Eur. J. Immunol. 1991. 21: 635-641
Marcella Sanottia, Vinay Kumar+, Michael Bennett+ and Garnett KelsoeO Department of Veterans Affairs Medical CenterA,Baltimore, Department of Pathology, University of Texas Southwestern Medical Center at Dallas+, Dallas and Department of Microbiology and Immunologyo, University of Maryland School of Medicine, Baltimore
Murine Tand NK cell colonies
635
Cloning of murine splenic T lymphocytes and natural killer (NK) cells on filter paper discs: detection of a novel NK/T phenotype* Discrete colonies of splenocytes were grown on filter paper discs in the presence of concanavalin A and interleukin 2. Phenotypic analysis of the colonies indicated that the majority expressed the Thy-1.2 marker and 72% of these co-expressed the CD3 molecule. Of the colonies 20%-25% were NK 1.1+ and they developed regardless of the presence of Con A in the culture medium, a property of the NK lineage. In addition, Thy-1.2+ colonies developed when splenocytes from scid mice, which lack mature Tand B cells, were grown both in the presence and absence of concanavalin A . These results demonstrate that colonies of murine splenic T lymphocytes and NK cells could be successfully grown on filter paper discs and phenotypically characterized.With this colonies technique, it was possible to identify a novel subset of NK 1.1+ colonies that co-expresses CD3 and shares growth properties with T cell colonies.
1 Introduction
2 Materials and methods
Peripheral T lymphocytes can be activated with mitogenic lectins, such as Con A [l], to express IL2R and to proliferate in the presence of IL 2 [2]. In contrast, NK cells can be stimulated to proliferate in the presence of I L 2 alone [3]. These properties have been exploited in LD assays to analyze clones of T lymphocytes or human NK cells [4-71. While this strategy has proved generally successful for Tcells the ability to establish and analyze clones of murine NK cells remains elusive. In fact, all reported cloned murine cell lines of presumed NK origin have proved to belong to theTcell lineage [8,9].This inability has served as an impediment to investigations of the NK cell lineage.
2.1 Mice C57BL/6J mice were purchased (The Jackson Laboratories, Bar Harbor, ME) and maintained at the local animal facility. C.B-l7/Icr mice homozygous for the severe combined immunodeficiency disease (scid) mutation (C.B-17 scid) and their normal counterparts (C.B-17) were bred at the University of Texas Southwestern Medical Center at Dallas. Splenic lymphocytes were obtained from age- (6-16 weeks) and sex-matched.mice.
2.2 Media
One alternative to LD methods is the cloning of lympho- Cells were cultured in a-MEM (cat. no. 410-1900, Gibco cytes on filter paper discs.This technique was developed for Laboratories, Grand Island, NY), supplemented with 10% cloning mitogen- and antigen-stimulated B cells, and FCS (Hyclone, Logan, UT), 5 x 10-5M 2-ME (Sigma permits the phenotypic and genotypic analysis of large Chemical Co., St. Louis, MO), 2 g/l Na2HC03 (Sigma), numbers of spatially isolated lymphocyte colonies in replica and a penicillin (50 U/ml) - streptomycin (50 pg/ml) assays [lo, 111. In the present report, we demonstrate that mixture (Flow Laboratories, McLean, VA). A washing colonies of murine splenic T lymphocytes and NK cells medium (WM) was prepared by diluting culture medium could be successfully grown on filter paper discs and 1 : 10 with a-MEM medium. phenotypically characterized. T cell colonies were characterized by the co-expression of Thy-1.2 and CD3, the signal-transducing molecule for the TcR [12]. NK cell 2.3 Reagents colonies were characterized by the expression of the NK 1.1 molecule [13] and the co-expression of the NK 1.1 marker Con A (Sigma) was used at 5 pg/ml. Partially purified with the CD3 molecule was investigated. Unexpectedly, a human IL 2 (Electro-Nucleonics, Inc., Silver Spring, MD) was used at 100 U/ml. Biotin-N-hydroxysuccinimide ester subset of NK 1.1+ colonies co-expressed CD3. (BNHS; Vector Laboratories, Burlingame, CA) was used to conjugate mAb as described by the manufacturer. [I 87561 Anti-Thy-1.2 mAb (Accurate Chemicals, Westbury, NY) was purified by protein A HPLC and biotinylated. Anti-NK * Supported in part by USPHS grants AG-08192 (G.K.) and 1.1 mAb was obtained from culture SN of clone PK136 [ 131, AI-20451 (V.K.). purified over Affi-Gel-protein A and biotinylated. Antiarsonate (anti-ars) mAb obtained from ascites fluid proCorrespondence: Marcella Sarzotti, Research Service (151), duced by the 22B5 hybridoma (kindly provided by Dr. D. D.V.A. Medical Center, 3900 Loch Raven Blvd., Baltimore, MD Capra, UTHSC Dallas, TX) was purified over Affi-Gel21218, USA protein A and biotinylated. Biotin-conjugated anti-CDS and antLCD8 mAb were purchased from Becton-DickinAbbreviations: am: Arsonate IF: Immunofluorescence WM: Washing medium son (Mountain View, CA). Anti-CD3 mAb was derived 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+ .251a 0014-298019110303-0635$3.~0
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Eur. J. Immunol. 1991. 21: 635-641
M. Sarzotti,V. Kumar, M. Bennett and G. Kelsoe
from culture SN of the 145-2C11 hybridoma, a cell line derived from hamster and specific for the E chain of the murine CD3 complex [ 141. FITC-labeled goat antibody specific for hamster IgG Hand L chains (OrganonTecknika Corp. ,Westchester, PA) used at a 1 : 40 dilution did not bind to mouse thymocytes or splenocytes. Lymphocyte colonies were grown on Whatman 54 filter paper discs (8.26 cm 0 ; Whatman Paper Division, Clifton, NJ).
extensively with PBS/BSA, treated with a fluorescence stabilizer (Amersham), and mounted on glass slides in a solution of 50% PBS and 50% glycerol for fluorescence microscopy.
3 Results 3.1 Kinetics of colony growth
2.4 Preparation and culture of splenocytes Before culture, filter paper discs were prepared as described [lo]. Spleens were taken aseptically from mice killed by cervical dislocation. Single-cell suspensions were prepared by standard methods and the cells washed twice in ice-cold WM. Five milliliters of culture medium containing splenocytes (5 x lo6 cells, unless otherwise specified) was pipetted evenly onto each washed filter paper disc. Next, 5 ml of culture medium containing IL 2 (200 U/ml) and Con A (10 pg/ml) (when indicated) was gently added. Cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO:! for 8 days (unless otherwise specified). 2.5 Visualization of colonies
Standard histological techniques were used to demonstrate colonies by microscopy [lo]. Discs were fixed in 10% neutral-buffered formalin for 5-7 min at room temperature and washed 2 x with 0.1 x PBS [ 101. Discs were then stained with Wright-Giemsa stain (Diagnostic Systems, Inc., Gibbstown, NJ), washed with distilled water, dehydrated in absolute ethanol, cleared in xylene and mounted on glass slides for microscopy.
2.6 RIA Discs were fixed with a solution of 0.5% glutaraldehyde (Polysciences, Inc.,Warrington, PA) in PBS for 10 min at 4°C. Discs were then washed 4 x in PBS and 3 x in PBS containing 1% BSA (Sigma; PBSBSA). Washed discs were overlayed with specific mAb diluted in PBS/BSA and incubated at 4°C for 5-6 h. Discs were then washed 5 x in PBS/BSA containing 0.05% Tween 20 and 0.01% SDS (RIA wash). Washed discs were overlayed with RIA wash containing '*'I-labeled streptavidin = 3.7-18.5 kBq/ml (0.1-0.05 pCi/ml; Amersham, Arlington Heights, IL), incubated overnight at 4"C, rinsed extensively in RIA, washed and air-dried. Autoradiography was performed as described [ 101.
Splenocytes seeded onto filter paper discs in medium containing IL 2 and Con A produced discrete colonies of lymphocytes that could be visualized by microscopy. Titration experiments were performed to determine the optimal number of plated splenocytes for the production of colonies and to ensure that colony formation was dependent upon proliferation of a single precursor cell (Fig. 1). Over a 50-fold range of input cell densities (1 x 105-5 X 106 splenocytes/disc), the production of colonies was a linear function of cell number with a slope of approximately 1, implying a first-order relationship between the generation of colonies and precursor cell number [16]. At plating densities > 5 x 106 splenocytes/disc, the plating efficiency decreased. Subsequent experiments were then carried out using 5 x lo6 input splenocytes/disc. Fig. 2 illustrates the growth of colonies of splenocytes during 12 days of culture with IL 2 and Con A . On day 6 of culture, 55% of the colonies contained between 5 and 16 cells, and 44% between 17 and 64 cells. By day 8 the size of the colonies had increased substantially: only 10% of the colonies contained 5-16 cells while 47% and 34% contained 17-64 and 65-256 cells, respectively. However, by day 12, colony size had not increased and cell death began to occur, as evidenced by an increased percentage of small colonies (5-16 cells) and a decreased percentage of colonies containing 65-256 cells (Fig. 2). Thus, colonies grew in rough synchrony and maximum colony size was achieved at 8 days of culture. Using the median colony size on each day of culture, we calculated the average doubling-time of cells to be 33 h at day 8. Similar kinetics of colony growth were obtained when spleen cells were plated at lower densities (data not shown). Thus, a plating number of 5 x 106
2.7 Immunofluorescence (IF)
Discs were fixed with a solution of 0.5% paraformaldehyde (Sigma) in PBS [15]. Discs were then washed as described above, subsequently overlayed with specific mAb diluted in PBS/BSA and incubated at room temperature for 1-2 h. Discs were then washed 5 x in PBSBSA and overlayed with PE-streptavidin (Southern Biotechnology Assoc., Birmingham, AL) or with FITC-labeled secondary Ab at room temperature for 1-2 h in the dark. Finally, discs were rinsed
Zl
1:o
510
1010
MILLIONS OF INPUT CELLS/DISC
Figure 1. Cell dose-dependency of colony formation. Colonybearing filter paper discs were fixed and stained with WrightGiemsa on day 8 of culture. Mean number of colonieddin (12-6 discs) (k SEM) was determined for each input number of splenocytes. The least-squares regression line generated from the arithmetic means of the points, 1 x 105-5 X 106, is shown.
Eur. J. Immunol. 1991. 21: 635-641
-
80 601
-
Murine Tand NK cell colonies
iments are summarized in Table 1 (RIA column). The highest number of colonies/disc (=42Ydisc) was identified with mAb directed against Thy-1.2, a marker present on all Tcells and on the majority of NK cells propagated in IL 2 [3, 17, 181. About 40% and 33% of the number of Thy-1.2+ colonies expressed the CD5 or CD8 determinants, respectively. Interestingly, a mAb specific for the NK 1.1 determinant, a marker present on NK cells [13], identified = 88 colonies/disc. Some 13 colonies/disc bound an mAb, 22B5, specific for ars. The 22B5 mAb bears the isotype (y2b) of PK136 (anti-NK 1.1) and was included to control for nonspecific binding of Ab to colonies.These results implied that in the presence of Con A and IL 2, splenocytes gave rise to discrete colonies of T lymphocytes and NK cells.
DAY 6
DAY 8
DAY 12
n
40
5-16
637
3.3 Coexpression of CD3 by Thy-1.2+ and by NK 1.1+ colonies
17-64 65-256 2 257
NUMBERS OF CELLS/COLONY
Figure2. Analysis of the kinetics of Con A-induced colony growth. Colony-bearing filter paper discs were fixed and stained with WrightIGiemsa on days 6, 8, and 12 of culture. Average numbers of celldcolony were determined (2200 colonies) for each day. Distribution of colony size is shown in the graph. Typical colonies from day 6, 8, and 12 of culture are illustrated. Small darkly stained cells at day 12 indicate cell death.
splenocytes/disc and 8 days of culture were chosen as standard conditions for all subsequent experiments. 3.2 Phenotypic analysis of colonies grown with IL 2 and Con A Phenotypes of colonies were initially analyzed using single radiolabeled mAb probes. Fig. 2 shows a typical autoradiograph of a filter paper disc labeled with mAb specific for CD5; each focus of label (dot) represents a CD5+ colony. Autoradiographic dots from a series of discs were visually enumerated to determine the average number of colonies/disc expressing each phenotype. Results of three exper-
TcR gene products are expressed with the CD3 molecule on the surface of all Tcells [ 12].The CD3 marker could be used to identify Tcell colonies, since theThy-1.2 molecule is not restricted to T cells [3, 17, 181. As NK cells are not associated with the expression of TcR and CD3 gene products [18,19], it was expected that co-expression of NK 1.1 and CD3 would be unlikely. Thus, we used dual IF to determine CD3 expression among Thy-1.2+ and NK 1.1+ colonies. Fig. 4 shows examples of colonies stained with anti-NK 1.1 and anti-CD3 fluorescent mAb to demonstrate that colonies could be easily identified by IF.The red color identifies a colony stained with PE-labeled anti-NK 1.1 while the green color indicates a colony stained with FITC-labeled antiCD3 (Fig. 4). Table 1 (IF column) summarizes the results of three such experiments. Although more colonies were revealed by IF than by RIA, the ratio of Thy-1.2+/NK 1.1+ colonies was essentially the same. The total number of CD3+ colonies (800 f 130) was equivalent to the number of Thy-
Table 1. Phenotypic analysis of colonies from C57BL/6 splenocytes grown in the presence of Con A and IL 2
MarkeP)
Number of positive colonieddisch) RIA
Thy-1.2 NK 1.1 cD5
CD8 ars cD3 Thy-l.ZCD3 NK 1.llCD3
425 f 55b’
88 f 15 171 f 11 141 f 20 13f 2 ND ND ND
IF 1075 f 174 267f 34 ND ND ND 800 130 778 f 162 91 f 2
*
a) Specificity of mAb used to label colonies. b) Numberof positive colonieddisc identified using RIA or IF (X f SEM). The total number of colonieddisc detected by WrightGiernsa staining was 885 f 133.
Figure3. Autoradiograph of colonies on a filter paper disc. Colony-bearing filter paper disc was fixed after 8 days of culture and incubated with biotin-conjugated anti-CD5 rnAb. Bound mAb was revealed with ‘2SI-labeledstreptavidin, followed by autoradiography. Each “dot” represents a CD5+ colony (163 CD5+ colonies).
638
M. Sarzotti,V. Kumar, M. Bennett and G. Kelsoe
1.2+/CD3+ colonies (778 f 162), indicating that dual IF detected the presence of the CD3 E molecule only on Thy-1.2+ colonies.Thy-1.2+/CD3+ cells are by definitionT cells [12]. Thus, dual IF established the mean number of T cell (Thy-1.2+/CD3+) colonies (= 778/disc), the mean number of NK cell (NK 1.1+/CD3-) colonies (= 176/disc), and the mean number of colonies co-expressing NK 1.1 and CD3 (=91/disc). Using these values, the precursor fre-
Eur. J. Immunol. 1991. 21: 635-641
quency (f) for T cells (calculated as mean number of Thy-1.2+/CD3+ colonieshumber of input cells) was deterof NK 1.1+/CD3- cells, f = 3.5 x mined to be f = 2 x and o ~ N K1.1+/CD3+ cells, f = 2 x low5.Thus, the CD3 molecule was co-expressed-by about 72% of all Thy-1.2+ colonies. Most NK 1.1+ colonies (66%) were CD3-; however, about 1/3 of all NK 1.1+ colonies also expressed CD3 E.
Figure 4. Determination of dual phenotype by immunofluorescence. Colony-bearing filter paper discs were fixed and incubated with anti-NK 1.1 and anti-CD3 mAb. Bound mAb were revealed by PE-labeled streptavidin and by FITC-labeled goat anti-hamster Ab, followed by fluorescence microscopy. Three colonies are illustrated: NK 1.1-/CD3+ (top) NK l.l+/CD3- (middle), NK 1.1+/CD3+ (bottom). Magnification: 100 x (top), 50 X (middle), 20 X (bottom). Subsequently, each figure was photographically enlarged to approximately equal final magnification.
Eur. J. Immunol. 1991. 21: 635-641
Murine Tand NK cell colonies
3.4 Are NK 1.1+colonies NK cell colonies? To assess whether colonies bearing the NK 1.1+ phenotype were in fact NK cells, we used two approaches: (a) deletion of Con A from the culture media, or (b) use of splenocytes from scid mice. As NK cells do not require Con A stimulation to grow in the presence of IL 2 [3], we plated C57BL/6 splenocytes onto filter paper discs in medium containing IL 2 alone. In the absence of Con A , maximum colony size (= 100 cells) was also achieved in 8 days, but the total number of colonies/disc was much lower (210 k 49) than in the presence of Con A (885 k 133). Colonies grown in the absence of Con A expressed the same phenotypic markers as those grown in the presence of Con A, but at different frequencies (Table 2). For instance, the frequency of Tcell (Thy-1.2+/CD3+)colonies grown in the absence of Con A almost 7-fold lower than in was calculated as f = 3 x the presence of C6n A (Table 1). Interestingly, the frequenwas decreased to cy of NK 1.1+/CD3+cells (f = 4 x background levels (5-fold) In the absence of Con A while the frequency (f = 1.5 x of NK cells (NK 1.1+/CD3-) was only reduced 2-fold. Thus, while the absence of Con A significantly reduced the total number of colonieddisc, the formation and growth of NK 1.1+/CD3- colonies was relatively spared. The RIA results also showed that some CD8+ colonies (117 k 4) developed in culture with IL 2 and without Con A , possibly accounting for the majority of CD3+ colonies recovered in these cultures. In a more strict approach to ensure that NK cells gave rise to colonies, we employed scid mice as splenocyte donors. Scid mice lack matureTand B cells [20]; the only Thy-1.2+ cells that are recovered from IL 2-enriched cultures of scid mouse splenocytes are NK cells [3]. As shown in Table 3, splenocytes from scid mice generated Thy-1.2" colonies equally well in both the presence or absence of Con A . Splenocytes from scid and phenotypically normal congenic C.B-17 mice generated similar numbers of Thy-1.2+ colonieddisc (151 2 scid vs. 180 f 19 normal) in the absence of Con A . As expected, the number of Thy-1.2+ colonies generated from spleens of normal mice was significantly higher in the presence of Con A . Since -50% of the
*
Table 2. Phenotypic analysis of colonies from C57BL16 splenocytes grown with IL 2 in the absence of Con A
Markers)
Thy-1.2a) NK 1.1 CD8 am cD3 Thy-l.ZCD3 NK l.llCD3
639
Thy-l.2+ colonies generated by normal splenocytes in the absence of Con A co-expressed CD3 (Table2) one would have expected a lower number of Thy-1.2+ colonies generated by scid mice splenocytes (which lack CD3+ cells). However, the absence of T and B lymphocytes and the consequent lower cellularity of scid spleens likely resulted in the higher frequency of Thy-1.2+ colonies of scid mice. Together, these experiments indicate that NK 1 . 1 + colonies from C57BL/6 splenocytes grown in the absence of Con A and Thy-1.2+ colonies derived from scid mice were indeed NK cell colonies. Unfortunately, as the C.B-17 strain is NK 1.1-, the confirming assay for NK 1 . If colonies could not be performed.
4 Discussion Our studies have shown that the filter paper disc method. originally described for the growth of B cell colonies [lo], effectively supports the short-term growth of other murine lymphoid lineages. Phenotypic analyses of colonies grown in the presence of Con A and IL2 revealed discrete colonies of T (CD3+) and NK (NK 1.1+) cells. Optimal colony growth was achieved at 8 days of culture with an average colony size of 100 cells. Colonies were identified by specific radiolabeled or fluorescent mAb reagents. The majority of colonies expressed the Thy-1.2 marker, and 72% of these co-expressed the CD3 E molecule (Table 1). About 20%-25%0 of the colonies were NK 1.1+ (Table 1). Interestingly, about 12% of the CD3+ colonies coexpressed NK 1.1 (Tables 1 and 2).
-
Cloning of T lymphocytes has been extensively performed using LD methods [4-61. The precursor frequency of murine splenic T lymphocytes, as calculated from LD assays,varies between f = 1 x and 3 x lo-' [4-6].The frequency of T h ~ - l . 2 ~ / C D 3cells + grown on filter paper discs with Con A and IL 2 (f = 2 x lo-'') was substantially lower than that achieved by LD. However, the ease in growing and screening colonies on filter paper discs may compensate for the low frequency of colony founding cells for many purposes. Moreover, the spacing (= 1 colony/S mm2)of these colonies and their direct cell-dose dependency (Fig. 1) likely precludes the generation of colonies from multiple precursors, a common hazard of LD techniques.
Number of positive colonieddisch) RIA
IF
179 f 16 74f 5 117 ? 4 19f 3 ND ND ND
283 f 34
83
* 10
ND ND 145 f 26 148 f 10 18f 6
Table3. Phenotypic analysis of colonies from C.B-17 scid or normal splenocytes grown with IL 2 in the presence and absence of Con A
Mouse strain
C.B-17 scid C.B-17 a) Specificity of mAb used to label colonies. b) Numberof positive colonieddisc identified using RIA or IF (X f SEM). The total number of colonies/disc detected by WrightGiemsa staining was 210 f 49.
Number of Thy-1.2+ colonieddisc
Con A
+ConA
-
175 f 9) 488 f 32
151 f 2 180 f 19
a) Number of Thy-1.2+ colonies/disc identified using R I A (f f SEM) .
640
M. Sarzotti,V. Kumar, M. Bennett and G. Kelsoe
The physical separation of colonies grown on filter paper discs with Con A and IL 2 does not support notions that cell contact between accessory cells and Tcells is required for T cell proliferation to occur after mitogen stimulation [21]. In fact, our results recall the findings of Palacios et al. [22], who showed that a combination of IL 1 and IL 2 efficiently replaced accessory cells in the growth of Tcells initiated by Con A. In our experiments, we used a partially purified IL 2 that may contain low levels of IL 1 and/or other lymphokines. Ancillary factors are likely necessary to support the growth of progeny clones on filter paper discs.
Eur. J. Immunol. 1991.21: 635441
unpublished data). TcR gene transcripts found in NK l.l+/CD3+ colonies would demonstrate that these are indeed Tcells expressing the NK 1.1 marker. It is not entirely clear why there are virtually no detectable NK l.l+/CD3+ cells in the normal spleen, yet 20-33% of the NK 1.1+ colonies coexpressed CD3. If NK 1.1+/CD3and NK 1.1 +/CD3+colonies do arise from different precursors, it may be that the expansion of the latter is highly regulated in vivo. Our studies also raise questions regarding the function of NK 1.1+/CD3+cells and the role of the NK 1.1 molecule in this population. In this respect, it is of interest to note that NK 1.1+/CD3+ thymocytes exhibit non-MHC-restricted, i.e. NK-like, cytotoxicity ([26] and V.K. and M.B., unpublished data). It is tempting to speculate that the NK 1.1 molecule may be associated with non-MHC-restrictedcytolysis. This notion is supported by two recent observations: (a) a subpopulation of F4/80+ monocytic cells that mediates NK-like killing also expresses NK 1.1 [29]; (b) certain NK functions in vivo are regulated by the NK 1.1 gene [30]. These studies indicate that although NK 1.1 can be used as a marker for endogenous NK cells [18, 251, its expression in cultured cells is not restricted to cells of the NK lineage.
Importantly, our results further showed that culture conditions used to cloneT lymphocytes on filter paper discs were also suitable for the growth of NK cell colonies. In contrast toTcells, cloning of murine NK cells (NK 1.1+/CD3-) has not been accomplished by any previous method. This has imposed a significant limitation on the study of NK cell differentiation.Todemonstrate that cells of the NK lineage were able to generate progeny colonies,we cultured cells in the absence of Con A [3].The frequencyof NK cell colonies (NK l.l+/CD3-) was relatively spared in the absence of Con A, while the frequency of Tcell colonies was reduced 7-fold (Table 2; IF column) when Con A was absent from the culture medium. Furthermore, we used scid mice which lack functional Tand B lymphocytes [20] but have normal We thank Dr. J. A . Bluestone for kindly providing the 145-2CIl NK cell precursors [23] and splenic NK activity [24]. Splenic hybridoma and Dr. D. H. Coppenhaver for performing the protein NK cells from scid mice areThy-1.2- when freshly isolated, A HPLC purification of mAb. The secretarial assistance of Ms. E . Young and Ms. J. Dailey is greatly appreciated. but become Thy-1.2+ after culture in IL2 and do not require Con A to proliferate in the presence of IL 2 [3]. Received July 23, 1990; in revised form October 20, 1990. Thus, our observation that Thy-1.2+ colonies developed from scid mice splenocytes, even in the absence of Con A (Table 3), indicates that colonies of NK cells could be 5 References grown. It is unknown whether the NK 1.1+/CD3+ phenotype
identifies colonies of NK cells that express the CD3 molecule or of T cells expressing the NK 1.1 marker. Previous studies with NK 1.1 mAb suggest that all NK activity in the murine spleen is contained within NK 1.1+ cells [25]. Furthermore, NK 1.1+ cells isolated from the spleen do not express functional transcripts of the TcR gene or the 6 and E chains of the CD3 complex [18,19]. Thus it appears unlikely that NK 1.1+/CD3+ cells were derived from NK 1.1+/CD3- (NK) cells. In contrast, in recent studies we observed that NK 1.1-/CD3+/Ly-2- thymocytes could give rise to NK 1.1+/CD3+ cells in culture (GarniWagner, B. A . , Bennett, M. and Kumar,V., manuscript in preparation). Apparently similar cells have been detected by others as well [26]. It is interesting to note that in our studies the frequency of both NK 1.1+/CD3+cells and Tcells decreased dramatically in the absence of Con A , suggesting that the growth requirements of NK 1.1+/CD3+cells were more like Tcells than NK cells. We concluded, therefore, that the NK 1.1 +/CD3+ colonies detected probably represent an unusual set of Tcells with NK cell markers [7, 27, 281. This, however, needs to be proven formally.
One way to address this issue will be to analyze the somatic genotype of colonies. Detection of TcR and lymphokine (IFN-y) gene transcripts in colonies have given positive results in preliminaj experiments (M. S, and 'G. K . ,
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Eur. J. Immunol. 1991. 21: 635-641 16 Paige. C. J., Gisler, R. H . , McKearn, J. I? and Iscove, N. N., Eur. J. Immunol. 1984. 14: 979. 17 Pollack, S. B..Tam, M. R.,Nowinski, R. C. andEmmons, S. L., J. Irnmunol. 1979. 123: 1818. 18 Tutt. M. M., Kuziel,W. A., Hackett, J. Jr., Bennett, M.,Tucker, F? W. and Kumar,V., J. Irnmunol. 1986. 137: 2998. 19 Biron. C. A.,Van den Elsen, F?,Tutt, M. M., Medveczky, F?, Kumar,V. and Terhorst, C., J. Irnmunol. 1987. 139: 1704. 20 Dorshkind, K., Keller, G . M., Phillips, R. A . , Miller, R. G., Bosma, G. C., OToole, M. and Bosma, M. J., J. Irnmunol. 1984. 132: 1804. 21 Hiinig,T., Eur. J. Immunol. 1983. 13: 596. 22 Palacios, R. and Leu,T., Cell. Immunol. 1985. 94: 369. 23 Hackett. J. Jr., Bosma, G . C., Bosma, M. J., Bennett, M. and Kumar,V., Proc. Natl. Acad. Sci. USA 1986. 83: 3427.
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24 Dorshkind, K., Pollack, S. B., Bosma, M. J. and Phillips, R. A.. J. Immunol. 1985. 134: 3798. 25 Hackett, J.,Tutt,M.,Lipscomb, M. F., Bennett. M.. Koo. G . C. and Kumar,V., J. Irnmunol. 1986. 136: 3124. 26 Ballas, Z. K. and Rasmussen,W., FASEB J. 1989. 3: A490. 27 Yankelevich, B., Knobloch, C., Nowicki, M. and Dennert. G . . J. Irnmunol. 1989. 142: 3423. 28 Mason, L., Giardina, S. L., Hecht. T.. Ortaldo. J. and Mathieson, B. J., J. Imrnunol. 1988. 140: 4403. 29 Li, H., Schwinzer, R . , Baccarini, M. and Lohmann-Mathes. M.-L., J. Exp. Med. 1989. 169: 973. 30 Sentman, C. L., Kumar,V., Koo, G . C. and Bennett. M.. J. Irnmunol. 1989. 142: 1847.
Marcella Sanottia, Vinay Kumar+, Michael Bennett+ and Garnett KelsoeO Department of Veterans Affairs Medical CenterA,Baltimore, Department of Pathology, University of Texas Southwestern Medical Center at Dallas+, Dallas and Department of Microbiology and Immunologyo, University of Maryland School of Medicine, Baltimore
Murine Tand NK cell colonies
635
Cloning of murine splenic T lymphocytes and natural killer (NK) cells on filter paper discs: detection of a novel NK/T phenotype* Discrete colonies of splenocytes were grown on filter paper discs in the presence of concanavalin A and interleukin 2. Phenotypic analysis of the colonies indicated that the majority expressed the Thy-1.2 marker and 72% of these co-expressed the CD3 molecule. Of the colonies 20%-25% were NK 1.1+ and they developed regardless of the presence of Con A in the culture medium, a property of the NK lineage. In addition, Thy-1.2+ colonies developed when splenocytes from scid mice, which lack mature Tand B cells, were grown both in the presence and absence of concanavalin A . These results demonstrate that colonies of murine splenic T lymphocytes and NK cells could be successfully grown on filter paper discs and phenotypically characterized.With this colonies technique, it was possible to identify a novel subset of NK 1.1+ colonies that co-expresses CD3 and shares growth properties with T cell colonies.
1 Introduction
2 Materials and methods
Peripheral T lymphocytes can be activated with mitogenic lectins, such as Con A [l], to express IL2R and to proliferate in the presence of IL 2 [2]. In contrast, NK cells can be stimulated to proliferate in the presence of I L 2 alone [3]. These properties have been exploited in LD assays to analyze clones of T lymphocytes or human NK cells [4-71. While this strategy has proved generally successful for Tcells the ability to establish and analyze clones of murine NK cells remains elusive. In fact, all reported cloned murine cell lines of presumed NK origin have proved to belong to theTcell lineage [8,9].This inability has served as an impediment to investigations of the NK cell lineage.
2.1 Mice C57BL/6J mice were purchased (The Jackson Laboratories, Bar Harbor, ME) and maintained at the local animal facility. C.B-l7/Icr mice homozygous for the severe combined immunodeficiency disease (scid) mutation (C.B-17 scid) and their normal counterparts (C.B-17) were bred at the University of Texas Southwestern Medical Center at Dallas. Splenic lymphocytes were obtained from age- (6-16 weeks) and sex-matched.mice.
2.2 Media
One alternative to LD methods is the cloning of lympho- Cells were cultured in a-MEM (cat. no. 410-1900, Gibco cytes on filter paper discs.This technique was developed for Laboratories, Grand Island, NY), supplemented with 10% cloning mitogen- and antigen-stimulated B cells, and FCS (Hyclone, Logan, UT), 5 x 10-5M 2-ME (Sigma permits the phenotypic and genotypic analysis of large Chemical Co., St. Louis, MO), 2 g/l Na2HC03 (Sigma), numbers of spatially isolated lymphocyte colonies in replica and a penicillin (50 U/ml) - streptomycin (50 pg/ml) assays [lo, 111. In the present report, we demonstrate that mixture (Flow Laboratories, McLean, VA). A washing colonies of murine splenic T lymphocytes and NK cells medium (WM) was prepared by diluting culture medium could be successfully grown on filter paper discs and 1 : 10 with a-MEM medium. phenotypically characterized. T cell colonies were characterized by the co-expression of Thy-1.2 and CD3, the signal-transducing molecule for the TcR [12]. NK cell 2.3 Reagents colonies were characterized by the expression of the NK 1.1 molecule [13] and the co-expression of the NK 1.1 marker Con A (Sigma) was used at 5 pg/ml. Partially purified with the CD3 molecule was investigated. Unexpectedly, a human IL 2 (Electro-Nucleonics, Inc., Silver Spring, MD) was used at 100 U/ml. Biotin-N-hydroxysuccinimide ester subset of NK 1.1+ colonies co-expressed CD3. (BNHS; Vector Laboratories, Burlingame, CA) was used to conjugate mAb as described by the manufacturer. [I 87561 Anti-Thy-1.2 mAb (Accurate Chemicals, Westbury, NY) was purified by protein A HPLC and biotinylated. Anti-NK * Supported in part by USPHS grants AG-08192 (G.K.) and 1.1 mAb was obtained from culture SN of clone PK136 [ 131, AI-20451 (V.K.). purified over Affi-Gel-protein A and biotinylated. Antiarsonate (anti-ars) mAb obtained from ascites fluid proCorrespondence: Marcella Sarzotti, Research Service (151), duced by the 22B5 hybridoma (kindly provided by Dr. D. D.V.A. Medical Center, 3900 Loch Raven Blvd., Baltimore, MD Capra, UTHSC Dallas, TX) was purified over Affi-Gel21218, USA protein A and biotinylated. Biotin-conjugated anti-CDS and antLCD8 mAb were purchased from Becton-DickinAbbreviations: am: Arsonate IF: Immunofluorescence WM: Washing medium son (Mountain View, CA). Anti-CD3 mAb was derived 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+ .251a 0014-298019110303-0635$3.~0
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M. Sarzotti,V. Kumar, M. Bennett and G. Kelsoe
from culture SN of the 145-2C11 hybridoma, a cell line derived from hamster and specific for the E chain of the murine CD3 complex [ 141. FITC-labeled goat antibody specific for hamster IgG Hand L chains (OrganonTecknika Corp. ,Westchester, PA) used at a 1 : 40 dilution did not bind to mouse thymocytes or splenocytes. Lymphocyte colonies were grown on Whatman 54 filter paper discs (8.26 cm 0 ; Whatman Paper Division, Clifton, NJ).
extensively with PBS/BSA, treated with a fluorescence stabilizer (Amersham), and mounted on glass slides in a solution of 50% PBS and 50% glycerol for fluorescence microscopy.
3 Results 3.1 Kinetics of colony growth
2.4 Preparation and culture of splenocytes Before culture, filter paper discs were prepared as described [lo]. Spleens were taken aseptically from mice killed by cervical dislocation. Single-cell suspensions were prepared by standard methods and the cells washed twice in ice-cold WM. Five milliliters of culture medium containing splenocytes (5 x lo6 cells, unless otherwise specified) was pipetted evenly onto each washed filter paper disc. Next, 5 ml of culture medium containing IL 2 (200 U/ml) and Con A (10 pg/ml) (when indicated) was gently added. Cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO:! for 8 days (unless otherwise specified). 2.5 Visualization of colonies
Standard histological techniques were used to demonstrate colonies by microscopy [lo]. Discs were fixed in 10% neutral-buffered formalin for 5-7 min at room temperature and washed 2 x with 0.1 x PBS [ 101. Discs were then stained with Wright-Giemsa stain (Diagnostic Systems, Inc., Gibbstown, NJ), washed with distilled water, dehydrated in absolute ethanol, cleared in xylene and mounted on glass slides for microscopy.
2.6 RIA Discs were fixed with a solution of 0.5% glutaraldehyde (Polysciences, Inc.,Warrington, PA) in PBS for 10 min at 4°C. Discs were then washed 4 x in PBS and 3 x in PBS containing 1% BSA (Sigma; PBSBSA). Washed discs were overlayed with specific mAb diluted in PBS/BSA and incubated at 4°C for 5-6 h. Discs were then washed 5 x in PBS/BSA containing 0.05% Tween 20 and 0.01% SDS (RIA wash). Washed discs were overlayed with RIA wash containing '*'I-labeled streptavidin = 3.7-18.5 kBq/ml (0.1-0.05 pCi/ml; Amersham, Arlington Heights, IL), incubated overnight at 4"C, rinsed extensively in RIA, washed and air-dried. Autoradiography was performed as described [ 101.
Splenocytes seeded onto filter paper discs in medium containing IL 2 and Con A produced discrete colonies of lymphocytes that could be visualized by microscopy. Titration experiments were performed to determine the optimal number of plated splenocytes for the production of colonies and to ensure that colony formation was dependent upon proliferation of a single precursor cell (Fig. 1). Over a 50-fold range of input cell densities (1 x 105-5 X 106 splenocytes/disc), the production of colonies was a linear function of cell number with a slope of approximately 1, implying a first-order relationship between the generation of colonies and precursor cell number [16]. At plating densities > 5 x 106 splenocytes/disc, the plating efficiency decreased. Subsequent experiments were then carried out using 5 x lo6 input splenocytes/disc. Fig. 2 illustrates the growth of colonies of splenocytes during 12 days of culture with IL 2 and Con A . On day 6 of culture, 55% of the colonies contained between 5 and 16 cells, and 44% between 17 and 64 cells. By day 8 the size of the colonies had increased substantially: only 10% of the colonies contained 5-16 cells while 47% and 34% contained 17-64 and 65-256 cells, respectively. However, by day 12, colony size had not increased and cell death began to occur, as evidenced by an increased percentage of small colonies (5-16 cells) and a decreased percentage of colonies containing 65-256 cells (Fig. 2). Thus, colonies grew in rough synchrony and maximum colony size was achieved at 8 days of culture. Using the median colony size on each day of culture, we calculated the average doubling-time of cells to be 33 h at day 8. Similar kinetics of colony growth were obtained when spleen cells were plated at lower densities (data not shown). Thus, a plating number of 5 x 106
2.7 Immunofluorescence (IF)
Discs were fixed with a solution of 0.5% paraformaldehyde (Sigma) in PBS [15]. Discs were then washed as described above, subsequently overlayed with specific mAb diluted in PBS/BSA and incubated at room temperature for 1-2 h. Discs were then washed 5 x in PBSBSA and overlayed with PE-streptavidin (Southern Biotechnology Assoc., Birmingham, AL) or with FITC-labeled secondary Ab at room temperature for 1-2 h in the dark. Finally, discs were rinsed
Zl
1:o
510
1010
MILLIONS OF INPUT CELLS/DISC
Figure 1. Cell dose-dependency of colony formation. Colonybearing filter paper discs were fixed and stained with WrightGiemsa on day 8 of culture. Mean number of colonieddin (12-6 discs) (k SEM) was determined for each input number of splenocytes. The least-squares regression line generated from the arithmetic means of the points, 1 x 105-5 X 106, is shown.
Eur. J. Immunol. 1991. 21: 635-641
-
80 601
-
Murine Tand NK cell colonies
iments are summarized in Table 1 (RIA column). The highest number of colonies/disc (=42Ydisc) was identified with mAb directed against Thy-1.2, a marker present on all Tcells and on the majority of NK cells propagated in IL 2 [3, 17, 181. About 40% and 33% of the number of Thy-1.2+ colonies expressed the CD5 or CD8 determinants, respectively. Interestingly, a mAb specific for the NK 1.1 determinant, a marker present on NK cells [13], identified = 88 colonies/disc. Some 13 colonies/disc bound an mAb, 22B5, specific for ars. The 22B5 mAb bears the isotype (y2b) of PK136 (anti-NK 1.1) and was included to control for nonspecific binding of Ab to colonies.These results implied that in the presence of Con A and IL 2, splenocytes gave rise to discrete colonies of T lymphocytes and NK cells.
DAY 6
DAY 8
DAY 12
n
40
5-16
637
3.3 Coexpression of CD3 by Thy-1.2+ and by NK 1.1+ colonies
17-64 65-256 2 257
NUMBERS OF CELLS/COLONY
Figure2. Analysis of the kinetics of Con A-induced colony growth. Colony-bearing filter paper discs were fixed and stained with WrightIGiemsa on days 6, 8, and 12 of culture. Average numbers of celldcolony were determined (2200 colonies) for each day. Distribution of colony size is shown in the graph. Typical colonies from day 6, 8, and 12 of culture are illustrated. Small darkly stained cells at day 12 indicate cell death.
splenocytes/disc and 8 days of culture were chosen as standard conditions for all subsequent experiments. 3.2 Phenotypic analysis of colonies grown with IL 2 and Con A Phenotypes of colonies were initially analyzed using single radiolabeled mAb probes. Fig. 2 shows a typical autoradiograph of a filter paper disc labeled with mAb specific for CD5; each focus of label (dot) represents a CD5+ colony. Autoradiographic dots from a series of discs were visually enumerated to determine the average number of colonies/disc expressing each phenotype. Results of three exper-
TcR gene products are expressed with the CD3 molecule on the surface of all Tcells [ 12].The CD3 marker could be used to identify Tcell colonies, since theThy-1.2 molecule is not restricted to T cells [3, 17, 181. As NK cells are not associated with the expression of TcR and CD3 gene products [18,19], it was expected that co-expression of NK 1.1 and CD3 would be unlikely. Thus, we used dual IF to determine CD3 expression among Thy-1.2+ and NK 1.1+ colonies. Fig. 4 shows examples of colonies stained with anti-NK 1.1 and anti-CD3 fluorescent mAb to demonstrate that colonies could be easily identified by IF.The red color identifies a colony stained with PE-labeled anti-NK 1.1 while the green color indicates a colony stained with FITC-labeled antiCD3 (Fig. 4). Table 1 (IF column) summarizes the results of three such experiments. Although more colonies were revealed by IF than by RIA, the ratio of Thy-1.2+/NK 1.1+ colonies was essentially the same. The total number of CD3+ colonies (800 f 130) was equivalent to the number of Thy-
Table 1. Phenotypic analysis of colonies from C57BL/6 splenocytes grown in the presence of Con A and IL 2
MarkeP)
Number of positive colonieddisch) RIA
Thy-1.2 NK 1.1 cD5
CD8 ars cD3 Thy-l.ZCD3 NK 1.llCD3
425 f 55b’
88 f 15 171 f 11 141 f 20 13f 2 ND ND ND
IF 1075 f 174 267f 34 ND ND ND 800 130 778 f 162 91 f 2
*
a) Specificity of mAb used to label colonies. b) Numberof positive colonieddisc identified using RIA or IF (X f SEM). The total number of colonieddisc detected by WrightGiernsa staining was 885 f 133.
Figure3. Autoradiograph of colonies on a filter paper disc. Colony-bearing filter paper disc was fixed after 8 days of culture and incubated with biotin-conjugated anti-CD5 rnAb. Bound mAb was revealed with ‘2SI-labeledstreptavidin, followed by autoradiography. Each “dot” represents a CD5+ colony (163 CD5+ colonies).
638
M. Sarzotti,V. Kumar, M. Bennett and G. Kelsoe
1.2+/CD3+ colonies (778 f 162), indicating that dual IF detected the presence of the CD3 E molecule only on Thy-1.2+ colonies.Thy-1.2+/CD3+ cells are by definitionT cells [12]. Thus, dual IF established the mean number of T cell (Thy-1.2+/CD3+) colonies (= 778/disc), the mean number of NK cell (NK 1.1+/CD3-) colonies (= 176/disc), and the mean number of colonies co-expressing NK 1.1 and CD3 (=91/disc). Using these values, the precursor fre-
Eur. J. Immunol. 1991. 21: 635-641
quency (f) for T cells (calculated as mean number of Thy-1.2+/CD3+ colonieshumber of input cells) was deterof NK 1.1+/CD3- cells, f = 3.5 x mined to be f = 2 x and o ~ N K1.1+/CD3+ cells, f = 2 x low5.Thus, the CD3 molecule was co-expressed-by about 72% of all Thy-1.2+ colonies. Most NK 1.1+ colonies (66%) were CD3-; however, about 1/3 of all NK 1.1+ colonies also expressed CD3 E.
Figure 4. Determination of dual phenotype by immunofluorescence. Colony-bearing filter paper discs were fixed and incubated with anti-NK 1.1 and anti-CD3 mAb. Bound mAb were revealed by PE-labeled streptavidin and by FITC-labeled goat anti-hamster Ab, followed by fluorescence microscopy. Three colonies are illustrated: NK 1.1-/CD3+ (top) NK l.l+/CD3- (middle), NK 1.1+/CD3+ (bottom). Magnification: 100 x (top), 50 X (middle), 20 X (bottom). Subsequently, each figure was photographically enlarged to approximately equal final magnification.
Eur. J. Immunol. 1991. 21: 635-641
Murine Tand NK cell colonies
3.4 Are NK 1.1+colonies NK cell colonies? To assess whether colonies bearing the NK 1.1+ phenotype were in fact NK cells, we used two approaches: (a) deletion of Con A from the culture media, or (b) use of splenocytes from scid mice. As NK cells do not require Con A stimulation to grow in the presence of IL 2 [3], we plated C57BL/6 splenocytes onto filter paper discs in medium containing IL 2 alone. In the absence of Con A , maximum colony size (= 100 cells) was also achieved in 8 days, but the total number of colonies/disc was much lower (210 k 49) than in the presence of Con A (885 k 133). Colonies grown in the absence of Con A expressed the same phenotypic markers as those grown in the presence of Con A, but at different frequencies (Table 2). For instance, the frequency of Tcell (Thy-1.2+/CD3+)colonies grown in the absence of Con A almost 7-fold lower than in was calculated as f = 3 x the presence of C6n A (Table 1). Interestingly, the frequenwas decreased to cy of NK 1.1+/CD3+cells (f = 4 x background levels (5-fold) In the absence of Con A while the frequency (f = 1.5 x of NK cells (NK 1.1+/CD3-) was only reduced 2-fold. Thus, while the absence of Con A significantly reduced the total number of colonieddisc, the formation and growth of NK 1.1+/CD3- colonies was relatively spared. The RIA results also showed that some CD8+ colonies (117 k 4) developed in culture with IL 2 and without Con A , possibly accounting for the majority of CD3+ colonies recovered in these cultures. In a more strict approach to ensure that NK cells gave rise to colonies, we employed scid mice as splenocyte donors. Scid mice lack matureTand B cells [20]; the only Thy-1.2+ cells that are recovered from IL 2-enriched cultures of scid mouse splenocytes are NK cells [3]. As shown in Table 3, splenocytes from scid mice generated Thy-1.2" colonies equally well in both the presence or absence of Con A . Splenocytes from scid and phenotypically normal congenic C.B-17 mice generated similar numbers of Thy-1.2+ colonieddisc (151 2 scid vs. 180 f 19 normal) in the absence of Con A . As expected, the number of Thy-1.2+ colonies generated from spleens of normal mice was significantly higher in the presence of Con A . Since -50% of the
*
Table 2. Phenotypic analysis of colonies from C57BL16 splenocytes grown with IL 2 in the absence of Con A
Markers)
Thy-1.2a) NK 1.1 CD8 am cD3 Thy-l.ZCD3 NK l.llCD3
639
Thy-l.2+ colonies generated by normal splenocytes in the absence of Con A co-expressed CD3 (Table2) one would have expected a lower number of Thy-1.2+ colonies generated by scid mice splenocytes (which lack CD3+ cells). However, the absence of T and B lymphocytes and the consequent lower cellularity of scid spleens likely resulted in the higher frequency of Thy-1.2+ colonies of scid mice. Together, these experiments indicate that NK 1 . 1 + colonies from C57BL/6 splenocytes grown in the absence of Con A and Thy-1.2+ colonies derived from scid mice were indeed NK cell colonies. Unfortunately, as the C.B-17 strain is NK 1.1-, the confirming assay for NK 1 . If colonies could not be performed.
4 Discussion Our studies have shown that the filter paper disc method. originally described for the growth of B cell colonies [lo], effectively supports the short-term growth of other murine lymphoid lineages. Phenotypic analyses of colonies grown in the presence of Con A and IL2 revealed discrete colonies of T (CD3+) and NK (NK 1.1+) cells. Optimal colony growth was achieved at 8 days of culture with an average colony size of 100 cells. Colonies were identified by specific radiolabeled or fluorescent mAb reagents. The majority of colonies expressed the Thy-1.2 marker, and 72% of these co-expressed the CD3 E molecule (Table 1). About 20%-25%0 of the colonies were NK 1.1+ (Table 1). Interestingly, about 12% of the CD3+ colonies coexpressed NK 1.1 (Tables 1 and 2).
-
Cloning of T lymphocytes has been extensively performed using LD methods [4-61. The precursor frequency of murine splenic T lymphocytes, as calculated from LD assays,varies between f = 1 x and 3 x lo-' [4-6].The frequency of T h ~ - l . 2 ~ / C D 3cells + grown on filter paper discs with Con A and IL 2 (f = 2 x lo-'') was substantially lower than that achieved by LD. However, the ease in growing and screening colonies on filter paper discs may compensate for the low frequency of colony founding cells for many purposes. Moreover, the spacing (= 1 colony/S mm2)of these colonies and their direct cell-dose dependency (Fig. 1) likely precludes the generation of colonies from multiple precursors, a common hazard of LD techniques.
Number of positive colonieddisch) RIA
IF
179 f 16 74f 5 117 ? 4 19f 3 ND ND ND
283 f 34
83
* 10
ND ND 145 f 26 148 f 10 18f 6
Table3. Phenotypic analysis of colonies from C.B-17 scid or normal splenocytes grown with IL 2 in the presence and absence of Con A
Mouse strain
C.B-17 scid C.B-17 a) Specificity of mAb used to label colonies. b) Numberof positive colonieddisc identified using RIA or IF (X f SEM). The total number of colonies/disc detected by WrightGiemsa staining was 210 f 49.
Number of Thy-1.2+ colonieddisc
Con A
+ConA
-
175 f 9) 488 f 32
151 f 2 180 f 19
a) Number of Thy-1.2+ colonies/disc identified using R I A (f f SEM) .
640
M. Sarzotti,V. Kumar, M. Bennett and G. Kelsoe
The physical separation of colonies grown on filter paper discs with Con A and IL 2 does not support notions that cell contact between accessory cells and Tcells is required for T cell proliferation to occur after mitogen stimulation [21]. In fact, our results recall the findings of Palacios et al. [22], who showed that a combination of IL 1 and IL 2 efficiently replaced accessory cells in the growth of Tcells initiated by Con A. In our experiments, we used a partially purified IL 2 that may contain low levels of IL 1 and/or other lymphokines. Ancillary factors are likely necessary to support the growth of progeny clones on filter paper discs.
Eur. J. Immunol. 1991.21: 635441
unpublished data). TcR gene transcripts found in NK l.l+/CD3+ colonies would demonstrate that these are indeed Tcells expressing the NK 1.1 marker. It is not entirely clear why there are virtually no detectable NK l.l+/CD3+ cells in the normal spleen, yet 20-33% of the NK 1.1+ colonies coexpressed CD3. If NK 1.1+/CD3and NK 1.1 +/CD3+colonies do arise from different precursors, it may be that the expansion of the latter is highly regulated in vivo. Our studies also raise questions regarding the function of NK 1.1+/CD3+cells and the role of the NK 1.1 molecule in this population. In this respect, it is of interest to note that NK 1.1+/CD3+ thymocytes exhibit non-MHC-restricted, i.e. NK-like, cytotoxicity ([26] and V.K. and M.B., unpublished data). It is tempting to speculate that the NK 1.1 molecule may be associated with non-MHC-restrictedcytolysis. This notion is supported by two recent observations: (a) a subpopulation of F4/80+ monocytic cells that mediates NK-like killing also expresses NK 1.1 [29]; (b) certain NK functions in vivo are regulated by the NK 1.1 gene [30]. These studies indicate that although NK 1.1 can be used as a marker for endogenous NK cells [18, 251, its expression in cultured cells is not restricted to cells of the NK lineage.
Importantly, our results further showed that culture conditions used to cloneT lymphocytes on filter paper discs were also suitable for the growth of NK cell colonies. In contrast toTcells, cloning of murine NK cells (NK 1.1+/CD3-) has not been accomplished by any previous method. This has imposed a significant limitation on the study of NK cell differentiation.Todemonstrate that cells of the NK lineage were able to generate progeny colonies,we cultured cells in the absence of Con A [3].The frequencyof NK cell colonies (NK l.l+/CD3-) was relatively spared in the absence of Con A, while the frequency of Tcell colonies was reduced 7-fold (Table 2; IF column) when Con A was absent from the culture medium. Furthermore, we used scid mice which lack functional Tand B lymphocytes [20] but have normal We thank Dr. J. A . Bluestone for kindly providing the 145-2CIl NK cell precursors [23] and splenic NK activity [24]. Splenic hybridoma and Dr. D. H. Coppenhaver for performing the protein NK cells from scid mice areThy-1.2- when freshly isolated, A HPLC purification of mAb. The secretarial assistance of Ms. E . Young and Ms. J. Dailey is greatly appreciated. but become Thy-1.2+ after culture in IL2 and do not require Con A to proliferate in the presence of IL 2 [3]. Received July 23, 1990; in revised form October 20, 1990. Thus, our observation that Thy-1.2+ colonies developed from scid mice splenocytes, even in the absence of Con A (Table 3), indicates that colonies of NK cells could be 5 References grown. It is unknown whether the NK 1.1+/CD3+ phenotype
identifies colonies of NK cells that express the CD3 molecule or of T cells expressing the NK 1.1 marker. Previous studies with NK 1.1 mAb suggest that all NK activity in the murine spleen is contained within NK 1.1+ cells [25]. Furthermore, NK 1.1+ cells isolated from the spleen do not express functional transcripts of the TcR gene or the 6 and E chains of the CD3 complex [18,19]. Thus it appears unlikely that NK 1.1+/CD3+ cells were derived from NK 1.1+/CD3- (NK) cells. In contrast, in recent studies we observed that NK 1.1-/CD3+/Ly-2- thymocytes could give rise to NK 1.1+/CD3+ cells in culture (GarniWagner, B. A . , Bennett, M. and Kumar,V., manuscript in preparation). Apparently similar cells have been detected by others as well [26]. It is interesting to note that in our studies the frequency of both NK 1.1+/CD3+cells and Tcells decreased dramatically in the absence of Con A , suggesting that the growth requirements of NK 1.1+/CD3+cells were more like Tcells than NK cells. We concluded, therefore, that the NK 1.1 +/CD3+ colonies detected probably represent an unusual set of Tcells with NK cell markers [7, 27, 281. This, however, needs to be proven formally.
One way to address this issue will be to analyze the somatic genotype of colonies. Detection of TcR and lymphokine (IFN-y) gene transcripts in colonies have given positive results in preliminaj experiments (M. S, and 'G. K . ,
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