, an equal volume of sterile, heat-inactivated fetal calf serum (Gibco BRL) was added to the sample for 1 min to quench the reaction. AIM-V medium was added and the samples were centrifuged and washed twice in 10 ml AIM-V medium. The cells were counted with a manual hemocytometer and adjusted to the appropriate concentration. Mixed Lymphocyte Cultures CFSE-labeled responder cells (PBLs, T cells, or T cells with APCs added back) were adjusted to cells per milliliter, and the stimulator cells were adjusted to cells per milliliter as previously described (19). APCs were added back to CFSE-labeled responder T cells in a 1:4 ratio. The stimulator cells, pig PBLs, or pig APCs, were irradiated with 25 Gy from a 137 Cs source. Responder cells (0.5 ml) were cultured with stimulator cells (0.5 ml) in

3 AIM-V medium supplemented with 2-mercaptoethanol ( M), NEAA (0.1 mm), and Na-pyruvate (1 mm) (all from Gibco BRL) in a conventional MLC. The mixed cultures were incubated for 1 7 days or 4 7 days in a 48-well plate (Corning Glass Works, Corning, NY) at 37 C and 5% CO 2. IMMUNE MONITORING IN XENOTRANSPLANTATION 279 Mitogen Stimulation Assay Cells were obtained as described above and kept overnight at 4 C before use. One milliliter of a cell suspension containing CFSE-labeled PBLs or T cells with autologous APCs added back was transferred to a single well of a 48-well plate, and phytohemagglutinin (PHA, Sigma Bio- Sciences, St. Louis, MO) was added at a final concentration 1 g/ml (20). The cells were incubated for 3 days at 37 C and 5% CO 2. 3 H-Thymidine Incorporation Assay The 3 H-thymidine incorporation assay was used as a standard measure of proliferation (4). At each time point studied, three aliquots (100 l each) were harvested and transferred to a round-bottom 96-well plate (Becton Dickinson). Each sample was pulsed with 1 Ci 3 H-thymidine (DuPont NEN, Boston, MA), in 25 l culture media, and incubated at 37 C in 5% CO 2. After 6hofincubation, the cultures were kept frozen at 30 C until analysis. Frozen cultures were thawed and immediately harvested (TOMTEC, Orange, CT) and 3 H-thymidine incorporation of the samples was measured using a Wallac 1205 Beta plate liquid scintillation system (Wallac Inc., Gaithersburg, MD). The radioactivity was expressed in counts per minute (cpm). Multiparameter Flow Cytometric Mixed Lymphocyte Culture Assay After the samples were taken for the 3 H-thymidine incorporation assay, the remaining mixed lymphocyte culture sample was used for flow cytometric analysis. The following monoclonal antibodies were used: phycoerythrin (PE) conjugated anti-human CD3, CD4 and CD8 monoclonal antibodies (Caltag, Burlingame, CA), and Cy- Chrome (Cyc) conjugated anti-human CD4 antibody (Pharmingen, San Diego, CA). In some assays, a viability probe, 7-amino-actinomycin D (ViaProbe, Pharmingen) was used as a viability probe. Briefly, 200 l cell culture was transferred to a plastic 4-ml tube (Falcon no , Becton Dickinson, Franklin Lakes, NJ) and 1 ml cold flow cytometry buffer (FB; dpbs with 1% heat-inactivated, sterile fetal calf serum) was added. The cells were centrifuged and the supernatant was discarded. The cell pellet was resuspended in 100 l FB containing PE and/or Cyc labeled anti-human monoclonal antibodies at a pretitrated concentration. The samples were incubated for at least 45 min at 4 C and washed again in 1 ml cold FB. The cells were finally resuspended in 400 l dpbs with 1% paraformaldehyde. The samples were stored at 4 C and analyzed within 1 3 days. If the viability probe was used, the cells were not fixed and were analyzed the same day. FIG. 1. Detection of proliferation at day 6 within a mixed lymphocyte culture of human CFSE-labeled T cells with APC added back and stimulated with pig PBL. An electronic gate was set around high FSC to visualize the dividing cells. A: The FSC versus SSC dot plot shows all events recorded. Region 1 (R1) marks all events with a high FSC containing proliferating lymphocytes. B: The histogram shows the CFSE signal for the ungated (light line) and R1 (bold line) population from (A). Flow Cytometric Data Acquisition and Analysis A FACScan (Becton Dickinson, San Jose, CA) was set up for single or multiparameter data acquisition. CFSE was measured in the FL-1 channel ( nm bandpass filter), PE in the FL-2 channel ( nm bandpass filter) and Cyc or ViaProbe in the FL-3 channel (650-nm long-pass filter). Compensation for CFSE in multiparameter flow cytometry is dose dependent. We used the following method to compensate CFSE in our labeling protocol: CFSE compensation was set at 95% and an equilibrium between the FL-1 and FL-2 signal was sought, by adjusting the FL-1 and FL-2 parameters, to properly scale both FL-1 and FL-2 in a dot plot. Finally, the FL-2 and FL-3 signals were compensated, typically 0.3% and 10%, respectively. For each sample, 50,000 or 100,000 events were acquired for analysis. Data acquisition and analysis was performed with CELLQuest software (Becton Dickinson). The analysis gating strategy was as follows: For singleparameter CFSE analysis, all events were plotted in a forward-side scatter plot and a gate was set around the high forward scatter events. These gated events were analyzed for CFSE contents and visualized in a histogram (Fig. 1). For multiparameter CFSE analysis, the acquired data were plotted in a contour plot with FL-2 on the ordinate and FL-3 on the abscissa. A gate was then set around the population to be analyzed for proliferation (for example, all CD4-positive events, Fig. 2A). The events within the gate were analyzed for CFSE and were plotted in a histogram (Fig. 2B). RESULTS Detection and Analysis of Cell Proliferation in Mitogen-Stimulated Cultures To verify that we could detect cell proliferation with flow cytometry, CFSE-labeled PBL and CFSE-labeled T cells with autologous unlabeled APC added back were cultured in the presence of PHA (1 g/ml). At day 3, the cultures were analyzed using flow cytometry and 3 H-thymidine incorporation in parallel. In a representative experiment, flow cytometric analysis of the high forward scatter events

4 280 POPMA ET AL. FIG. 2. MFC-MLC assays can be analyzed in different ways, as shown with this representative culture of CFSE-labeled human T cells responding against porcine APCs at day 5. Based on a CD4 versus ViaProbe contour plot (A), the CD4-positive events (R1, events, 52% of total cells acquired) were analyzed for cell proliferation as shown in the CFSE histogram (B). The histogram shows a parental peak (M1) and multiple generations of CD4 positive, proliferated cells (M2). M1 contains 19,272 events (39% of total) and M2 contains 6,755 events (14% of total). Additionally, generations of divided cells can be analyzed. A generation (R2) can be selected from a histogram (C) and analyzed for phenotype (CD4) and viability (ViaProbe) as shown in a dot plot (D). Analysis of the R2 showed 347 live CD4-positive events (upper left quadrant), 89 apoptotic and/or dead CD4-positive events (upper right quadrant) and 1,411 CD4 negative events (lower left quadrant plus lower right quadrant). showed that four rounds of cell division had occurred. The 3 H-thymidine data showed an average response of 158,249 cpm (n 2) for PHA-stimulated APC cultures with CFSE-labeled T cells added back and a response of 100,602 cpm (n 3) for PHA-stimulated CFSE-labeled PBL cultures (Fig. 3). These results demonstrated that dividing cells could be detected in the mitogen-stimulated cultures using the multiparameter flow cytometry. Monitoring Cell Proliferation in MLC by Flow Cytometry MLCs were designed to monitor cell proliferative responses in our heterogeneous, xenogeneic human antiporcine cell culture system. The cultures were harvested daily for days 5 7 and flow cytometric and 3 H-thymidine incorporation data were acquired. To detect cell proliferation by CFSE, an electronic gate was set on the events with a high forward scatter in a forward-side scatter plot. Events within this gate were analyzed for CFSE content as described (Fig. 1). A representative MFC-MLC assay of CFSE-labeled human T cells and irradiated porcine APC, inducing a potent T-cell response, is shown in Figure 4. The dividing cells detected in this MFC-MLC assay increased from 5% to 21% of the total during the 3-day FIG. 3. Representing a flow cytometric analysis of CFSE-labeled T cells with autologous APCs added back, cultured in the presence of PHA (1 g/ml) for 3 days. The CFSE signal is depicted in a histogram showing the parent generation M1, 14,019 events (28% of total 50,000 analyzed cells) and four generations of divided cells, M2 M5, M2 8,818 events (18%), M3 7,902 events (16%), M4 5,416 events (11%), and M5 8,989 events (18%).

5 IMMUNE MONITORING IN XENOTRANSPLANTATION 281 FIG. 4. Analysis of CFSE-labeled T cells, stimulated with pig APCs for days 5 7, shows the increasing number of divided cells. 3 H-thymidine incorporation is shown in cpm (mean of triplicates) and M1 represents the number of dividing cells in an ungated histogram with their percentage of the total acquired events in parentheses. detected by an increase in the number of dividing cells in a CFSE histogram. Similarly, low levels of 3 H-thymidine incorporation are detected at days 1 3 ( 1,000 cpm). The levels of 3 H-thymidine incorporation increased rapidly during days 4 6, yielding up to 33,000 cpm at day 6. In these experiments, CFSE analysis showed an increase of dividing cells coinciding with increased 3 H-thymidine incorporation starting at day 4 of the MLC. FIG. 5. Comparison of 3 H-thymidine incorporation expressed in cpm (left y-axis, solid bars) and MFC-MLC expressed in number of divided cells (right y-axis, solid line) for days 1 7 (average S.D.; n 4). Similar proliferation kinetics between the two methods are demonstrated in heterogeneous cultures of CFSE-labeled human PBL and porcine PBL. period. The 3 H-thymidine incorporation peaked on day 6, with the MFC-MLC assay peaking on day 7. This experiment is representative of four independent similar studies. CFSE Data Correlate with the Conventional MLR We also analyzed more heterogeneous cultures of CFSElabeled human PBL cultured with irradiated porcine PBL for days 1 7. This to compare our MFC-MLC assay with 3 H-thymidine incorporation for the ability to detect cell proliferation. The data shown in Figure 5 are the results of four independent experiments. The CFSE data revealed few dividing cells ( 500) for days 1 3. At day 4, the kinetics changed and cells proliferated rapidly, which was Multiparameter Analysis Is Possible in the MFC-MLC Assay To determine the phenotype of the dividing cells in a MLC, we used CFSE labeling in combination with one or two phenotypic markers in the MFC-MLC assay. CFSElabeled cultures were counterstained with anti-human CD8-PE and anti-human CD4-Cyc to determine CD4/CD8 ratios within the cultures (data not shown). In most assays, a live gate with ViaProbe (FL-3) was included and samples were counterstained with anti-human PE-labeled anti-cd3, anti-cd4, or anti-cd8 (all FL-2). With these approaches, subpopulations, such as CD4-positive cells, could be analyzed for cell division, as demonstrated in Figure 2A,B. Although it is possible to analyze each generation for phenotype and viability (Fig. 2C,D), we did not analyze all the generations within these cultures. Reproducibility of the MFC-MLC Analysis Analysis of six independent cultures of human CFSElabeled T cells cultured with irradiated porcine APC stimulators showed that CD3-, CD4-, and CD8-positive T cells could be detected reliably. The analysis showed that the cultures contained 12 4% CD3, 10% 5% CD4, and 5 1% CD8 (average of n 6, S.D.) proliferating T cells (Fig. 6).

6 282 POPMA ET AL. FIG. 6. Six cultures of CFSE-labeled human T cells stimulated with porcine APCs were harvested at day 5 and stained with PE labeled phenotypic antibodies against CD3, CD4, and CD8 and finally, ViaProbe was added to allow viability assessment. The acquired data was plotted in a phenotype against ViaProbe dot plot, and the phenotype-positive events were gated and analyzed for CFSE in a histogram. Percent dividing cells per 50,000 acquired events; average S.D.; n 4). DISCUSSION With the advent of xenotransplantation, we must develop sensitive assays to measure the immune responses between host and recipient. The success of the transplant is dependent on determining what are positive and negative responses to guide patient management. The mixed lymphocyte reaction has long been used to measure host/ donor cellular interactions in vitro (4). Our goal was to develop a sensitive assay to detect and analyze xenogeneic responses. The use of CFSE, in combination with other fluorescent antibodies in a multiparameter setting, allowed a detailed analysis of dividing T-cell populations within our xenogeneic MLC. The multiparameter flow cytometric MLC assay provides a feasible method to clinically monitor immune status of patients receiving xenotransplants. Fluorescent dyes are routinely used in analyzing cell proliferation or cell migration. Although dyes such as Hoechst 33342, CFDA-AM, FDA, etc. (14) have been used, the PKH dyes have shown more utility in this area. PKH derivatives can be used to detect cell migration and cell proliferation in murine (7,11,21,22) and human systems (8,23 28), and have been used in cytotoxicity assays to replace 51 Cr release techniques (9). More recently, CFSE has been used in similar assays such as cell tracking, lymphocyte proliferation in murine (18) and human systems (16,17) in vitro, replacement of the 51 Cr release assay (NA Bos, unpublished data), and measurement of in vivo proliferation of adoptively transferred cells (12,13) in mice. Based on these studies, we designed the MFC-MLC assay to use CFSE in our xenogeneic mixed lymphocyte cultures. CFSE labeling is inexpensive, simple, and fast, with similar results as reported with PKH labeling. In our experience, cell proliferation analysis with CFSE resulted in sharp, distinct generations of cells with replicable results in our MFC-MLC assay. The measurement of cell proliferation with CFSE in a xenogeneic MLC offers many advantages over conventional 3 H-thymidine incorporation assays. Labeling cells with CFSE is a simple procedure, eliminates the use of radioactive materials, and results in an extremely bright fluorescence signal that is easily detected by tabletop flow cytometers. Intracellular esterases hydrolyze CFSE into a fluorescent dye that binds covalently to cytoplasmic protein residues such as lysine (29); therefore, only viable cells are labeled. However, labeled cells that die during the culture period remain detectable until they disintegrate. These late apoptotic cells and cell fragments can cause an overall increased, less defined, CFSE signal that may artifactually affect results; thus, we gate on the high FSC events as shown in Figure 1. The labeled proteins have a low turnover, allowing long-term in vivo cell tracking of up to 11 weeks (14), and in vitro cell proliferation can be tracked for up to at least 7 days. Labeling cells with CFSE enabled us to detect dividing cells in our heterogeneous xenogeneic MLC. CFSE-labeled divided cells show clearly defined narrow peaks for each subsequent generation of divided cells, as demonstrated in our mitogenstimulated cultures and as others have shown in murine systems (12). We were able to detect as few as 500 divided cells; however, larger numbers of dividing cells significantly improved the ease and quality of the analysis. Multiparameter flow cytometry required compensation to eliminate bleeding of the CFSE signal (FL-1) into the red channel (FL-2). In our experience, the compensation was typically 95%, due to the intensity of the CFSE signal, and required adjustments of the FL-1 and/or FL-2 to scale the CFSE peak over the 3rd log in a dot plot. However, CFSE compensation is dose dependent, and using less CFSE for labeling will ease compensation. The addition of a ViaProbe, a viability and apoptosis marker (30,31) measured in the FL-3 channel, allowed us to assess the viability of the cell populations, which added another parameter to the analysis. Therefore, in our setup we were able to monitor three parameters in the MFC- MLC, as compared to one parameter in the 3 H-thymidine incorporation assay, in one culture sample. Our preliminary studies in a xenogeneic MLC demonstrated increased cellular proliferation over time with a rapid expansion of T cells starting on day 3. Proliferating cells could be phenotyped by CD3, CD4, and CD8 antibodies within our heterogeneous MLC. Furthermore, the combination of CFSE, phenotypic labels, and ViaProbe allowed us to examine the viability of T-cell populations within each generation. Interestingly, we detected apoptotic and dead cells in each generation of our MFC-MLC assay (data not shown). Therefore, precursor frequency calculations, as done by others (18), within MFC-MLC

7 assays might not be possible because these calculations are based on the assumption that each generation consists of proliferating, viable cells. In addition, the proliferation patterns seen in our histograms vary significantly. This probably reflects the random matching of the human and pig blood. Human anti-pig responses are major histocompatibility complex restricted (5), similarly to allotransplantation; thus, the responses vary considerably as in human allogeneic MLC, reflecting the compatibility between the donor and recipient. At early time points, generations of divided cells are visible; however, at later time points, the proliferating cells appear as one high peak at the left side of the histogram, as can be seen in Figure 4. This is most likely due to the accumulation proliferating cells in logarithmic phase and because these cells contain little CFSE, so division will not cause a visible shift in CFSE intensity histogram. Thus, MFC-MLC assay allowed detailed analysis of proliferating cell populations within a heterogeneous culture system and is theoretically only limited by the availability of appropriate cell markers. Flow cytometry with CFSE labeling allowed us to study generations of proliferated cells, a method that is, as we showed, kinetically comparable to the conventional 3 H-thymidine incorporation assay. We demonstrated that CFSE labeling could be used to detect cell proliferation of xenogeneic-stimulated human lymphocytes in heterogeneous culture samples. Detection of cell division within a heterogeneous cell culture allowed cell proliferation to be studied within a more intact cellular environment, avoiding the use of radioactivity and the need to irradiate the stimulator cells. In combination with surface phenotypic antibodies, this assay was used to dissect proliferative responses of subpopulations of T cells in xenogeneic MLC. 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