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Characterization of mitogen-stimulated porcine lymphocytes using a stable fluorescent dye (PKH2) and multicolor flow cytometry
Veterinary Immunology and Immunopathology 87 (2002) 1±10
Characterization of mitogen-stimulated porcine lymphocytes using a stable ¯uorescent dye (PKH2) and multicolor ¯ow cytometry A.D. Dorna,*, W.R. Watersb, V.M. Byersa, B.A. Peschc, M.J. Wannemuehlera a
Veterinary Medical Research Institute, Iowa State University, 1802 Elwood Drive, Ames, IA 50011, USA US Department of Agriculture, Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, P.O. Box 70, Ames, IA 50010-0070, USA c US Department of Agriculture, Periparturient Diseases of Cattle Research Unit, National Animal Disease Center, Agricultural Research Service, P.O. Box 70, Ames, IA 50010-0070, USA b
Received 9 August 2001; received in revised form 28 December 2001; accepted 28 January 2002
Abstract Stimulation of lymphocyte proliferation using mitogens or speci®c antigens is a method that is used frequently to assess immune responsiveness. While useful, lymphocyte blastogenesis, or 3 H-thymidine incorporation, provides little information regarding the response of speci®c subsets to the stimulant. Here, we report that the ¯uorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multicolor ¯ow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using 3 Hthymidine incorporation as well as a ¯ow cytometric-based proliferation assay. From the 3 H-thymidine incorporation data alone, it was observed that PBMC stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and ¯ow cytometric analysis, the responsiveness of speci®c lymphocyte subsets to mitogen stimulation was detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4 CD8 , CD4 CD8ahi , CD4 CD8alo and gd TCR cells. PWM stimulation induced responses by CD4 CD8, CD4CD8ahi but not by CD4 CD8alo or gd TCR cells. Con A stimulation resulted in a sustained proliferation of CD8ahi cells over the 5-day period while PHA stimulation resulted in proliferation that peaked within the ®rst 3 days. Little or no proliferative responses were detected within the IgM population (e.g. B cells). This is the ®rst study to de®ne the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Porcine lymphocytes; PKH2; Mitogens; Flow cytometry; Blastogenesis; Lymphoproliferation
1. Introduction Abbreviations: cRPMI, complete tissue culture medium; CO2, carbon dioxide; PE, phycoerythrin; FACS, ¯uorescent activated cell sorter; SI, stimulation index; 7AAD, 7-amino-actinomycin D; CFSE, carboxy¯uorescein succinimidyl ester * Corresponding author. Tel.: 1-515-294-1025; fax: 1-515-294-1401. E-mail address: [email protected] (A.D. Dorn).
Lymphocyte proliferation is a useful tool for measuring immune function and mitogens have long been used to induce proliferation in lymphocyte cultures (Resch et al., 1976; Janossy et al., 1977; Flaming et al., 1989; Morrison et al., 1990; Waters et al., 1999). Standard assays of lymphocyte proliferation include
0165-2427/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 0 2 2 - 3
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measurement of 3 H-thymidine incorporation or analysis of cell surface markers on lymphoblasts by ¯ow cytometry (Zuckermann and Husmann, 1996). Analysis of lymphoblasts for cell surface marker expression, however, does not determine if these cells have actually divided, only the relative size and granularity. More recently, vital ¯uorescent stains such as carboxy¯uorescein succinimidyl ester (CFSE) and PKH dyes have been used to determine proliferation of in vitro stimulated lymphocytes (Wells et al., 1997; Gudmundsdottir et al., 1999; Waters et al., 2000a). Use of vital ¯uorescent stains to determine proliferation affords the capability to determine proliferation over the entire culture period as well as the contribution of individual lymphocyte subsets to the proliferative response (Ashley et al., 1993; Quade and Roth, 1999). Previously, to study speci®c lymphocyte subset proliferation, the cells would have to be physically separated (e.g. by magnetic or ¯ow cytometric separation) and then cultured in vitro (Zuckermann and Husmann, 1996). This type of assay does not account for the importance of cell-to-cell interactions in the proliferative process (Hanrahan et al., 1997). Using the vital ¯uorescent stains, proliferation of speci®c cell subsets can be identi®ed in unfractionated cell populations. The objective of the present study was to measure the proliferation of porcine lymphocyte subsets to mitogen stimulation using the vital ¯uorescent stain, PKH2. Lymphocyte proliferation was measured by 3 H-thymidine incorporation and the PKH2 ¯ow cytometry-based assay in order to compare the results obtained by the two methods and at two time points. This is the ®rst study to determine the contribution of individual lymphocyte subsets to the proliferative response of mitogen-stimulated porcine peripheral blood mononuclear cells (PBMCs). 2. Materials and methods 2.1. Pigs Eight 4±5-week-old crossbred pigs from a breeding herd maintained at the College of Veterinary Medicine, Iowa State University, Ames, IA were used for the study. No clinical signs of disease were detected during the study period. The Iowa State University Committee on Animal Care approved all procedures.
2.2. Harvesting of PBMCs Peripheral blood was obtained from the anterior vena cava and PBMC were isolated by gradient centrifugation. Brie¯y, whole blood was diluted 1:3 in sterile phosphate buffered saline (0.05 M, pH 7.4; PBS), overlaid onto ®coll-hypaque (Histopaque 1077; Sigma Chemical Company, St. Louis, MO), and centrifuged at 550 g for 40 min. PBMC were obtained from the ®coll/plasma interface, washed two times with sterile PBS, and standardized to 2 107 /ml for PKH2 staining. 2.3. PKH2 assay Isolated PBMC were stained with the green ¯uorescent dye, PKH2 (Sigma) according to the manufacturer's recommendations. Brie¯y, 2 107 PBMC were centrifuged at 150 g for 10 min, supernatants decanted completely and 1 ml of diluent (provided in the kit) added to each tube of cells. Cells were thoroughly resuspended in diluent using a P1000 Pipetman and a large bore pipette tip then transferred to the tube containing 1 ml of PKH2 green ¯uorescent dye (4 10 6 M). Because the cells are more fragile after the dye is added, a Pipetman (P1000) was used between every wash step rather than a vortexer to be certain the cells were thoroughly resuspended between washings. Cells were then incubated in the dye solution for 5 min, followed by a 1 min incubation with 2 ml of fetal bovine serum (Hyclone Laboratories, Logan, UT, FBS) to stop the reaction. After 1 min incubation, 4 ml of RPMI 1640 with L-glutamine (Mediatech, Huntingford, VA) was added and the cells were centrifuged at 150 g for 10 min. The supernatant was decanted and the cells were washed three more times with RPMI 1640 and resuspended in culture medium for counting. Following enumeration, the PKH2stained cells were used for both the ¯ow cytometric assay and the 3 H-thymidine incorporation assay. In this way, any effects of the PKH2 dye on subsequent cellular proliferation would be constant to both assays. Culture medium (complete RPMI medium, cRPMI) consisted of RPMI 1640 with L-glutamine (Mediatech) supplemented with 1% non-essential amino acids (Sigma), 1% sodium pyruvate (Sigma), 2% essential amino acids (Mediatech), 25 mM HEPES buffer (Sigma), 100 units/ml penicillin, 0.1 mg/ml streptomycin, 1% L-glutamine (Difco, Detroit, MI),
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5 10 5 M 2-mercaptoethanol (Sigma), and 10% heat-inactivated FBS. Wells of 96-well ¯at-bottomed microtiter plates were seeded with 2 105 PKH2stained mononuclear cells in cRPMI at a total volume of 200 ml per well. Wells also contained either concanavalin A (Con A, 5 mg/ml, Sigma), puri®ed phytohemagglutinin (PHA, 5 mg/ml, Murex Biotech Ltd., Dartford, UK), pokeweed mitogen (PWM, 5 mg/ml, Sigma) or cRPMI alone (e.g. no stimulation). These concentrations were previously determined to be optimal for lymphocyte blastogenesis (data not shown). Plates were then incubated for 3 or 5 days at 37 8C in 5% CO2 in air. Cells from replicate wells of each treatment group were pooled and analyzed by ¯ow cytometry for PKH2 staining intensity as well as cell surface marker expression. 2.4. Preparation of cells and analysis by ¯ow cytometry Cultured mononuclear cells were analyzed for expression of cell surface antigens and PKH2 intensity as previously described (Waters et al., 2000b). Brie¯y, cell suspensions were harvested from the 96-well culture plates and like wells were pooled according to treatment. Cell suspensions (100 ml) were transferred to appropriate wells of a 96-well, round-bottomed microtiter plate and stained with 50 ml of primary antibody to swine leukocyte surface antigens. The monoclonal antibodies used to detect cell surface markers were anti-CD3 (8E62, VMRD, Pullman, WA), PE-conjugated anti-CD4 (74-12-4), biotinylated anti-CD8 (76-2-11), anti-gd T cells (PGBL22A, VMRD), anti-IgM (PG145A, VMRD). PBS containing 1% FBS and 0.04% sodium azide was used as a FACS buffer. Wells with appropriate isotype control antibodies (all diluted in the FACS buffer) were used. Cells were labeled using the following combinations of monoclonal antibodies and analyzed using two- or three-color ¯ow cytometry. T cells (CD3) were differentiated from B cells (IgM) using 8E6 (PElabeled secondary antibody) and PG145A (PE-labeled secondary antibody) in separate tubes. T cell subsets were evaluated for expression of CD4 and CD8a using 74-12-4[PE 76-2-11 (biotinylated streptavidinconjugated CyChrome). The percentage of gdT cells were measured using PGBL22A (PE-labeled secondary antibody).
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Following a 15 min incubation, cells were washed twice with FACS buffer and resuspended in 50 ml of secondary antibody. Appropriate secondary antibodies used were PE-conjugated goat anti-mouse IgM for PG145A, PE-conjugated goat anti-mouse IgG1 for 8E62 and PGBL22A, and streptavidin-conjugated CyChrome (Sigma) for the biotinylated 76-2-11. Cells were incubated for 15 min with the secondary antibodies, washed twice and resuspended in FACS buffer. Cells were then analyzed using a Becton Dickinson FACScan ¯ow cytometer (10,000 events from each sample). The number of cells proliferating was calculated by the following formula: % proliferation to mitogen no: of cells in the R1 gate % proliferation with no stimulation no: of cells in the R1 gate and presented as the mean number of proliferative cells S:E:M: per 10,000 PBMC. R1 is the region containing live lymphocytes based upon typical forward and side light scatter properties of porcine lymphocytes (Fig. 1). The excitation and emission wavelengths for PKH2 are 490 and 504 nm, respectively. Percent proliferation is de®ned as the percent of cells (e.g. PKH2 dim cells) that are not within the parent generation (e.g. PKH2 bright cells). The stability of the dye incorporation into the lipid membrane of the cell ensures that when cells divide the dye is distributed equally between daughter cells. PKH2 staining intensity diminishes with each cell division resulting in a decreased mean ¯uorescence intensity (Ashley et al., 1993). Mod®t Proliferation Wizard (Verity Software House Inc., Topsham, Maine) was used for cell proliferation analysis and CellQuest software (Becton Dickinson, San Jose, CA) was used for phenotype analysis. 2.5. Lymphocyte blastogenesis Wells of 96-well ¯at-bottomed microtiter plates were seeded with 2 105 PKH2-stained mononuclear cells in cRPMI at a total volume of 200 ml per well. Wells also contained either 5 mg/ml Con A, 5 mg/ml PHA, 5 mg/ml PWM, or cRPMI alone (e.g. no stimulation). Plates were then incubated for 3 or 5 days at 37 8C in 5% CO2 in air. After 2 or 4 days, 0.5 mCi of
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methyl-3 H-thymidine (speci®c activity 6.7 Ci/mmol, Amersham Life Science, Arlington Heights, IL) at a concentration of 50 mCi/ml in 10 ml of RPMI 1640 with L-glutamine (Mediatech) was added to each well, and plates incubated for an additional 16±18 h. Well contents were harvested onto glass ®ber ®lters, and incorporated radioactivity measured by liquid scintillation counting. Treatments were run in triplicate and stimulation indices (SIs) calculated by dividing counts per minute of stimulated wells by counts per minute from non-stimulated wells. Data are presented as mean SI S:E:M: 2.6. Statistics Data were analyzed by one-way analysis of variance followed by Tukey±Kramer multiple comparisons test. Differences between groups were considered signi®cant if probability values of P < 0:05 were obtained. 3. Results 3.1. Induction of lymphoblasts by mitogen stimulation
Fig. 1. Forward and side scatter analysis of control and mitogenstimulated PBMC. Mitogen stimulation of PBMC results in increased number of lymphoblasts in the live lymphocyte gate (R1) as compared to (A) non-stimulated PBMC. Note fewer number of lymphoblasts in (D) PWM-stimulated cultures as compared to the number of lymphoblasts detected in (B) Con Aand (C) PHA-stimulated cultures. Cells located in gate R2 are predominantly dead cells and debris or apoptotic cells as determined by staining with Annexin V and 7AAD. Data presented is from a single animal and is representative of all animals n 8.
Proliferation of lymphocytes results in the formation of lymphoblasts. These larger cells can be detected by ¯ow cytometry (e.g. lymphocytes with increased forward (size) and side (granularity) light scatter properties). As shown in Fig. 1, increased number of lymphoblasts were detected in the live lymphocyte gate (R1) after stimulation with Con A (Fig. 1B), PHA (Fig. 1C), or PWM (Fig. 1D) as compared to non-stimulated PBMC (Fig. 1A). Fewer number of lymphoblasts were detected for PWM-stimulated cultures as compared to the number of lymphoblasts detected in Con A- or PHA-stimulated cultures. Cells that are located in gate R2 are predominantly dead cells and debris or apoptotic cells as determined by staining with Annexin V and 7-amino-actinomycin D (7AAD). 3.2. Lymphocyte proliferation, effects of time and assay Proliferative responses of PBMC recovered from healthy, young pigs were assessed by two methods: a standard 3 H-thymidine incorporation assay and the
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Fig. 2. Assessment of cellular proliferation by blastogenesis or ¯ow cytometry. Proliferation data comparing (A) 3-day vs. (B) 5-day cultured PBMC with mitogen stimulation, as described in Section 2, using two different assays. Proliferative responses as measured by the PKH2 assay were increased on day 5 of culture as compared to day 3 of culture, whereas 3 H-thymidine incorporation was decreased on day 5 of culture as compared to day 3 of culture. Counts per minute (e.g. for the 3 H-thymidine assay) for non-stimulated cultures ranged from 428 to 5004. Data represents the mean S:E:M: for eight pigs. (a) Signi®cantly P < 0:05 greater as compared to stimulation index for Con A response at day 5 of culture (e.g. vertical comparisons). Although not statistically signi®cant, proliferation induced by PHA or PWM stimulation as measured by 3 H-thymidine incorporation was also greater on day 3 of culture as compared to day 5 of culture. (b) Signi®cantly P < 0:05 less than responses by PBMC stimulated with either Con A or PHA (e.g. horizontal comparisons) as measured by the PKH2 assay for either 3 or 5 days of culture.
PKH2 ¯ow cytometric-based assay. Proliferation induced by Con A, as measured by 3 H-thymidine incorporation was signi®cantly P < 0:05 greater on day 3 of culture as compared to day 5 (Fig. 2). Although not statistically signi®cant, proliferation induced by PHA or PWM stimulation, as measured by 3 H-thymidine incorporation, was also greater on day 3 of culture as compared to day 5 of culture. The
counts per minute for non-stimulated cultures ranged from 428 to 5004. Total cell proliferative responses as measured by the PKH2 assay were greater on day 5 of culture as compared to day 3, although not statistically signi®cant. Proliferative responses to PWM stimulation at both 3 and 5 days of culture were signi®cantly P < 0:05 less than responses to Con A or PHA stimulation as measured by the PKH2 assay.
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3.3. Lymphocyte subset proliferation Three-color ¯ow cytometry was used to assess proliferation of lymphocyte subsets (Fig. 3). The proliferative response of CD3 cells was signi®cantly P < 0:05 greater than responses by IgM cells for each mitogen at both 3 and 5 days of culture (Table 1). Five days of Con A (Fig. 3B) or PHA (Fig. 3C) stimulation resulted in increased number of cells detected in the CD8ahi (R3 gate), CD4 CD8 (R4 gate), and CD4 CD8alo (R5 gate) populations as compared to non-stimulated cells (Fig. 3A). After 5 days of PWM stimulation, increased number of cells were detected in the CD4 CD8 (R4 gate) as compared to non-stimulated cells (Fig. 3A). The distribution of proliferation of individual subsets is shown in Table 1. Although analysis of this table is complex, several observations can be made. First, cells stimulated with either Con A or PHA, demonstrated a proliferative response by each of the individual lymphocyte subsets analyzed (except for IgM cells) at each time point. Second, few CD4 single positive (i.e. CD4 CD8 ) cells had proliferated in comparison to other subsets examined. Third, CD8a cells (e.g. CD4 CD8alo , CD4 CD8ahi , CD4 CD8 ) and gd TCR cells contributed to the proliferative response with the exception that gd TCR cells did not proliferate to PWM stimulation. Statistical comparisons between individual subsets, days of culture, and mitogen stimulation are provided in Table 1. Proliferation of gd TCR cells is demonstrated in Fig. 4. Using Mod®t Proliferation Wizard software, live cells (e.g. R1 gate in Fig. 1) were analyzed for PKH2 staining intensity and histograms generated. The histograms demonstrate that as cells divide there is a generational shift from right to left (i.e. PKH2bright to PKH2dim) corresponding to parental and daughter populations, respectively. Each peak to the left of the parental peak (black peak in Fig. 4) is equivalent to a divisional generation (gray peaks in Fig. 4). Over time, following mitogen stimulation, the percentage of the total population of stained cells in the parental peak decreases and more generational peaks appear. Fig. 4 further corroborates the data shown in Table 1 which demonstrate a lack of proliferation of gd TCR cells to PWM stimulation.
Fig. 3. Three-color ¯ow cytometric analysis of porcine PBMC stimulated by mitogen: (A) no stimulation; (B) Con A stimulation; (C) PHA stimulation; (D) PWM stimulation. Distribution of porcine lymphocyte subsets as de®ned by CD4 and CD8 expression demonstrating effects of mitogen stimulation. R3: CD8ahi cells; R4: CD4 CD8 cells; R5: CD4 CD8alo cells; R6: CD4CD8 cells. Data represents the response of cells from a single pig and is representative of the group n 8.
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Table 1 Measurement of mitogen-stimulated lymphocyte proliferation using three-color ¯ow cytometrya,b Days of culture
CD3
IgM
CD4 CD8
CD4 CD8alo
CD4 CD8ahi
CD4 CD8
gd TCR
Con A 3 5
2571 268 3659 324d
33 15c 35 14c
113 32 abd 92 48 abcd
621 94 b 708 106 b
1066 165 1723 146d
461 54 b 1024 201d b
641 110 971 134
PHA 3 5
2177 230 2851 444
16 11c 8 4c
118 47 abd 113 51 bc
599 80 485 97 c
931 121 919 121
450 53 b 1220 176d
650 110 687 153 c
32 15c 24 13c
23 12 bc 0 10 bc
174 30 478 89d
171 34 524 128d
33 19 c 0 10 c
PWM 3 5
336 93 818 128d
44 19 bc 6 3 bc
a
Three-color ¯ow cytometry was used to analyze proliferating PBMCs and data represent the mean number of cells in gate R1 that had proliferated per 10,000 events analyzed in response to stimulation with 5 mg/ml mitogen n 8. b Comparisons of letters (a±d) do not include data representing responses of CD3 and IgM cells: signi®cantly P < 0:05 less than (letter `a') CD8lo, (letter `b') CD8hi, (letter `c') CD4 CD8 and (letter `d') gd T cells for the same mitogenic stimulation and day of culture (e.g. horizontal comparisons). c Signi®cantly P < 0:05 less than CD3 cells for the same mitogenic stimulation and day of culture (i.e. horizontal comparison between CD3 cells and IgM cells). d Signi®cantly P < 0:05 greater than data representing responses at 3 days of culture for the same lymphocyte subset and the same mitogenic stimulation (i.e. vertical comparison for the same mitogenic stimulation).
4. Discussion Proliferative responses of lymphocytes have been used to evaluate the immunological status of the host following onset of disease or vaccination with a speci®c immunogen. While useful, proliferative assays (i.e. 3 H-thymidine or BrdU incorporation) provide limited information with respect to the responding cell types. These approaches only facilitate detection of cells capable of responding during the pulse period following the addition of the reagent to the culture. In addition, traditional blastogenesis assays or the incorporation of BrdU would not permit the accurate detection of daughter generations accumulated over the entire culture period. Unless cumbersome cell separation techniques are utilized, the responsiveness of speci®c subsets cannot be ascertained. This weakness in traditional proliferation assays has been overcome by the use of ¯ow cytometry (Caruso et al., 1997; Lyons, 2000; Schmid et al., 2000). Flow cytometry has been used to evaluate the proliferation of human and murine lymphocytes (Lyons, 2000). In particular, the use of vital dyes in combination with analysis of cell surface markers allows the evaluation of speci®c subset analysis in unfractionated populations of lymphocytes. This is particularly important because cell-to-cell interactions (e.g. T cell and anti-
gen-presenting cells) are often required for optimal response. In this regard, the proliferative responses of porcine PBMC to speci®c mitogens were analyzed using multicolor ¯ow cytometry. While subset responsiveness for mouse and human lymphocytes to speci®c mitogens has been described, there is little or no published information describing either the subsets of porcine lymphocytes that respond to Con A, PHA, or PWM nor the kinetics of the subset responses. Flow cytometry has been used to evaluate the recall response of porcine subpopulations to viral antigens (Summer®eld et al., 1996; Zuckermann and Husmann, 1996). These previous studies used fractionated subpopulations of PBMCs while the present study examined the response of speci®c lymphocyte subsets in unfractionated populations to mitogen stimulation. This allowed for dynamic cell-to-cell interactions, which may be important in overall response. For instance, mouse and human B cells have been shown to be dependent on the presence of T cells for proliferation to PWM stimulation (Keightley et al., 1976; Janossy et al., 1977). Likewise, the proliferative responses of gd T cells have been shown to be dependent upon the presence of accessory cells (e.g. antigen-presenting cells) or soluble factors from accessory cells (Hanrahan et al., 1997). Use of the PKH assay, however, allows for the quanti®cation of
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Fig. 4. PKH2 ¯uorescence intensity of porcine gd TCR T cells following stimulation with mitogens. Porcine PBMC were stained with PKH2 dye, cultured and labeled with appropriate mAb and analyzed by ¯ow cytometry as described in Section 2. Representation of PKH2 intensity histograms showing mitogen-stimulated ((B) Con A, (C) PHA, (D) PWM) vs. (A) non-stimulated cells. As cells divide there is a generational shift (i.e. PKH2bright (right, black peak) to PKH2dim (left, gray peaks)) from parent to daughter populations. Each peak to the left of the parental peak is equivalent to a divisional generation. Note lack of proliferation of gd TCR T cells in response to PWM stimulation. Data represents the response of peripheral blood gd T cells from a single pig. The response was representative of all pigs n 8. Data analyzed using Mod®t software.
lymphocyte proliferation when subsets are allowed to interact in a mixed culture. This study illustrates the quanti®cation of porcine lymphocyte subset proliferation. Total T cell, CD4 CD8 , CD8alo, CD8ahi and CD4 CD8 T cell
subsets all responded to stimulation by Con A, PHA or PWM. The PKH assay also demonstrated signi®cant P < 0:05 proliferative responses to Con A or PWM within the CD3, CD8ahi and CD4 CD8 populations. Cultures stimulated with PHA or Con A, CD4 CD8 T cells demonstrated signi®cant increases in cell numbers between days 3 and 5 of culture. Conversely, proliferation of single positive CD4 cells in response to mitogen was not detected at either time point. This, along with the increase in CD4 CD8 T cells over time, may indicate that activated or proliferating CD4 T cells express CD8a. Zuckermann and Gaskins (1996) have described this observation using cell separation techniques. Using PKH2 ¯uorescence intensity and monoclonal antibodies speci®c to lymphocyte subsets, contributions of individual lymphocyte subsets to the overall proliferative response were measured. Traditional lymphocyte proliferation assays (e.g. 3 H-thymidine incorporation, MTT assay), however, have been used to assess the relative responsiveness to mitogens or speci®c antigens. Utilization of ¯uorescent cell tracking dyes allows for the measurement of cumulative and individual proliferation of all cell types over the entire culture period. This is a distinct advantage over traditional methods of assessing cellular proliferation that only measure 3 H-thymidine incorporation at a speci®c endpoint. One difference between the two assays would be that signi®cant decreases can be observed in the proliferative response over time using 3 H-thymidine incorporation while samples analyzed by the PKH2 method would continue to indicate increasing or stable responses. In this study, while the results of 3 H-thymidine incorporation revealed that the peak response to mitogen stimulation was on day 3 of culture, the ¯ow cytometric assay indicated that proliferation continued within the culture for at least 5 days (Fig. 4). This data emphasizes that although traditional 3 H-thymidine incorporation can demonstrate the proliferative responses at a speci®c time point, the use of vital dyes like PKH2 permits the measurement of cumulative proliferation over time. In addition, analysis of the number of cells proliferating to a given stimulus may more accurately re¯ect the magnitude of the response than does traditional blastogenesis. This is shown in Fig. 2 by comparing the stimulation index induced by PWM (day 3) to that for the other two mitogens. While the
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stimulation index was similar for all three mitogens, there were signi®cantly fewer cells responding to PWM. This suggests that 3 H-thymidine incorporation is less likely to detect differences in lymphocyte responsiveness than is the PKH2 assay. While gd TCR lymphocytes did proliferate to Con A and PHA stimulation, they did not proliferate in response to PWM stimulation. The data in Table 1 shows that there was no signi®cant increase in the gd T cell population between days 3 and 5. This is in contrast to the proliferation of the CD4 CD8 T cells. Further studies are needed to determine whether the gd TCR lymphocytes are responding directly to the mitogens or whether this response is dependent upon factors (e.g. cytokines) secreted from other cell types (e.g. CD4 CD8 T cells). It should also be noted that the CD8ahi population demonstrated differential proliferative kinetics depending on the mitogen used for stimulation (Table 1). While the Con A-stimulated CD8ahi T cells continued to proliferate through 5 days of culture, stimulation with PHAinduced proliferation of the CD8ahi T cells only within the ®rst 3 days of culture. Unlike observations in bovine studies (Quade and Roth, 1999), porcine B cells did not appear to proliferate in response to mitogen stimulation. One explanation for the inability to detect B cell proliferative responses is that porcine IgM cells (i.e. B cells) are smaller than the CD3 cells and were excluded from the analysis because of their location in the R2 gate (as shown in Fig. 1). In conclusion, this study is the ®rst to document the proliferative response of porcine T cell subsets to Con A, PHA or PWM. Assays that allow for cell-tocell interactions will provide a more relevant depiction of whole population as well as subset responses than assays employing cell separation techniques. The use of ¯ow cytometric assays in addition to standard 3 H-thymidine incorporation assays will allow investigators to accurately evaluate the immunologic status of PBMC collected from healthy or ill individuals. Acknowledgements The authors wish to acknowledge Dr. L.E. Evans for providing the pigs for this study and Dr. Kathleen
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Mullin and Jackie Jens for kindly collecting peripheral blood samples. Thanks also to Dr. Kristi Harkins for lending her expertise and advice and to Dr. Kevan Flaming for editorial assistance. This work was supported, in part, by grants funded by the Healthy Livestock Initiative, Iowa State University and USDA-NRI Grant #960-2438.
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¯uorescence and DNA ¯ow cytometry. J. Immunol. Methods 235, 121±131. Summer®eld, A., Rziha, H., SaalmuÈller, A., 1996. Functional characterization of porcine CD4 CD8 extrathymic T lymphocytes. Cell. Immunol. 168, 291±296. Waters, W.R., Sacco, R.E., Dorn, A.D., Hontecillas, R., Zuckermann, F.A., Wannemuehler, M.J., 1999. Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin. Vet. Immunol. Immunopathol. 69, 75±87. Waters, W.R., Hontecillas, R., Sacco, R.E., Zuckermann, F.A., Harkins, K.R., Bassaganya-Riera, J., Wannemuehler, M.J., 2000a. Antigen-speci®c proliferation of porcine CD8aa cells to an extracellular bacterial pathogen. Immunology 101, 333±341.
Waters, W.R., Pesch, B.A., Hontecillas, R., Sacco, R.E., Zuckermann, F.A., Wannemuehler, M.J., 2000b. Cellular immune responses of pigs induced by vaccination with either a whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin. Vaccine 18, 711±719. Wells, A.D., Gudmundsdottir, H., Turka, L.A., 1997. Following the fate of individual T cells throughout activation and clonal expansion. J. Clin. Invest. 100, 3173±3183. Zuckermann, F.A., Gaskins, H.R., 1996. Distribution of porcine CD4/CD8 double-positive T lymphocytes in mucosa-associated lymphoid tissues. Immunology 87, 493±499. Zuckermann, F.A., Husmann, R.J., 1996. Functional and phenotypic analysis of porcine peripheral blood CD4/CD8 doublepositive T cells. Immunology 87, 500±512.
Characterization of mitogen-stimulated porcine lymphocytes using a stable ¯uorescent dye (PKH2) and multicolor ¯ow cytometry A.D. Dorna,*, W.R. Watersb, V.M. Byersa, B.A. Peschc, M.J. Wannemuehlera a
Veterinary Medical Research Institute, Iowa State University, 1802 Elwood Drive, Ames, IA 50011, USA US Department of Agriculture, Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, P.O. Box 70, Ames, IA 50010-0070, USA c US Department of Agriculture, Periparturient Diseases of Cattle Research Unit, National Animal Disease Center, Agricultural Research Service, P.O. Box 70, Ames, IA 50010-0070, USA b
Received 9 August 2001; received in revised form 28 December 2001; accepted 28 January 2002
Abstract Stimulation of lymphocyte proliferation using mitogens or speci®c antigens is a method that is used frequently to assess immune responsiveness. While useful, lymphocyte blastogenesis, or 3 H-thymidine incorporation, provides little information regarding the response of speci®c subsets to the stimulant. Here, we report that the ¯uorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multicolor ¯ow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using 3 Hthymidine incorporation as well as a ¯ow cytometric-based proliferation assay. From the 3 H-thymidine incorporation data alone, it was observed that PBMC stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and ¯ow cytometric analysis, the responsiveness of speci®c lymphocyte subsets to mitogen stimulation was detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4 CD8 , CD4 CD8ahi , CD4 CD8alo and gd TCR cells. PWM stimulation induced responses by CD4 CD8, CD4CD8ahi but not by CD4 CD8alo or gd TCR cells. Con A stimulation resulted in a sustained proliferation of CD8ahi cells over the 5-day period while PHA stimulation resulted in proliferation that peaked within the ®rst 3 days. Little or no proliferative responses were detected within the IgM population (e.g. B cells). This is the ®rst study to de®ne the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Porcine lymphocytes; PKH2; Mitogens; Flow cytometry; Blastogenesis; Lymphoproliferation
1. Introduction Abbreviations: cRPMI, complete tissue culture medium; CO2, carbon dioxide; PE, phycoerythrin; FACS, ¯uorescent activated cell sorter; SI, stimulation index; 7AAD, 7-amino-actinomycin D; CFSE, carboxy¯uorescein succinimidyl ester * Corresponding author. Tel.: 1-515-294-1025; fax: 1-515-294-1401. E-mail address: [email protected] (A.D. Dorn).
Lymphocyte proliferation is a useful tool for measuring immune function and mitogens have long been used to induce proliferation in lymphocyte cultures (Resch et al., 1976; Janossy et al., 1977; Flaming et al., 1989; Morrison et al., 1990; Waters et al., 1999). Standard assays of lymphocyte proliferation include
0165-2427/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 0 2 2 - 3
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measurement of 3 H-thymidine incorporation or analysis of cell surface markers on lymphoblasts by ¯ow cytometry (Zuckermann and Husmann, 1996). Analysis of lymphoblasts for cell surface marker expression, however, does not determine if these cells have actually divided, only the relative size and granularity. More recently, vital ¯uorescent stains such as carboxy¯uorescein succinimidyl ester (CFSE) and PKH dyes have been used to determine proliferation of in vitro stimulated lymphocytes (Wells et al., 1997; Gudmundsdottir et al., 1999; Waters et al., 2000a). Use of vital ¯uorescent stains to determine proliferation affords the capability to determine proliferation over the entire culture period as well as the contribution of individual lymphocyte subsets to the proliferative response (Ashley et al., 1993; Quade and Roth, 1999). Previously, to study speci®c lymphocyte subset proliferation, the cells would have to be physically separated (e.g. by magnetic or ¯ow cytometric separation) and then cultured in vitro (Zuckermann and Husmann, 1996). This type of assay does not account for the importance of cell-to-cell interactions in the proliferative process (Hanrahan et al., 1997). Using the vital ¯uorescent stains, proliferation of speci®c cell subsets can be identi®ed in unfractionated cell populations. The objective of the present study was to measure the proliferation of porcine lymphocyte subsets to mitogen stimulation using the vital ¯uorescent stain, PKH2. Lymphocyte proliferation was measured by 3 H-thymidine incorporation and the PKH2 ¯ow cytometry-based assay in order to compare the results obtained by the two methods and at two time points. This is the ®rst study to determine the contribution of individual lymphocyte subsets to the proliferative response of mitogen-stimulated porcine peripheral blood mononuclear cells (PBMCs). 2. Materials and methods 2.1. Pigs Eight 4±5-week-old crossbred pigs from a breeding herd maintained at the College of Veterinary Medicine, Iowa State University, Ames, IA were used for the study. No clinical signs of disease were detected during the study period. The Iowa State University Committee on Animal Care approved all procedures.
2.2. Harvesting of PBMCs Peripheral blood was obtained from the anterior vena cava and PBMC were isolated by gradient centrifugation. Brie¯y, whole blood was diluted 1:3 in sterile phosphate buffered saline (0.05 M, pH 7.4; PBS), overlaid onto ®coll-hypaque (Histopaque 1077; Sigma Chemical Company, St. Louis, MO), and centrifuged at 550 g for 40 min. PBMC were obtained from the ®coll/plasma interface, washed two times with sterile PBS, and standardized to 2 107 /ml for PKH2 staining. 2.3. PKH2 assay Isolated PBMC were stained with the green ¯uorescent dye, PKH2 (Sigma) according to the manufacturer's recommendations. Brie¯y, 2 107 PBMC were centrifuged at 150 g for 10 min, supernatants decanted completely and 1 ml of diluent (provided in the kit) added to each tube of cells. Cells were thoroughly resuspended in diluent using a P1000 Pipetman and a large bore pipette tip then transferred to the tube containing 1 ml of PKH2 green ¯uorescent dye (4 10 6 M). Because the cells are more fragile after the dye is added, a Pipetman (P1000) was used between every wash step rather than a vortexer to be certain the cells were thoroughly resuspended between washings. Cells were then incubated in the dye solution for 5 min, followed by a 1 min incubation with 2 ml of fetal bovine serum (Hyclone Laboratories, Logan, UT, FBS) to stop the reaction. After 1 min incubation, 4 ml of RPMI 1640 with L-glutamine (Mediatech, Huntingford, VA) was added and the cells were centrifuged at 150 g for 10 min. The supernatant was decanted and the cells were washed three more times with RPMI 1640 and resuspended in culture medium for counting. Following enumeration, the PKH2stained cells were used for both the ¯ow cytometric assay and the 3 H-thymidine incorporation assay. In this way, any effects of the PKH2 dye on subsequent cellular proliferation would be constant to both assays. Culture medium (complete RPMI medium, cRPMI) consisted of RPMI 1640 with L-glutamine (Mediatech) supplemented with 1% non-essential amino acids (Sigma), 1% sodium pyruvate (Sigma), 2% essential amino acids (Mediatech), 25 mM HEPES buffer (Sigma), 100 units/ml penicillin, 0.1 mg/ml streptomycin, 1% L-glutamine (Difco, Detroit, MI),
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5 10 5 M 2-mercaptoethanol (Sigma), and 10% heat-inactivated FBS. Wells of 96-well ¯at-bottomed microtiter plates were seeded with 2 105 PKH2stained mononuclear cells in cRPMI at a total volume of 200 ml per well. Wells also contained either concanavalin A (Con A, 5 mg/ml, Sigma), puri®ed phytohemagglutinin (PHA, 5 mg/ml, Murex Biotech Ltd., Dartford, UK), pokeweed mitogen (PWM, 5 mg/ml, Sigma) or cRPMI alone (e.g. no stimulation). These concentrations were previously determined to be optimal for lymphocyte blastogenesis (data not shown). Plates were then incubated for 3 or 5 days at 37 8C in 5% CO2 in air. Cells from replicate wells of each treatment group were pooled and analyzed by ¯ow cytometry for PKH2 staining intensity as well as cell surface marker expression. 2.4. Preparation of cells and analysis by ¯ow cytometry Cultured mononuclear cells were analyzed for expression of cell surface antigens and PKH2 intensity as previously described (Waters et al., 2000b). Brie¯y, cell suspensions were harvested from the 96-well culture plates and like wells were pooled according to treatment. Cell suspensions (100 ml) were transferred to appropriate wells of a 96-well, round-bottomed microtiter plate and stained with 50 ml of primary antibody to swine leukocyte surface antigens. The monoclonal antibodies used to detect cell surface markers were anti-CD3 (8E62, VMRD, Pullman, WA), PE-conjugated anti-CD4 (74-12-4), biotinylated anti-CD8 (76-2-11), anti-gd T cells (PGBL22A, VMRD), anti-IgM (PG145A, VMRD). PBS containing 1% FBS and 0.04% sodium azide was used as a FACS buffer. Wells with appropriate isotype control antibodies (all diluted in the FACS buffer) were used. Cells were labeled using the following combinations of monoclonal antibodies and analyzed using two- or three-color ¯ow cytometry. T cells (CD3) were differentiated from B cells (IgM) using 8E6 (PElabeled secondary antibody) and PG145A (PE-labeled secondary antibody) in separate tubes. T cell subsets were evaluated for expression of CD4 and CD8a using 74-12-4[PE 76-2-11 (biotinylated streptavidinconjugated CyChrome). The percentage of gdT cells were measured using PGBL22A (PE-labeled secondary antibody).
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Following a 15 min incubation, cells were washed twice with FACS buffer and resuspended in 50 ml of secondary antibody. Appropriate secondary antibodies used were PE-conjugated goat anti-mouse IgM for PG145A, PE-conjugated goat anti-mouse IgG1 for 8E62 and PGBL22A, and streptavidin-conjugated CyChrome (Sigma) for the biotinylated 76-2-11. Cells were incubated for 15 min with the secondary antibodies, washed twice and resuspended in FACS buffer. Cells were then analyzed using a Becton Dickinson FACScan ¯ow cytometer (10,000 events from each sample). The number of cells proliferating was calculated by the following formula: % proliferation to mitogen no: of cells in the R1 gate % proliferation with no stimulation no: of cells in the R1 gate and presented as the mean number of proliferative cells S:E:M: per 10,000 PBMC. R1 is the region containing live lymphocytes based upon typical forward and side light scatter properties of porcine lymphocytes (Fig. 1). The excitation and emission wavelengths for PKH2 are 490 and 504 nm, respectively. Percent proliferation is de®ned as the percent of cells (e.g. PKH2 dim cells) that are not within the parent generation (e.g. PKH2 bright cells). The stability of the dye incorporation into the lipid membrane of the cell ensures that when cells divide the dye is distributed equally between daughter cells. PKH2 staining intensity diminishes with each cell division resulting in a decreased mean ¯uorescence intensity (Ashley et al., 1993). Mod®t Proliferation Wizard (Verity Software House Inc., Topsham, Maine) was used for cell proliferation analysis and CellQuest software (Becton Dickinson, San Jose, CA) was used for phenotype analysis. 2.5. Lymphocyte blastogenesis Wells of 96-well ¯at-bottomed microtiter plates were seeded with 2 105 PKH2-stained mononuclear cells in cRPMI at a total volume of 200 ml per well. Wells also contained either 5 mg/ml Con A, 5 mg/ml PHA, 5 mg/ml PWM, or cRPMI alone (e.g. no stimulation). Plates were then incubated for 3 or 5 days at 37 8C in 5% CO2 in air. After 2 or 4 days, 0.5 mCi of
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methyl-3 H-thymidine (speci®c activity 6.7 Ci/mmol, Amersham Life Science, Arlington Heights, IL) at a concentration of 50 mCi/ml in 10 ml of RPMI 1640 with L-glutamine (Mediatech) was added to each well, and plates incubated for an additional 16±18 h. Well contents were harvested onto glass ®ber ®lters, and incorporated radioactivity measured by liquid scintillation counting. Treatments were run in triplicate and stimulation indices (SIs) calculated by dividing counts per minute of stimulated wells by counts per minute from non-stimulated wells. Data are presented as mean SI S:E:M: 2.6. Statistics Data were analyzed by one-way analysis of variance followed by Tukey±Kramer multiple comparisons test. Differences between groups were considered signi®cant if probability values of P < 0:05 were obtained. 3. Results 3.1. Induction of lymphoblasts by mitogen stimulation
Fig. 1. Forward and side scatter analysis of control and mitogenstimulated PBMC. Mitogen stimulation of PBMC results in increased number of lymphoblasts in the live lymphocyte gate (R1) as compared to (A) non-stimulated PBMC. Note fewer number of lymphoblasts in (D) PWM-stimulated cultures as compared to the number of lymphoblasts detected in (B) Con Aand (C) PHA-stimulated cultures. Cells located in gate R2 are predominantly dead cells and debris or apoptotic cells as determined by staining with Annexin V and 7AAD. Data presented is from a single animal and is representative of all animals n 8.
Proliferation of lymphocytes results in the formation of lymphoblasts. These larger cells can be detected by ¯ow cytometry (e.g. lymphocytes with increased forward (size) and side (granularity) light scatter properties). As shown in Fig. 1, increased number of lymphoblasts were detected in the live lymphocyte gate (R1) after stimulation with Con A (Fig. 1B), PHA (Fig. 1C), or PWM (Fig. 1D) as compared to non-stimulated PBMC (Fig. 1A). Fewer number of lymphoblasts were detected for PWM-stimulated cultures as compared to the number of lymphoblasts detected in Con A- or PHA-stimulated cultures. Cells that are located in gate R2 are predominantly dead cells and debris or apoptotic cells as determined by staining with Annexin V and 7-amino-actinomycin D (7AAD). 3.2. Lymphocyte proliferation, effects of time and assay Proliferative responses of PBMC recovered from healthy, young pigs were assessed by two methods: a standard 3 H-thymidine incorporation assay and the
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Fig. 2. Assessment of cellular proliferation by blastogenesis or ¯ow cytometry. Proliferation data comparing (A) 3-day vs. (B) 5-day cultured PBMC with mitogen stimulation, as described in Section 2, using two different assays. Proliferative responses as measured by the PKH2 assay were increased on day 5 of culture as compared to day 3 of culture, whereas 3 H-thymidine incorporation was decreased on day 5 of culture as compared to day 3 of culture. Counts per minute (e.g. for the 3 H-thymidine assay) for non-stimulated cultures ranged from 428 to 5004. Data represents the mean S:E:M: for eight pigs. (a) Signi®cantly P < 0:05 greater as compared to stimulation index for Con A response at day 5 of culture (e.g. vertical comparisons). Although not statistically signi®cant, proliferation induced by PHA or PWM stimulation as measured by 3 H-thymidine incorporation was also greater on day 3 of culture as compared to day 5 of culture. (b) Signi®cantly P < 0:05 less than responses by PBMC stimulated with either Con A or PHA (e.g. horizontal comparisons) as measured by the PKH2 assay for either 3 or 5 days of culture.
PKH2 ¯ow cytometric-based assay. Proliferation induced by Con A, as measured by 3 H-thymidine incorporation was signi®cantly P < 0:05 greater on day 3 of culture as compared to day 5 (Fig. 2). Although not statistically signi®cant, proliferation induced by PHA or PWM stimulation, as measured by 3 H-thymidine incorporation, was also greater on day 3 of culture as compared to day 5 of culture. The
counts per minute for non-stimulated cultures ranged from 428 to 5004. Total cell proliferative responses as measured by the PKH2 assay were greater on day 5 of culture as compared to day 3, although not statistically signi®cant. Proliferative responses to PWM stimulation at both 3 and 5 days of culture were signi®cantly P < 0:05 less than responses to Con A or PHA stimulation as measured by the PKH2 assay.
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3.3. Lymphocyte subset proliferation Three-color ¯ow cytometry was used to assess proliferation of lymphocyte subsets (Fig. 3). The proliferative response of CD3 cells was signi®cantly P < 0:05 greater than responses by IgM cells for each mitogen at both 3 and 5 days of culture (Table 1). Five days of Con A (Fig. 3B) or PHA (Fig. 3C) stimulation resulted in increased number of cells detected in the CD8ahi (R3 gate), CD4 CD8 (R4 gate), and CD4 CD8alo (R5 gate) populations as compared to non-stimulated cells (Fig. 3A). After 5 days of PWM stimulation, increased number of cells were detected in the CD4 CD8 (R4 gate) as compared to non-stimulated cells (Fig. 3A). The distribution of proliferation of individual subsets is shown in Table 1. Although analysis of this table is complex, several observations can be made. First, cells stimulated with either Con A or PHA, demonstrated a proliferative response by each of the individual lymphocyte subsets analyzed (except for IgM cells) at each time point. Second, few CD4 single positive (i.e. CD4 CD8 ) cells had proliferated in comparison to other subsets examined. Third, CD8a cells (e.g. CD4 CD8alo , CD4 CD8ahi , CD4 CD8 ) and gd TCR cells contributed to the proliferative response with the exception that gd TCR cells did not proliferate to PWM stimulation. Statistical comparisons between individual subsets, days of culture, and mitogen stimulation are provided in Table 1. Proliferation of gd TCR cells is demonstrated in Fig. 4. Using Mod®t Proliferation Wizard software, live cells (e.g. R1 gate in Fig. 1) were analyzed for PKH2 staining intensity and histograms generated. The histograms demonstrate that as cells divide there is a generational shift from right to left (i.e. PKH2bright to PKH2dim) corresponding to parental and daughter populations, respectively. Each peak to the left of the parental peak (black peak in Fig. 4) is equivalent to a divisional generation (gray peaks in Fig. 4). Over time, following mitogen stimulation, the percentage of the total population of stained cells in the parental peak decreases and more generational peaks appear. Fig. 4 further corroborates the data shown in Table 1 which demonstrate a lack of proliferation of gd TCR cells to PWM stimulation.
Fig. 3. Three-color ¯ow cytometric analysis of porcine PBMC stimulated by mitogen: (A) no stimulation; (B) Con A stimulation; (C) PHA stimulation; (D) PWM stimulation. Distribution of porcine lymphocyte subsets as de®ned by CD4 and CD8 expression demonstrating effects of mitogen stimulation. R3: CD8ahi cells; R4: CD4 CD8 cells; R5: CD4 CD8alo cells; R6: CD4CD8 cells. Data represents the response of cells from a single pig and is representative of the group n 8.
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Table 1 Measurement of mitogen-stimulated lymphocyte proliferation using three-color ¯ow cytometrya,b Days of culture
CD3
IgM
CD4 CD8
CD4 CD8alo
CD4 CD8ahi
CD4 CD8
gd TCR
Con A 3 5
2571 268 3659 324d
33 15c 35 14c
113 32 abd 92 48 abcd
621 94 b 708 106 b
1066 165 1723 146d
461 54 b 1024 201d b
641 110 971 134
PHA 3 5
2177 230 2851 444
16 11c 8 4c
118 47 abd 113 51 bc
599 80 485 97 c
931 121 919 121
450 53 b 1220 176d
650 110 687 153 c
32 15c 24 13c
23 12 bc 0 10 bc
174 30 478 89d
171 34 524 128d
33 19 c 0 10 c
PWM 3 5
336 93 818 128d
44 19 bc 6 3 bc
a
Three-color ¯ow cytometry was used to analyze proliferating PBMCs and data represent the mean number of cells in gate R1 that had proliferated per 10,000 events analyzed in response to stimulation with 5 mg/ml mitogen n 8. b Comparisons of letters (a±d) do not include data representing responses of CD3 and IgM cells: signi®cantly P < 0:05 less than (letter `a') CD8lo, (letter `b') CD8hi, (letter `c') CD4 CD8 and (letter `d') gd T cells for the same mitogenic stimulation and day of culture (e.g. horizontal comparisons). c Signi®cantly P < 0:05 less than CD3 cells for the same mitogenic stimulation and day of culture (i.e. horizontal comparison between CD3 cells and IgM cells). d Signi®cantly P < 0:05 greater than data representing responses at 3 days of culture for the same lymphocyte subset and the same mitogenic stimulation (i.e. vertical comparison for the same mitogenic stimulation).
4. Discussion Proliferative responses of lymphocytes have been used to evaluate the immunological status of the host following onset of disease or vaccination with a speci®c immunogen. While useful, proliferative assays (i.e. 3 H-thymidine or BrdU incorporation) provide limited information with respect to the responding cell types. These approaches only facilitate detection of cells capable of responding during the pulse period following the addition of the reagent to the culture. In addition, traditional blastogenesis assays or the incorporation of BrdU would not permit the accurate detection of daughter generations accumulated over the entire culture period. Unless cumbersome cell separation techniques are utilized, the responsiveness of speci®c subsets cannot be ascertained. This weakness in traditional proliferation assays has been overcome by the use of ¯ow cytometry (Caruso et al., 1997; Lyons, 2000; Schmid et al., 2000). Flow cytometry has been used to evaluate the proliferation of human and murine lymphocytes (Lyons, 2000). In particular, the use of vital dyes in combination with analysis of cell surface markers allows the evaluation of speci®c subset analysis in unfractionated populations of lymphocytes. This is particularly important because cell-to-cell interactions (e.g. T cell and anti-
gen-presenting cells) are often required for optimal response. In this regard, the proliferative responses of porcine PBMC to speci®c mitogens were analyzed using multicolor ¯ow cytometry. While subset responsiveness for mouse and human lymphocytes to speci®c mitogens has been described, there is little or no published information describing either the subsets of porcine lymphocytes that respond to Con A, PHA, or PWM nor the kinetics of the subset responses. Flow cytometry has been used to evaluate the recall response of porcine subpopulations to viral antigens (Summer®eld et al., 1996; Zuckermann and Husmann, 1996). These previous studies used fractionated subpopulations of PBMCs while the present study examined the response of speci®c lymphocyte subsets in unfractionated populations to mitogen stimulation. This allowed for dynamic cell-to-cell interactions, which may be important in overall response. For instance, mouse and human B cells have been shown to be dependent on the presence of T cells for proliferation to PWM stimulation (Keightley et al., 1976; Janossy et al., 1977). Likewise, the proliferative responses of gd T cells have been shown to be dependent upon the presence of accessory cells (e.g. antigen-presenting cells) or soluble factors from accessory cells (Hanrahan et al., 1997). Use of the PKH assay, however, allows for the quanti®cation of
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Fig. 4. PKH2 ¯uorescence intensity of porcine gd TCR T cells following stimulation with mitogens. Porcine PBMC were stained with PKH2 dye, cultured and labeled with appropriate mAb and analyzed by ¯ow cytometry as described in Section 2. Representation of PKH2 intensity histograms showing mitogen-stimulated ((B) Con A, (C) PHA, (D) PWM) vs. (A) non-stimulated cells. As cells divide there is a generational shift (i.e. PKH2bright (right, black peak) to PKH2dim (left, gray peaks)) from parent to daughter populations. Each peak to the left of the parental peak is equivalent to a divisional generation. Note lack of proliferation of gd TCR T cells in response to PWM stimulation. Data represents the response of peripheral blood gd T cells from a single pig. The response was representative of all pigs n 8. Data analyzed using Mod®t software.
lymphocyte proliferation when subsets are allowed to interact in a mixed culture. This study illustrates the quanti®cation of porcine lymphocyte subset proliferation. Total T cell, CD4 CD8 , CD8alo, CD8ahi and CD4 CD8 T cell
subsets all responded to stimulation by Con A, PHA or PWM. The PKH assay also demonstrated signi®cant P < 0:05 proliferative responses to Con A or PWM within the CD3, CD8ahi and CD4 CD8 populations. Cultures stimulated with PHA or Con A, CD4 CD8 T cells demonstrated signi®cant increases in cell numbers between days 3 and 5 of culture. Conversely, proliferation of single positive CD4 cells in response to mitogen was not detected at either time point. This, along with the increase in CD4 CD8 T cells over time, may indicate that activated or proliferating CD4 T cells express CD8a. Zuckermann and Gaskins (1996) have described this observation using cell separation techniques. Using PKH2 ¯uorescence intensity and monoclonal antibodies speci®c to lymphocyte subsets, contributions of individual lymphocyte subsets to the overall proliferative response were measured. Traditional lymphocyte proliferation assays (e.g. 3 H-thymidine incorporation, MTT assay), however, have been used to assess the relative responsiveness to mitogens or speci®c antigens. Utilization of ¯uorescent cell tracking dyes allows for the measurement of cumulative and individual proliferation of all cell types over the entire culture period. This is a distinct advantage over traditional methods of assessing cellular proliferation that only measure 3 H-thymidine incorporation at a speci®c endpoint. One difference between the two assays would be that signi®cant decreases can be observed in the proliferative response over time using 3 H-thymidine incorporation while samples analyzed by the PKH2 method would continue to indicate increasing or stable responses. In this study, while the results of 3 H-thymidine incorporation revealed that the peak response to mitogen stimulation was on day 3 of culture, the ¯ow cytometric assay indicated that proliferation continued within the culture for at least 5 days (Fig. 4). This data emphasizes that although traditional 3 H-thymidine incorporation can demonstrate the proliferative responses at a speci®c time point, the use of vital dyes like PKH2 permits the measurement of cumulative proliferation over time. In addition, analysis of the number of cells proliferating to a given stimulus may more accurately re¯ect the magnitude of the response than does traditional blastogenesis. This is shown in Fig. 2 by comparing the stimulation index induced by PWM (day 3) to that for the other two mitogens. While the
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stimulation index was similar for all three mitogens, there were signi®cantly fewer cells responding to PWM. This suggests that 3 H-thymidine incorporation is less likely to detect differences in lymphocyte responsiveness than is the PKH2 assay. While gd TCR lymphocytes did proliferate to Con A and PHA stimulation, they did not proliferate in response to PWM stimulation. The data in Table 1 shows that there was no signi®cant increase in the gd T cell population between days 3 and 5. This is in contrast to the proliferation of the CD4 CD8 T cells. Further studies are needed to determine whether the gd TCR lymphocytes are responding directly to the mitogens or whether this response is dependent upon factors (e.g. cytokines) secreted from other cell types (e.g. CD4 CD8 T cells). It should also be noted that the CD8ahi population demonstrated differential proliferative kinetics depending on the mitogen used for stimulation (Table 1). While the Con A-stimulated CD8ahi T cells continued to proliferate through 5 days of culture, stimulation with PHAinduced proliferation of the CD8ahi T cells only within the ®rst 3 days of culture. Unlike observations in bovine studies (Quade and Roth, 1999), porcine B cells did not appear to proliferate in response to mitogen stimulation. One explanation for the inability to detect B cell proliferative responses is that porcine IgM cells (i.e. B cells) are smaller than the CD3 cells and were excluded from the analysis because of their location in the R2 gate (as shown in Fig. 1). In conclusion, this study is the ®rst to document the proliferative response of porcine T cell subsets to Con A, PHA or PWM. Assays that allow for cell-tocell interactions will provide a more relevant depiction of whole population as well as subset responses than assays employing cell separation techniques. The use of ¯ow cytometric assays in addition to standard 3 H-thymidine incorporation assays will allow investigators to accurately evaluate the immunologic status of PBMC collected from healthy or ill individuals. Acknowledgements The authors wish to acknowledge Dr. L.E. Evans for providing the pigs for this study and Dr. Kathleen
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Mullin and Jackie Jens for kindly collecting peripheral blood samples. Thanks also to Dr. Kristi Harkins for lending her expertise and advice and to Dr. Kevan Flaming for editorial assistance. This work was supported, in part, by grants funded by the Healthy Livestock Initiative, Iowa State University and USDA-NRI Grant #960-2438.
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