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Increased number of Leu-2-bearing non-T cells with natural killer activity in chimpanzees
CELLULAR
IMMUNOLOGY
97, 164-172 ( 1986)
Increased Number of Leu-ZBearing Non-T Cells with Natural Killer Activity in Chimpanzees THOMAS
M. FOLKS,’ THOMAS M. CHUSED,DIANE PORTNOY,LINETTE EDISON, WILLIAM
LEISERSON, AND KENNETH
W. SELL
Ofice of the ScientiJic Director and the Laboratory of Microbial Immunity, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205 Received August 12, 1985; acceptedSeptember 23, I985 Peripheral blood lymphocytes from adult and adolescentchimpanzees,as well asadult humans, were studied for phenotypic surfacemarkers by tlow cytometry. Lymphocytes from chimpanzees were found to have increased numbers of Leu-l-, LX-~+ cells as compared to humans. These cells, following preparative electronic cell sorting, were shown to possessnatural killer function. Further analysis of this subpopulation indicated that they lacked responsivenessto a number of T-cell mitogens. The differencesin lymphocyte subpopulations between chimpanzeesand humans can almost be totally accounted for by the Leu-l-, Leu-2+ cells. Phylogenetic disparity between these two speciesmay aho be found within this population. 0 1986 Academic Press,IIIC.
INTRODUCTION Regulation of the immune response is greatly affected by the interactions of helper and suppressor cells (l-5). Functionally distinct yet phenotypically overlapping cytotoxic/suppressor Leu-2+ peripheral blood mononuclear lymphocytes (PBML)2 in the human are Leu-l+ T cells (6). Recently, new monoclonal antibodies have been shown to distinguish the suppressorsubpopulation from the cytotoxic (7). In addition, suppressorcells (Leu-2+) have been shown to be subpopulated into natural killer (NK) cells and high-density T cells lacking Fc receptors (8). In other studies, HNK-l+ (Leu7) cells were shown to function as suppressorcells in both pokeweed mitogen-induced IgG production and mixed lymphocyte reaction (MLR) (9). However, I-AX-~+ cells were shown to be distinct from the HNK- 1+ cells since no immune-complex activation was required for their suppressor function. Less correlation between phenotype and function is known among the nonhuman primates. In a phylogenetic study examining human and nonhuman primate T-lymphocyte surface structures with anti-human T-cell monoclonal antibodies a number of phenotypes were shown to be shared (10). In the present study, we have examined lymphocyte cell surface markers (Leu-1 and Leu-2) in chimpanzees by two-color flu’ To whom correspondence should be addressedat: National Institutes of Health, Building 10, Room I 1B- 13, Bethesda, Md. 20205. 2Abbreviations used:PBML, peripheral blood mononuclear lymphocytes; NK, natural kiheq MLR, mixed lymphocyte reaction; FITC, fluorescein isothiocyanate; PHA, phytohemagghrtinin; Con A, concanavalin A; PWM, pokeweed mitogen. 164 OOOS-8749186 $3.00 Copyri&t Q 1986 by Academic Rss, Inc. All ri@s of reproduction in any form reserved.
165
LEU-2-BEARING NON-T CELLS
orescent analysis. Preparative two-parameter isolation of Leu- 1-, Leu-2+ lymphocytes was carried out by electronic cell sorting. Lymphocytes, isolated by this method, were shown to possessNK function. In addition, two-parameter analysis showed Leu-l-, Leu-2+ cells to be circulating in greater numbers in chimpanzees than humans. These studies suggesta possible phylogenetic link between NK and suppressormarked lymphocytes. MATERIALS
AND METHODS
Preparation of peripheral blood mononuclear leukocytes. Heparinized whole blood was obtained by venipuncture from either 10 normal human adults from our laboratory or 8 chimpanzees (3-20 kg) at the National Institute of Allergy and Infectious Diseases primate facility, Rockville, Maryland (Meloy Laboratories). PBML were isolated by Ficoll-Hypaque; washed three times in RPMI-1640, with glutamine, Hepes, gentamicin, and 10% fetal calf serum (medium); and cryopreserved until further use. Panning andfluorescent antibody staining for sorting. For enrichment by panning, cryopreserved chimpanzee cells were thawed and washed three times in medium, and 1 X lo* PBML were incubated with 100 ~1 of OKT-3 (Table 1) (Ortho Diagnostics, Raritan, N.J.) for 30 min at 4°C. Cells were then washed and added to two Costar Petri dishes precoated with goat anti-mouse Ig (Cappel, Court Malvern, Pa.). After 1 hr at 25°C the nonadherent cells were removed, washed, and stained with 50 ~1 of Leu-3-fluorescein isothiocyanate (FITC) (Becton-Dickinson Monoclonal Center, Mountain View, Calif.). After staining, cells were washed and preparatively sorted on a modified FACS II (Becton-Dickinson). Procedure for two-color analysis and cell sorting. Chimpanzee or human PBML were thawed, washed three times in either RPMI- 1640 for cell sorting or Hanks’ buffer containing 0.05% Na azide and 1% BSA (Sorter Buffer) for cell analysis. Cells were stained with Leu- 1-FITC and Leu-2-P-phycoerythrin (Table 1) and resuspended in 3 ml of RPMI- 1640 at 1 X log/ml for cell sorting or in 1 ml of sorter buffer at 1 X 106/ml for cell analysis. All staining and cell analysis were carried out at 4’C. Cells were analyzed and sorted using a modified FACS II exciting fluorescein with 200~nW, 488-nm output from an Argon laser and exciting phycoerythrin with 150-NW, 568nm output from a krypton laser. Monocytes, erythrocytes, and dead cells were excluded from analysis using a 90” light scatter by forward-angle light scatter gate. Green and red fluorescent signals were stored in list mode using a PDP-11 computer system. Data from two-parameter analysis were collected in matrix form and presented as three-dimensional maps. By retrieving data from list storage, contour plots were used TABLE 1 Specificity of Antibodies Used Antibody
Specificity
Anti-Leu- 1 Anti-Leu-2 (OKT-8) Anti-OKT-3 Anti-human Ig (polyvalent) Anti-Leu-3a
T cells and B-cell subset Suppressor:cytotoxic T subset T cells Human Ig Helper:inducer T subset
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FOLKS ET AL.
to determine the percentage of cells within each contour grouping. Four populations were isolated: Leu-l+, Leu-2+; Leu-l+, Leu-2-; Leu-l-, Leu-2+; and Leu-l-, Leu-2-. The flow rate was 750-1000 cells/set. Analysis of the sorted populations showed 85-97% of the cells to have the selected phenotypes after sorting. NK assay. NK cell assayswere performed by determination of 51Crrelease from target K562 cells. The assaywas carried out in 200~Ccl-volumeLinbro 96 U-bottom multiwell plates. K562 cells (1 X 104/well) were incubated with chimpanzee cells at varying concentrations (E:T ratios from O.l:l to 40:1) for 3 hr at 37°C. Plates were then spun and 100 ~1of supematant was counted on a Beckman 9000 gamma counter for releasedradioactivity. Percentagecytotoxicity was basedon the following formula: [(test cpm) - (spontaneous releasecpm)]/[(total releasecpm) - (spontaneous release cpm)] X 100. Spontaneous releasecpm was never more than 10%of the total release cpm. Standard deviation among triplicates was also never more than 10%. Mitogen assays.Test populations of cells (1 X 105/well) were added to Costar 3596 plates in medium to a total volume of 200 &well. The mitogens phytohemagglutinin (PHA; Difco, Detroit, Mich.), concanavalin A (Con A), and pokeweedmitogen (PWM; Sigma Chemical Co., St. Louis, MO.) were used at concentrations of 10, 1, and 0.1 pg/ml. PHA- and Con A-treated cells were incubated for 4 days while PWM-treated cells were incubated for 5 days at 37’C and 7% COZ. All cells were pulsed with [3H]thymidine (sp act 6.7; New England Nuclear, Boston Mass.) and harvested on a PHD Harvester (Cambridge, Mass.) 12 hr later. Filter disks were counted for radioactivity using 2 ml Econofluor (New England Nuclear) on a Beckman LS7000 beta counter. RESULTS Loss of mitogenic proliferation and enhancement of NK activity after panning and one-color sorting. Chimpanzee PBML were fractionated into OKT-3- cells by panning and further enriched by Leu- 1-FITC preparative sorting. Unfmctionated and Leu- 1-enriched populations of cells were assayedfor their ability to proliferate in the presence of the T-cell mitogens PHA, Con A, and PWM. The data in Fig. 1 indicate that removal of Leu-1 chimpanzee lymphocytes eliminates the ability of the remaining cells (Leu-1 -) to respond to these mitogens. An additional question concerning the function of the Leu- l- population was the type of NK activity it might contain. It was not too surprising to find (Fig. 2) that the Leu-l- population contained an enriched number of cytotoxic cells for K562 over the unfmctionated cells. At all effector-to-targetratios testedthere was an approximately sixfold increase in cytotoxic activity. Two-color qualitative analysis. Since it is well established in the human that NK cells reside in the non-T-cell lymphocyte subpopulation our next question was to determine if the Leu-l- population could be further fractionated in the chimpanzee as it has in the human using Leu-2. Chimpanzee and human PBML were analyzed by two-color fluorescence. Figures 3 and 4 show representative two-parameter histograms of chimpanzee and human unfractionated PBML, respectively. Four populations were defined when red Leu-2 and green Leu-l were compared: (I) Leu-l-, Leu-2-; (II) Leu-l-, Leu-2+; (III) Leul+, Leu-2+; (IV) Leu-l+, Leu-2-. Major differences in cell subpopulation were found especially in the Leu-l-, Leu-2+ staining. Table 2 shows the percentagesof all com-
167
LEU-2-BEARING NON-T CELLS
30000
20000 i
PIG. 1. Unfractionated and OKT-3 panned and preparatively sorted Leu-l- chimpanzee PBML were tested for mitogen responsivenessto Con A, PHA, and PWM. Cells were assayed 4-5 days later for [‘Hlthymidine uptake.
binations of subpopulations while comparing young (~9 kg) chimpanzees, older (>9 kg) chimpanzees, and adult humans. Chimpanzees >9 kg had 2.4 times more Leul-, Leu-2+ (subpopulation II) cells than humans. This difference was almost totally reflected in the percentage of Leu-2-, Leu- 1- cells between chimpanzees and humans (7.0%). Young vs older chimpanzeesshowed major differencesbetween subpopulations Leu-l+, Leu-2- (9.5%) and Leu-l-, Leu-2+ (5.7%).
50
C+SW
CELLS
0 0.01:1
0.1:1
1:l
10:1
lo&l
EFFECTORTARGET
FIG. 2. Unfmctionated and Leu-I- chimpanzee PBML were assayedfor NK activity on KS62 target cells. Varying numbers of NK cells were incubated for 3 hr with 51Cr-labeledK562 cells (1 x 104).
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FOLKS ET AL.
L E U 2 IV
LEU 1 FIG. 3. PBML from eight chimpanzees were stained with Leu-I-FITC and Leu-2-PE and analyzed by two-color flow cytometry. The average percentage for each population is represented.(I) 18.32, (II) 13.996, (III) 17.52, (IV) 50.3%.
Two-color preparative analysis and NKfunction. The finding of increased numbers in subpopulation II (Leu 1-, Leu2+) from chimpanzeesled us to the step of preparatively isolating this cell by two-color flow cytometry and assessingits NK activity. Figure 5
100
E’ u 2 ----.I 10 -----_
I
LEU 1 FIG. 4. PBML from 10 normal human subjectswere stained with Leu-I-FITC and Leu-2-PE and analyzed by two-color Bow cytometry. The averagepercentagefor each population is represented.(I) 25.396,(II) 5.896, (III) 16.96, (IV) 5 1.9%.
LEU-ZBEARING
169
NON-T CELLS
TABLE 2 Leu- 1 X Leu-2 Two-Color Analysis of PBML from Chimpanzees and Humans Percentage* SW
Lab2+
Leu-1+ Leu-1+ + Leu-2+ Leu- 1-, Lell-2Leu- 1+, Leu-2-
Adult chimpanzee
Adolescent chimpanzee
Adult human
31.4 + 10.6 67.8 81.7
24.4 74.7 82.9
22.1 * 7.5 68.8 74.6 25.3 + 7.2 51.9 5.8 + 1.6
18.3 f
17.1
1.2
59.5 8.2 15.2
50.3 13.9 f 4.5 17.5
Leu- I-, Lab2+ Leu- 1+, Leu-2+
16.9
Note. Adult and adolescent chimpanzee and adult human PBML were compared for the percentagesof Leu- 1 and Leu-2 lymphocyte subsetsby two-color analysis. ’ Average percentageswere derived from 10 human subjects and 8 chimpanzees.
shows the relative purity (85.6%) of subpopulation II following preparative isolation. All four subpopulations were isolated by similar techniques and were tested for NK activity. Unfractionated chimp cells were stained in a similar way but were not sorted. The results of the NK assay(Fig. 6) showed a lo- to 1Zfold enrichment in cytotoxicity for K562 cells by subpopulation II (Leu-l-, LAX-~+) over unfractionated. However, the slope and percentage kill of subpopulation I (Leu- 1-, Leu-23 appeared to be very similar to those for unfractionated cells (approximately 7% at 10:1 effector:target ratio) while subpopulations III (Leu- l+, Leu-2+) and IV (Leu-l+, Leu-23 showed less than 1%cytotoxicity at all effector:target ratios tested. Helper:suppressor ratio determination by two-color analysis. In order to determine how much the Leu-2 marked non-T cell (Leu-l-) contributed to the helper:suppressor
loo0 I---------
“a
k
U 2
LEU 1
FIG. 5. Chimpanzee PBML were stained with Leu- 1-FITC and Leu-2-PE and preparatively sorted. Four populations were isolated. Population II (shown above) was enriched 85.6%.
170
FOLKS ET AL. 15 “NF --.^
t
I /
I./
. k
g:
= e
T-
g
5-
I.*. L,+ -..-_.L2- I.,.-.-
/
_
s
3-
/ / ,
l-1
I
,
I11111
1
I
I,,,,11
O.l:l
I 1:1
I
1llll lo:1
EFFECTORTARGET
FIG. 6. Unfractionated population and four Leu-I-FITC, Leu-2-PE isolated subpopulations were tested for NK activity. Effector cells were titrated onto “Cr-labeled K562 cells (1 X IO’) and assayed3 hr later for percent lysis.
cell ratio, two-color analysiswith Leu-2 and Leu- 1was usedasa method of quantitation. It was found that, when the Leu-l-, Leu-2+ subpopulation was eliminated from the calculation of helper:suppressor cells, the ratios of human cells change from 3.0: 1 to 2.2: 1 while the ratios of chimpanzee cells change even more from 2.9:1 to 1.6:1 (Table 3). DISCUSSION Phenotypic differences between human and nonhuman primate lymphocyte subpopulations have previously been reported (10). In these studies, the differences in molecular structure between chimpanzee and human, termed “antigenic distance,” were shown to possibly be related to divergence of phenotypic surface markers. In addition, these differences might also indicate the origin of functional subgroups in humans. Our results with two-color flow cytometric analysis showed greater numbers of Leu-2 (suppressor/cytotoxic cells) in the chimpanzee than in the human. These additional cells appeared, however, to be Leu-l- implying that they were either not T cells or had lost or never developed the Leu-1 surface molecule. In an earlier report (6), the percentageof Leu-l-, Leu-2+ cells in a single reported normal human volunteer TABLE 3 Changes in Helper:Suppressor Ratios Depending on Population Inclusive of the Leu-2+, Leu-lHelperxuppressor
Adult chimpanzee
Adolescent chimpanzee
Adult human
Leu- 1+, Leu-2-:Leu-2+, Leu- 1+ Leu- I +, Leu-2-:Leu-2+
2.9: 1 1.6:1
3.9: I 2.4: I
3.0:1 2.2: 1
Note. Adult and adolescentchimpanzee and adult human PBML were analyzed by two-color Bow cytometry for Leu-I and Leu-2 surface markers. Ratios were determined from the averagesof 8 chimpanzees and 10 human subjects.
LEU-2-BEARING NON-T CELLS
171
was 14.1. This was in contrast to our findings where the average percentage was 5.8. This difference may be due to the fact that in the previous report only one individual was evaluated. In unpublished studies we have observed only one in 13 individuals where the Leu-l-, Leu-2+ population was as high as 14%. Mitogen studies following Leu- 1 cell depletion indicated that the Leu- 1- chimpanzee cells lacked responsiveness to the classical T-cell mitogens. These results could be explained, however, by the possible elimination of adherent monocytes which were lost during the panning procedure. From this same experiment, it was found that NK activity in the Leu-l- fraction was enhanced sixfold over unfractionated cells, confirming the finding that in humans the NK active population is Leu-l-. To actually determine which fraction possessedthe NK activity each of the four groups was isolated by electronic cell sorting. The Leu-l-, Leu-2+ population contained the most potent NK activity of the four with the only residual activity being present in the Leu-l-, Leu-2- population. This would indicate at least two subpopulations of NK cells in the chimpanzee which can be defined by Leu-2. The chimpanzee was observed to have twice the number of these Leu-l-, Leu-2+ cells as the human. Several phenotypically distinct non-T cells possessNK activity in the human (11). All are stained by Leu- 11, which recognize an Fc receptor, some by Leu-7, and some by Leu-2. It seemsclear, however, from data presented in this study that NK activity is more concentrated in Leu-l-, Leu-2+ cells in chimpanzees than is reported in humans. Since phenotype and function using anti-human monoclonal antibodies directed at chimpanzee PMBL have not as yet been firmly established, conclusions cannot be made concerning interspecies comparisons. Two reports showing large granular lymphocytes or NK cells having suppressor function in the human have been described (8,9). However, one report indicated that the Leu-7 marked suppressor cell in the human was distinct from Leu-2+ cells. This may represent a divergence in the chimpanzee whereby Leu-2+ NK cells lost the Leu2 marker during phylogenetic development. Interestingly, in human cord blood lymphocytes, it was found that OKT-8+ (Leu-2) cells were 8.4% increased over adults and the sum of helper and suppressor marked cells from cord blood was approximately 18% greater than the percentage of OKT-3+ T cells ( 12). This indicates a substantial difference in OKT-8+, OKT-3- subpopulations (suppressor marked non-T cells) between neonatal and adult humans. In our examination of human cord blood (unpublished results) by two-color analysis, we found 12.5% Leu-l-, Leu-2+ cells. Appearance of Leu-l-, Leu-2+ cells early in ontogeny with their subsequent disappearanceis consistent with the increased number of such cells in the phylogenetically less mature chimpanzee. The results presented here indicate that the chimpanzee is different from the human when compared by lymphocyte subset analysis. In addition, NK subsetsin the chimpanzee appear to be different from those reported in the human. Since we have no evidence that the same Leu-2+ NK cells in the chimpanzee can mediate suppression or what other functions the Leu-2+, Leu- I- cells may have, it is difficult to extrapolate these data directly into information about the human. In both species,the basis for the expression of Leu-2 on both NK and non-NK cells remains a fascinating enigma. ACKNOWLEDGMENTS The authors thank M. Belgrove for the processingand cryopreservation of chimpanzee blood and Meioy Laboratories for outstanding animal maintenance. In addition, we thank Ms. Ginny Shaw, Mary Rust, and Ann London for excellent editorial assistance.
172
FOLKS ET AL.
REFERENCES 1. Damle, K. N., and Engleman, E. G., J. Exp. Med. 158, 159, 1983. 2. Morimoto, C., Reinherz, E. L., Borel, Y., and Scholssman,S. F., J. Immunol. 130, 157, 1983. 3. Gatenby, P. A., Kansas, G. S., Xian, C. Y., Evans, R. L., and Engleman, E. G., J. Immunol. 129, 1997, 1982. 4. Damle, N. K., Mohagheghpour, N., and Engleman, E. G., J. Immunol. 132,644, 1984. 5. Lauday, A., Gartland, G. L., and Clement, L. T., J. Immunol. 131,2757, 1984. 6. Lanier, L. L., Le., A. M., Phillips, J. H., Warner, N. L., and Babcock, G. F., J. Immunol. 131, 1789, 1983. 7. Damle, N. K., Mohagheghpour, N., Hansen, J. A., and Engleman, E. G., J. Immunol. 131,2296, 1983. 8. Brieva, J. A., Targan, S., and Stevens, R. H., J. Immunol. 132,611, 1983. 9. Tilden, A. B., Abo, T., and Balch, C. M., J. Immunol. 130, 1171, 1983. 10. Letvin, N. L., King, N. W., Reinherz, E. L., Hunt, R. D., Lane, H., and Schlossman, S. F., Eur. J. Immunol. 13, 345, 1984. 11. Lanier, L. L., and Loken, M. R., J. Immunol. 132, 151, 1984. 12. Maccario, R., Nespoli, L., Mingrat, G., Vitiello, A., Ugazio, A. G., and Burgio, G. R., J. Immunol. 130, 1129, 1983.
IMMUNOLOGY
97, 164-172 ( 1986)
Increased Number of Leu-ZBearing Non-T Cells with Natural Killer Activity in Chimpanzees THOMAS
M. FOLKS,’ THOMAS M. CHUSED,DIANE PORTNOY,LINETTE EDISON, WILLIAM
LEISERSON, AND KENNETH
W. SELL
Ofice of the ScientiJic Director and the Laboratory of Microbial Immunity, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205 Received August 12, 1985; acceptedSeptember 23, I985 Peripheral blood lymphocytes from adult and adolescentchimpanzees,as well asadult humans, were studied for phenotypic surfacemarkers by tlow cytometry. Lymphocytes from chimpanzees were found to have increased numbers of Leu-l-, LX-~+ cells as compared to humans. These cells, following preparative electronic cell sorting, were shown to possessnatural killer function. Further analysis of this subpopulation indicated that they lacked responsivenessto a number of T-cell mitogens. The differencesin lymphocyte subpopulations between chimpanzeesand humans can almost be totally accounted for by the Leu-l-, Leu-2+ cells. Phylogenetic disparity between these two speciesmay aho be found within this population. 0 1986 Academic Press,IIIC.
INTRODUCTION Regulation of the immune response is greatly affected by the interactions of helper and suppressor cells (l-5). Functionally distinct yet phenotypically overlapping cytotoxic/suppressor Leu-2+ peripheral blood mononuclear lymphocytes (PBML)2 in the human are Leu-l+ T cells (6). Recently, new monoclonal antibodies have been shown to distinguish the suppressorsubpopulation from the cytotoxic (7). In addition, suppressorcells (Leu-2+) have been shown to be subpopulated into natural killer (NK) cells and high-density T cells lacking Fc receptors (8). In other studies, HNK-l+ (Leu7) cells were shown to function as suppressorcells in both pokeweed mitogen-induced IgG production and mixed lymphocyte reaction (MLR) (9). However, I-AX-~+ cells were shown to be distinct from the HNK- 1+ cells since no immune-complex activation was required for their suppressor function. Less correlation between phenotype and function is known among the nonhuman primates. In a phylogenetic study examining human and nonhuman primate T-lymphocyte surface structures with anti-human T-cell monoclonal antibodies a number of phenotypes were shown to be shared (10). In the present study, we have examined lymphocyte cell surface markers (Leu-1 and Leu-2) in chimpanzees by two-color flu’ To whom correspondence should be addressedat: National Institutes of Health, Building 10, Room I 1B- 13, Bethesda, Md. 20205. 2Abbreviations used:PBML, peripheral blood mononuclear lymphocytes; NK, natural kiheq MLR, mixed lymphocyte reaction; FITC, fluorescein isothiocyanate; PHA, phytohemagghrtinin; Con A, concanavalin A; PWM, pokeweed mitogen. 164 OOOS-8749186 $3.00 Copyri&t Q 1986 by Academic Rss, Inc. All ri@s of reproduction in any form reserved.
165
LEU-2-BEARING NON-T CELLS
orescent analysis. Preparative two-parameter isolation of Leu- 1-, Leu-2+ lymphocytes was carried out by electronic cell sorting. Lymphocytes, isolated by this method, were shown to possessNK function. In addition, two-parameter analysis showed Leu-l-, Leu-2+ cells to be circulating in greater numbers in chimpanzees than humans. These studies suggesta possible phylogenetic link between NK and suppressormarked lymphocytes. MATERIALS
AND METHODS
Preparation of peripheral blood mononuclear leukocytes. Heparinized whole blood was obtained by venipuncture from either 10 normal human adults from our laboratory or 8 chimpanzees (3-20 kg) at the National Institute of Allergy and Infectious Diseases primate facility, Rockville, Maryland (Meloy Laboratories). PBML were isolated by Ficoll-Hypaque; washed three times in RPMI-1640, with glutamine, Hepes, gentamicin, and 10% fetal calf serum (medium); and cryopreserved until further use. Panning andfluorescent antibody staining for sorting. For enrichment by panning, cryopreserved chimpanzee cells were thawed and washed three times in medium, and 1 X lo* PBML were incubated with 100 ~1 of OKT-3 (Table 1) (Ortho Diagnostics, Raritan, N.J.) for 30 min at 4°C. Cells were then washed and added to two Costar Petri dishes precoated with goat anti-mouse Ig (Cappel, Court Malvern, Pa.). After 1 hr at 25°C the nonadherent cells were removed, washed, and stained with 50 ~1 of Leu-3-fluorescein isothiocyanate (FITC) (Becton-Dickinson Monoclonal Center, Mountain View, Calif.). After staining, cells were washed and preparatively sorted on a modified FACS II (Becton-Dickinson). Procedure for two-color analysis and cell sorting. Chimpanzee or human PBML were thawed, washed three times in either RPMI- 1640 for cell sorting or Hanks’ buffer containing 0.05% Na azide and 1% BSA (Sorter Buffer) for cell analysis. Cells were stained with Leu- 1-FITC and Leu-2-P-phycoerythrin (Table 1) and resuspended in 3 ml of RPMI- 1640 at 1 X log/ml for cell sorting or in 1 ml of sorter buffer at 1 X 106/ml for cell analysis. All staining and cell analysis were carried out at 4’C. Cells were analyzed and sorted using a modified FACS II exciting fluorescein with 200~nW, 488-nm output from an Argon laser and exciting phycoerythrin with 150-NW, 568nm output from a krypton laser. Monocytes, erythrocytes, and dead cells were excluded from analysis using a 90” light scatter by forward-angle light scatter gate. Green and red fluorescent signals were stored in list mode using a PDP-11 computer system. Data from two-parameter analysis were collected in matrix form and presented as three-dimensional maps. By retrieving data from list storage, contour plots were used TABLE 1 Specificity of Antibodies Used Antibody
Specificity
Anti-Leu- 1 Anti-Leu-2 (OKT-8) Anti-OKT-3 Anti-human Ig (polyvalent) Anti-Leu-3a
T cells and B-cell subset Suppressor:cytotoxic T subset T cells Human Ig Helper:inducer T subset
166
FOLKS ET AL.
to determine the percentage of cells within each contour grouping. Four populations were isolated: Leu-l+, Leu-2+; Leu-l+, Leu-2-; Leu-l-, Leu-2+; and Leu-l-, Leu-2-. The flow rate was 750-1000 cells/set. Analysis of the sorted populations showed 85-97% of the cells to have the selected phenotypes after sorting. NK assay. NK cell assayswere performed by determination of 51Crrelease from target K562 cells. The assaywas carried out in 200~Ccl-volumeLinbro 96 U-bottom multiwell plates. K562 cells (1 X 104/well) were incubated with chimpanzee cells at varying concentrations (E:T ratios from O.l:l to 40:1) for 3 hr at 37°C. Plates were then spun and 100 ~1of supematant was counted on a Beckman 9000 gamma counter for releasedradioactivity. Percentagecytotoxicity was basedon the following formula: [(test cpm) - (spontaneous releasecpm)]/[(total releasecpm) - (spontaneous release cpm)] X 100. Spontaneous releasecpm was never more than 10%of the total release cpm. Standard deviation among triplicates was also never more than 10%. Mitogen assays.Test populations of cells (1 X 105/well) were added to Costar 3596 plates in medium to a total volume of 200 &well. The mitogens phytohemagglutinin (PHA; Difco, Detroit, Mich.), concanavalin A (Con A), and pokeweedmitogen (PWM; Sigma Chemical Co., St. Louis, MO.) were used at concentrations of 10, 1, and 0.1 pg/ml. PHA- and Con A-treated cells were incubated for 4 days while PWM-treated cells were incubated for 5 days at 37’C and 7% COZ. All cells were pulsed with [3H]thymidine (sp act 6.7; New England Nuclear, Boston Mass.) and harvested on a PHD Harvester (Cambridge, Mass.) 12 hr later. Filter disks were counted for radioactivity using 2 ml Econofluor (New England Nuclear) on a Beckman LS7000 beta counter. RESULTS Loss of mitogenic proliferation and enhancement of NK activity after panning and one-color sorting. Chimpanzee PBML were fractionated into OKT-3- cells by panning and further enriched by Leu- 1-FITC preparative sorting. Unfmctionated and Leu- 1-enriched populations of cells were assayedfor their ability to proliferate in the presence of the T-cell mitogens PHA, Con A, and PWM. The data in Fig. 1 indicate that removal of Leu-1 chimpanzee lymphocytes eliminates the ability of the remaining cells (Leu-1 -) to respond to these mitogens. An additional question concerning the function of the Leu- l- population was the type of NK activity it might contain. It was not too surprising to find (Fig. 2) that the Leu-l- population contained an enriched number of cytotoxic cells for K562 over the unfmctionated cells. At all effector-to-targetratios testedthere was an approximately sixfold increase in cytotoxic activity. Two-color qualitative analysis. Since it is well established in the human that NK cells reside in the non-T-cell lymphocyte subpopulation our next question was to determine if the Leu-l- population could be further fractionated in the chimpanzee as it has in the human using Leu-2. Chimpanzee and human PBML were analyzed by two-color fluorescence. Figures 3 and 4 show representative two-parameter histograms of chimpanzee and human unfractionated PBML, respectively. Four populations were defined when red Leu-2 and green Leu-l were compared: (I) Leu-l-, Leu-2-; (II) Leu-l-, Leu-2+; (III) Leul+, Leu-2+; (IV) Leu-l+, Leu-2-. Major differences in cell subpopulation were found especially in the Leu-l-, Leu-2+ staining. Table 2 shows the percentagesof all com-
167
LEU-2-BEARING NON-T CELLS
30000
20000 i
PIG. 1. Unfractionated and OKT-3 panned and preparatively sorted Leu-l- chimpanzee PBML were tested for mitogen responsivenessto Con A, PHA, and PWM. Cells were assayed 4-5 days later for [‘Hlthymidine uptake.
binations of subpopulations while comparing young (~9 kg) chimpanzees, older (>9 kg) chimpanzees, and adult humans. Chimpanzees >9 kg had 2.4 times more Leul-, Leu-2+ (subpopulation II) cells than humans. This difference was almost totally reflected in the percentage of Leu-2-, Leu- 1- cells between chimpanzees and humans (7.0%). Young vs older chimpanzeesshowed major differencesbetween subpopulations Leu-l+, Leu-2- (9.5%) and Leu-l-, Leu-2+ (5.7%).
50
C+SW
CELLS
0 0.01:1
0.1:1
1:l
10:1
lo&l
EFFECTORTARGET
FIG. 2. Unfmctionated and Leu-I- chimpanzee PBML were assayedfor NK activity on KS62 target cells. Varying numbers of NK cells were incubated for 3 hr with 51Cr-labeledK562 cells (1 x 104).
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L E U 2 IV
LEU 1 FIG. 3. PBML from eight chimpanzees were stained with Leu-I-FITC and Leu-2-PE and analyzed by two-color flow cytometry. The average percentage for each population is represented.(I) 18.32, (II) 13.996, (III) 17.52, (IV) 50.3%.
Two-color preparative analysis and NKfunction. The finding of increased numbers in subpopulation II (Leu 1-, Leu2+) from chimpanzeesled us to the step of preparatively isolating this cell by two-color flow cytometry and assessingits NK activity. Figure 5
100
E’ u 2 ----.I 10 -----_
I
LEU 1 FIG. 4. PBML from 10 normal human subjectswere stained with Leu-I-FITC and Leu-2-PE and analyzed by two-color Bow cytometry. The averagepercentagefor each population is represented.(I) 25.396,(II) 5.896, (III) 16.96, (IV) 5 1.9%.
LEU-ZBEARING
169
NON-T CELLS
TABLE 2 Leu- 1 X Leu-2 Two-Color Analysis of PBML from Chimpanzees and Humans Percentage* SW
Lab2+
Leu-1+ Leu-1+ + Leu-2+ Leu- 1-, Lell-2Leu- 1+, Leu-2-
Adult chimpanzee
Adolescent chimpanzee
Adult human
31.4 + 10.6 67.8 81.7
24.4 74.7 82.9
22.1 * 7.5 68.8 74.6 25.3 + 7.2 51.9 5.8 + 1.6
18.3 f
17.1
1.2
59.5 8.2 15.2
50.3 13.9 f 4.5 17.5
Leu- I-, Lab2+ Leu- 1+, Leu-2+
16.9
Note. Adult and adolescent chimpanzee and adult human PBML were compared for the percentagesof Leu- 1 and Leu-2 lymphocyte subsetsby two-color analysis. ’ Average percentageswere derived from 10 human subjects and 8 chimpanzees.
shows the relative purity (85.6%) of subpopulation II following preparative isolation. All four subpopulations were isolated by similar techniques and were tested for NK activity. Unfractionated chimp cells were stained in a similar way but were not sorted. The results of the NK assay(Fig. 6) showed a lo- to 1Zfold enrichment in cytotoxicity for K562 cells by subpopulation II (Leu-l-, LAX-~+) over unfractionated. However, the slope and percentage kill of subpopulation I (Leu- 1-, Leu-23 appeared to be very similar to those for unfractionated cells (approximately 7% at 10:1 effector:target ratio) while subpopulations III (Leu- l+, Leu-2+) and IV (Leu-l+, Leu-23 showed less than 1%cytotoxicity at all effector:target ratios tested. Helper:suppressor ratio determination by two-color analysis. In order to determine how much the Leu-2 marked non-T cell (Leu-l-) contributed to the helper:suppressor
loo0 I---------
“a
k
U 2
LEU 1
FIG. 5. Chimpanzee PBML were stained with Leu- 1-FITC and Leu-2-PE and preparatively sorted. Four populations were isolated. Population II (shown above) was enriched 85.6%.
170
FOLKS ET AL. 15 “NF --.^
t
I /
I./
. k
g:
= e
T-
g
5-
I.*. L,+ -..-_.L2- I.,.-.-
/
_
s
3-
/ / ,
l-1
I
,
I11111
1
I
I,,,,11
O.l:l
I 1:1
I
1llll lo:1
EFFECTORTARGET
FIG. 6. Unfractionated population and four Leu-I-FITC, Leu-2-PE isolated subpopulations were tested for NK activity. Effector cells were titrated onto “Cr-labeled K562 cells (1 X IO’) and assayed3 hr later for percent lysis.
cell ratio, two-color analysiswith Leu-2 and Leu- 1was usedasa method of quantitation. It was found that, when the Leu-l-, Leu-2+ subpopulation was eliminated from the calculation of helper:suppressor cells, the ratios of human cells change from 3.0: 1 to 2.2: 1 while the ratios of chimpanzee cells change even more from 2.9:1 to 1.6:1 (Table 3). DISCUSSION Phenotypic differences between human and nonhuman primate lymphocyte subpopulations have previously been reported (10). In these studies, the differences in molecular structure between chimpanzee and human, termed “antigenic distance,” were shown to possibly be related to divergence of phenotypic surface markers. In addition, these differences might also indicate the origin of functional subgroups in humans. Our results with two-color flow cytometric analysis showed greater numbers of Leu-2 (suppressor/cytotoxic cells) in the chimpanzee than in the human. These additional cells appeared, however, to be Leu-l- implying that they were either not T cells or had lost or never developed the Leu-1 surface molecule. In an earlier report (6), the percentageof Leu-l-, Leu-2+ cells in a single reported normal human volunteer TABLE 3 Changes in Helper:Suppressor Ratios Depending on Population Inclusive of the Leu-2+, Leu-lHelperxuppressor
Adult chimpanzee
Adolescent chimpanzee
Adult human
Leu- 1+, Leu-2-:Leu-2+, Leu- 1+ Leu- I +, Leu-2-:Leu-2+
2.9: 1 1.6:1
3.9: I 2.4: I
3.0:1 2.2: 1
Note. Adult and adolescentchimpanzee and adult human PBML were analyzed by two-color Bow cytometry for Leu-I and Leu-2 surface markers. Ratios were determined from the averagesof 8 chimpanzees and 10 human subjects.
LEU-2-BEARING NON-T CELLS
171
was 14.1. This was in contrast to our findings where the average percentage was 5.8. This difference may be due to the fact that in the previous report only one individual was evaluated. In unpublished studies we have observed only one in 13 individuals where the Leu-l-, Leu-2+ population was as high as 14%. Mitogen studies following Leu- 1 cell depletion indicated that the Leu- 1- chimpanzee cells lacked responsiveness to the classical T-cell mitogens. These results could be explained, however, by the possible elimination of adherent monocytes which were lost during the panning procedure. From this same experiment, it was found that NK activity in the Leu-l- fraction was enhanced sixfold over unfractionated cells, confirming the finding that in humans the NK active population is Leu-l-. To actually determine which fraction possessedthe NK activity each of the four groups was isolated by electronic cell sorting. The Leu-l-, Leu-2+ population contained the most potent NK activity of the four with the only residual activity being present in the Leu-l-, Leu-2- population. This would indicate at least two subpopulations of NK cells in the chimpanzee which can be defined by Leu-2. The chimpanzee was observed to have twice the number of these Leu-l-, Leu-2+ cells as the human. Several phenotypically distinct non-T cells possessNK activity in the human (11). All are stained by Leu- 11, which recognize an Fc receptor, some by Leu-7, and some by Leu-2. It seemsclear, however, from data presented in this study that NK activity is more concentrated in Leu-l-, Leu-2+ cells in chimpanzees than is reported in humans. Since phenotype and function using anti-human monoclonal antibodies directed at chimpanzee PMBL have not as yet been firmly established, conclusions cannot be made concerning interspecies comparisons. Two reports showing large granular lymphocytes or NK cells having suppressor function in the human have been described (8,9). However, one report indicated that the Leu-7 marked suppressor cell in the human was distinct from Leu-2+ cells. This may represent a divergence in the chimpanzee whereby Leu-2+ NK cells lost the Leu2 marker during phylogenetic development. Interestingly, in human cord blood lymphocytes, it was found that OKT-8+ (Leu-2) cells were 8.4% increased over adults and the sum of helper and suppressor marked cells from cord blood was approximately 18% greater than the percentage of OKT-3+ T cells ( 12). This indicates a substantial difference in OKT-8+, OKT-3- subpopulations (suppressor marked non-T cells) between neonatal and adult humans. In our examination of human cord blood (unpublished results) by two-color analysis, we found 12.5% Leu-l-, Leu-2+ cells. Appearance of Leu-l-, Leu-2+ cells early in ontogeny with their subsequent disappearanceis consistent with the increased number of such cells in the phylogenetically less mature chimpanzee. The results presented here indicate that the chimpanzee is different from the human when compared by lymphocyte subset analysis. In addition, NK subsetsin the chimpanzee appear to be different from those reported in the human. Since we have no evidence that the same Leu-2+ NK cells in the chimpanzee can mediate suppression or what other functions the Leu-2+, Leu- I- cells may have, it is difficult to extrapolate these data directly into information about the human. In both species,the basis for the expression of Leu-2 on both NK and non-NK cells remains a fascinating enigma. ACKNOWLEDGMENTS The authors thank M. Belgrove for the processingand cryopreservation of chimpanzee blood and Meioy Laboratories for outstanding animal maintenance. In addition, we thank Ms. Ginny Shaw, Mary Rust, and Ann London for excellent editorial assistance.
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