Immunology 1977 32 875
Detection of mitogen-activated T and non-T lymphocytes by virus plaque assay VIRUS PLAQUE ASSAY ON THE CELLS FRACTIONATED BY UNIT GRAVITY SEDIMENTATION
T. KASAHARA, K. SHIOIRI-NAKANO & A. SUGIURA Department of Medical Biology and Parasitology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken and Department of Microbiology, the Institute of Public Health, Minato-ku, Tokyo, Japan
Received 27 August 1976; accepted for publication
November 1976
considered 'to be general properties panying lymphocyte activation.
Summary. Virus plaque assay (VPA) was utilized for the quantitative evaluation of activated lymphocytes. We examined what types of cells, especially which of activated T and non-T lymphocytes, were detected as infective centres after infection with vesicular stomatitis virus. Marked increases in DNA synthesis and in virus-plaque forming cells (V-PFC) were observed not only during the activation of T lymphocytes with Con A, but also, though to a lesser extent, during the activation with lipopolysaccharide (LPS) of non-T lymphocyte preparations of nude spleen from which 0-positive lymphocytes and macrophages were completely depleted. The latter observation was further confirmed by the VPA on the populations enriched in LPS-activated non-T lymphocytes fractionated by the unit gravity sedimentation method. Fast sedimenting cells were found to be more active in DNA synthesis and contained more infective centres after infection than those sedimenting slowly and original unfractionated cells. Both the capacity for DNA synthesis and virus-replication
were
accom-
INTRODUCTION Lymphocytes, when transformed by appropriate stimulation, have been known to become permissive to the replication of certain viruses (Edelman & Wheelock, 1966; Willems, Melnick & Rawls, 1969; Bloom, Jimenez & Marcus, 1970; Joseph, Lampert & Oldstone, 1975). Based on this phenomenon, Bloom and co-workers (Bloom et al., 1970; Jimenez & Bloom, 1970) developed a method, comparable to Jerne's haemolytic plaque assay for antibodyforming cells (Jerne & Nordin, 1963), to enumerate sensitized T lymphocytes by virus plaque assay (VPA). It was reported that VPA detected activated T lymphocytes but not activated B lymphocytes (Kano, Bloom & Howe, 1973; Nowakowski, Feldman, Kano & Bloom, 1973). It was also found that continuous lymphoblastoid cell lines derived from T lymphocytes supported the replication of vesicular stomatitis virus (VSV), while cell lines derived from B lymphocytes were non-permissive except MOPC 104, a plasmacytoma presumed to be
* This work was supported in part by research grants from the Ministry of Education of Japan (010703, 087155). Correspondence: Dr T. Kasahara, Department of Medical Biology and Parasitology, Jichi Medical School, Minamikawachi-machi Tochigi-ken, 329-04, Japan.
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T. Kasahara, K. Shioiri-Nakano & A. Sugiura
of B-cell origin (Nowakowski et al., 1973). We found, however, that lymphocytes from mice with thymic deficiency became permissive to the replication of VSV, when appropriately activated. This was evident particularly when activated lymphocytes were separated according to their size by unit gravity sedimentation. The results indicate that non-T splenic cells, which are considered mainly to be B lymphocytes, as well as T lymphocytes are detected by VPA.
by Dr M. Nakano, Jichi Medical School) were used at the concentration of 3 pg/ml. Cells incubated in the presence of Con A were washed twice with 0.1 M a-methyl-D-mannoside to remove Con A. Depletion of adherent cells from splenic cell suspensions The method is described elsewhere (Kasahara et al., 1975). Briefly, nonadherent cells were obtained after spleen cells suspended in MEM containing 2% FCS were adhered to plastic dishes (Lux, 5220) at 370 twice for 30 min.
MATERIALS AND METHODS
Animals Male and female BALB/c, C3H/He and AKR mice, maintained in our laboratory, were used at 6-12 weeks of age. Congenitally athymic nude (nu/nu) mice derived from BALB/c were purchased from the Center of Laboratory Experimental Animals, Tokyo, Japan, and used at 4-6 weeks of age. Neonatally thymectomized (NTX) mice were also used and the mice with visible thymus remnants were not employed.
Cell cultures L cells were propagated in Eagle's MEM supplemented with 5 % heat-inactivated calf serum. Monolayer cultures for VPA were prepared in 60-mm LUX petri dishes (no. 5220, LUX Scientific Corp. Thousand Oaks, California) in humidified atmosphere containing 5% CO2 (CO2-incubator).
Lymphocytes Spleens were removed aseptically and teased. Viable lymphocytes were separated from granulocytes, erythrocytes, dead cells and cell debris by velocity sedimentation through a Ficoll-Urografin gradient (Kasahara, Shioiri-Nakano & Osawa, 1975). Cells were washed, resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS, Flow Laboratories, Rockville, Maryland) at the concentration of 2.0 x 106 viable cells/ml. In small scale culture, 1.0 ml of cell suspension was incubated in a sterile glass tube with or without mitogens in a C02-incubator at 370 for 2-3 days. In large scale culture, 15-20 ml of cell suspension was incubated in a glass bottle with a flat bottom. The mitogens concanavalin A (Con A, Pharmacia, Uppsala, Sweden) and lipopolysaccharide (LPS, from Salmonella typhimurium LT2, kindly provided
Unit gravity sedimentation through Ficoll gradient The methods described by Miller & Phillips (1969) and Everson, Buell & Rogentine (1973) were modified. By these methods, cells were separated mainly on the basis of their cell size, according to the equation of s = r2/4, in which s is sedimentation velocity (mm/h) and r is the radius of a cell (pm). Ficoll was dissolved in MEM and sterilized by autoclaving. Starting from the 20% stock solution, various concentrations of Ficoll solution were prepared by dilution in MEM. Discontinuous gradient was prepared in a 50 x 80-mm glass bottle by successively layering 15 ml of 12, 10, 8, 6 and 4% Ficoll. Lymphocytes that had been incubated with or without mitogens in large scale culture, usually 5060 ml of cell suspension, were washed, resuspended in 2 ml of 50% FCS, layered carefully on the top of a gradient and allowed to sediment at 40 for 5 h at the unit gravity. Fractions were collected from above through a glass capillary tubing with lower end upwardly curved in J shape. The tip of the glass tubing was placed about 1 mm below the meniscus and the fluid above was gently aspirated by a peristaltic micropump connected to the other end of the glass tubing. Usually six to ten fractions were collected. 60-90% of layered lymphocytes were recovered after sedimentation. Virus plaque assay The method developed by Jimenez & Bloom (1970) was followed. Stocks of VSV, Indiana strain, were prepared in L cells and stored at - 700. Lymphocytes, either unfractionated or fractionated as described above, were washed twice with MEM and resuspended in MEM supplemented with 2% FCS at the concentration of 2-0 x 106 viable cells/ml. Lymphocytes suspended in 0-2 ml were infected VSV at a dose with of 20 PFU/cell. Virus adsorption
877
Virus plaque assay on T and non-T cells carried out for 1.5-2 h in a C02-incubator at 370. Excess virus was removed by two washings with MEM, followed by neutralization with 0.15 ml of adequately diluted rabbit anti-VSV serum (final dilution (1:50) for 1 h at 4°. At this dilution, antiserum was not toxic to lymphocytes. Infected cells were washed once and diluted. From 5 x 104 to 5 x 103 viable cells suspended in 0.15 ml were plated onto a confluent monolayer culture of L cells. Immediately after the plating of lymphocytes, 0.5 ml of agar overlay medium (MEM with 0 7% Ionagar no. 2, 1 % calf serum, 10 ,4g/ml DEAE dextran, and 2 4ug/ml fungisone) was added and plated lymphocytes were thoroughly mixed with the overlay medium by pipetting. After this layer had solidified, a second 4 ml of agar overlay medium was added. Three monolayer cultures of L cells were used per dilution of lymphocytes. After incubation for 24 to 36 h in a CO2 incubator at 370, the agar overlay was removed, monolayers were stained with 0.1 % crystal violet in 20% ethanol and plaques were counted. was
Assay of DNA synthesis Lymphocytes incubated for 2-3 days were collected and 4 x 105 viable lymphocytes in a volume of 0.2 ml were reincubated for 2 h in the presence of 0.5 pCi 3H-labelled thymidine ([3H]Tdr, specific activity 5 Ci/mmol, Radiochemical Centre, Amersham). The radioactivity incorporated into trichloroacetic acidinsoluble materials was determined as described previously (Kasahara et al., 1975).
RESULTS Correlation between DNA synthesis and VPA in lymphocytes activated by mitogens The applicability of VPA for the quantitative study of lymphocyte activation was confirmed by simultaneous determination of DNA synthesis and virus plaque-forming cells (V-PFC) in various lymphocyte preparations (Table 1). When spleen cells from normal mice were incubated in the presence of Con
Table 1. DNA synthesis and virus plaque assay in lymphocyte preparations incubated with
mitogens*
Expt no. 1. 2.
Source of lymphocytes
Normal spleen Normal spleen treated with
anti-O+ complement 3.
NTX spleen§
4.
nu/nu spleen¶
5.
nu/nu spleen treated with anti-O+ complement
[3H]Tdr uptake
VPA
c.p.m./106 cells
V-PFC/106 cells
3363+ 157 35,952+2,724 9921+ 1,346 3349+ 289 6453+ 890 16,557+ 1,057 1396+60
2866+ 363 19,310+2,204 5190+ 1,125 4700+420 6050+450 9160+ 1,150 2180+380 3760+ 520 6720+ 1,780 1725+ 280 2280+ 350 6470+ 1,650 2600+390 3155+ 150 8550+ 1,850
Mitogenst None Con A LPS None Con A LPS None Con A LPS None Con A LPS None Con A LPS
3350+ 115 8732+650 815+ 75 1138+ 31 8906+ 242 1423+ 100 2032+ 116 7197+ 38
* Lymphocytes prepared as described in the Materials and Methods section were incubated in a glass tube (small scale culture) with or without mitogens. t The optimal dose of mitogens was added, which was 3 ,pg/ml for both Con A and LPS. I Spleen cells were treated with anti-0 serum (rabbit anti-serum to brain-associated 0 antigen of C3H mice, final dilution 1:20) and agar-absorbed normal guinea-pig complement. Viable lymphocytes were purified by Ficoll-Urografin and incubated. § 6-13Y4 of 0-positive lymphocytes existed at the initiation of incubation. T 2-9%. of 6-positive lymphocytes existed and Ig-positive lymphocytes were determined to be 45-50%. by FITC-conjugated rabbit anti-mouse IgG (MBL, Fujizoki, Tokyo, Japan).
T. Kasahara, K. Shioiri-Nakano & A. Sugiura
878
Table 2. Effects of depletion of adherent cells from splenic preparations
Expt no. 1.
Source of lymphocytes
BALB/c spleen, untreated BALB/c spleen, non-adherentt BALB/c spleen, non-adherent
2.
nu/nu spleen, untreated nu/nu spleen, non-adherentt
*3
[3H]Tdr uptake
VPA
c.p.m./106 cells
V-PFC/106 cells
880+ 34 1726+ 24 1326+ 12 2642+ 160 380+ 26 1998+ 184 16,967+ 806 415+30 447+ 21 4870+425 578+ 15 967+ 142 2331+218
960+ 169 1780+ 244 880+ 63 2387+ 390 813+ 162 2533+ 192 9660+ 260 914+ 106 951+ 201 1470+ 197 460+ 105 478+ 102 2011+ 137
Mitogen* None LPS None LPS
F-1t F-3 F-5
None Con A LPS None Con A LPS
4ug/ml for both LPS and Con A was used.
t See the Materials and Methods section. Cell recovery was 15-30%4 after plastic-adherent cells were extensively removed. t Non-adherent cells were incubated with LPS for 3 days and fractionated by Ficoll unit gravity sedimentation into five fractions. For other details, see legends for Figs 1 and 2.
A, both DNA synthesis and the number of V-PFC markedly increased. The increment after incubation with LPS was also appreciable but less pronounced. This could be either due to a low mitogenic activity of LPS compared with Con A, or to inherently low reactivity of B lymphocytes. Stimulation of normal spleen cells treated with anti-@ serum and complement, cells from NTX and nude mice, with Con A was marginal. Their reactivity to LPS, however, did not differ from that of normal lymphocytes. Since nude mouse spleen was considered to contain T-cell precursors in a negligible percentage (Roelants, Kathleen, Hagg & Loor, 1976), the cells from nude mice were treated with anti-l serum and complement. The response to LPS in DNA synthesis and the number of V-PFC was not decreased (Table 1, Expts 4 and 5). At least two different types of leucocytes can be mentioned in non-T splenic preparations which formed V-PFC, that is, macrophages and non-T lymphocytes. The former is known to be permissive for VSV (Edelman & Wheelock, 1967; Nowakowski et al., 1973). We examined therefore, the effect of depletion of adherent cells from nude splenic leucocytes. Cells adherent to plastic surface were removed extensively. As shown in Table 2, a nonadherent population free from monocytes and
macrophages remains to make V-PFC when stimulated with LPS. V-PFC derived, at least, from macrophages existing in non-T splenic leucocytes could be excluded (Table 2, Expt 2). It was concluded that lymphocyte preparations actively synthesizing DNA after incubation with mitogens contained a correspondingly greater number of V-PFC when infected with VSV, and that, when appropriately activated, non-T lymphocytes as well as T lymphocytes became permissive to the replication of VSV. The latter fact was further substantiated by the experiments described below. DNA synthesis and VPA in lymphocytes fractionated by unit gravity sedimentation In our experiments the number of V-PFC did not reach more than 2-3%. of the infected lymphocytes with VSV, while a considerable number of small lymphocytes from mouse spleen was observed to be transformed to large blastoid cells activated by Con A. If the activated lymphocytes allow the replication of VSV, more V-PFC must be detected in the preparations containing relatively more of activated lymphocytes. After 2 days' incubation with Con A, cells from normal mouse spleen were fractionated by unit gravity sedimentation (Fig. 1). In these large
879
Virus plaque assay on T and non-T cells
I 0
c'0 0()
0
6. E L
0D
H-
CL
I0
0. I3
>
BL CON A +) unfractionated
0
2
3
i- -F IF-F-i F-2 F-3 F-4 F
5
4
Sedimentation velocity -
F-5
6
(mm / h) -
F-F
F-I F-7
Figure 1. DNA synthesis and virus plaque assay in fractions of Con A-activated spleen cells. Spleen cells from normal mice were incubated with Con A in large scale culture for 46 h and fractionated by unit gravity sedimentation. Fraction number was named from upper of the gradient. The inset shows the distribution of viable lymphocytes among fractions. For other details, see the Materials and Methods section.
I
6.0
c
E
4.5 0
ci
C
-
30 0 "L--
n
-F
1.5 ..I-
I FL CON A LPS
crnrcct1oncated
4 3 5 2 Sed mentotion velocity (mm/h) - -i - i m I-i F-. F-2 F-3 F-4 F-5 8-6 F-7
Figure 2. DNA synthesis and virus plaque assay in fractions of LPS-activated non-T lymphocytes. Spleen cells from NTX mice were incubated with LPS in large scale culture for 42 h and fractionated by unit gravity sedimentation. The left ordinate is [3H]thymidine incorporated into 106 viable lymphocytes.
T. Kasahara, K. Shioiri-Nakano & A. Sugiura
880
Table 3. DNA synthesis and virus plaque assay in fractions of LPS-activated non-T lymphocytes from nu/nu mice*
Fraction number
Sedimentation velocity
Distribution of viable cells
[3H]Tdr uptake
VPA
(mm/hr)
(0)
c.p.m./106 cells
V-PFC/106 cells
Unfractionated None LPS (3,ug/ml) F-I F-2 F-3 F-4 F-5 F-6
0-80 1-3 2-1 2-9 3-8 52
230 3230
725 1680
Fractionated (LPS 3 ug/ml) 150 12-3 19-7 310 27-0 2180 16-8 6672 15-0 9980 90 9172
450 1800 5085 5220 3645 1485
* Spleen cells from athymic nu/nu mice were incubated with LPS in large scale culture for 46 h and fractionated by unit gravity sedimentation as described in the Materials and Methods section.
scale cultures, the activity of DNA synthesis and the number of V-PFC of the unfractionated cells were far less than those in small scale cultures. In microscopic examination, 80-90% of large blastoid cells were recovered in the bottom three fractions. The incorporation of [3H]Tdr was also marked in these fractions. The number of V-PFC per 106 viable cells inoculated was highest in F-6, the fraction most active in DNA synthesis, which was four to five times as high as that in the unfractionated preparation. There were, however, also V-PFC in the fractions sedimenting more slowly. The number of V-PFC relative to [3H]Tdr incorporation was always greater among cells sedimenting slowly (F-2-F-4) than among faster sedimenting cells. The disproportionality between the two parameters suggested that V-PFC comprised, in addition to large blastoid cells actively synthesizing DNA, cells smaller and relatively inactive in DNA synthesis. Non-T lymphocytes incubated with LPS for 2 days were fractionated in the same way (Fig. 2, Table 3). Again, faster sedimenting cells were more active in [3H]Tdr incorporation and contained a higher proportion of V-PFC. This was additional evidence that activated non-T lymphocytes could become V-PFC upon infection with VSV. The number of V-PFC relative to the rate of DNA synthesis was greater in the upper half of the gradient (Fig. 2, F-1-F-4). While the proportion of V-PFC was highest in the fraction of activated T lympho-
cytes in which DNA synthesis was also most active (Fig. 1, F-6), the V-PFC peak usually preceded the peak of DNA synthesis by one to two fractions in activated non-T lymphocytes (Fig. 2; Table 3). Activated T lymphocytes were fractionated more precisely into more than ten fractions (data not shown), but the peak of DNA synthesis and that of V-PFC were not completely separated from each other. It might be that non-T lymphocytes which supported the replication of VSV were generally smaller in size than the corresponding T lymphocytes.
V-PFC detected in unstimulated lymphocyte preparations Lymphocytes incubated in the absence of mitogens always contained a small number of V-PFC upon infection with VSV. The nature of these background V-PFC was examined by fractionation. Spleen cells from normal mice were incubated for 72 h in the absence of added mitogens and were separated by unit gravity sedimentation (Fig. 3). Being unstimulated, even the fraction with the highest rate of DNA synthesis (F-4) incorporated only twice as much [3H]Tdr as the whole population. On the other hand, V-PFC were found in ten-fold higher proportion in F-2 than in unfractionated preparation. The separation of V-PFC peak from the peak of DNA synthesis indicated that lymphocytes actively synthesizing
881
Virus plaque assay on T and non-T cells
I
0 C-) AD
C-
b C3a
I L
'O -!
LL.
-r >~
2
0
5
_
4
Sedimentation velocity (mm/h) F-I F-2 F-3
F-4
F-5
F-6
Figure 3. DNA synthesis and virus plaque assay in fractions of normal spleen cells incubated without mitogens. Spleen cells from normal mice were incubated for 72 h in the absence of added mitogens. For other details, see the legend for Figs 1 and 2. Note the scale in ordinate.
DNA were not necessarily susceptible to VSV. It has been reported that FCS was slightly mitogenic for B lymphocytes (Kreeftenberg, Leerling & Loggen, 1975). V-PFC found in a supposedly unstimulated preparation of lymphocytes might have resulted from B lymphocytes activated by FCS contained in the medium. An alternative possibility that cells other than lymphocytes, e.g. monocytes and macrophages, gave rise to V-PFC is also considered (Edelman & Wheelock, 1967; Nowakowski et al., 1973). The contribution of the latter to the background V-PFC can be estimated from Table 2. When adherent cells were completely removed and incubated without mitogens, there remained more than half of the original background V-PFC, indicating that the amount of V-PFC derived from monocytes and macrophages co-existing in our splenic preparation is not so large. It can be seen from Fig. 3 that the sum of V-PFC contained in fractions of lymphocytes exceeded the V-PFC present in the original unfractionated preparation. This phenomenon, though occasionally observed, is unexplicable at present. DISCUSSION The main purpose of this study was to determine what subtypes of lymphocytes were detected by VPA.
The incubation of normal spleen cells with Con A, i.e. activation of T lymphocytes, resulted in a sharp increase in the number of V-PFC, whereas the incubation with LPS generated fewer V-PFC. It is worth pointing out that the relative augmentation of VSV permissive cells after LPS stimulation in our experiment (Table 1, Expt 1: 2860-5190 V-PFC/106 cells) was of the same order of magnitude as that reported by Kano et al. (1973) and Bloom, Nowakowski & Kano (1974b) (1 800-3800 V-PFC/106 cells). The interpretation of this finding by earlier investigators was that VPA detected primarily activated T lymphocytes, but not activated B lymphocytes (Kano et al., 1973; Nowakowski et al., 1973; Bloom, Stoner, Fischetti, Nowakowski, Muschel & Rubinstein, 1974a). The extent of increase, however, would depend upon the particular combination of a mitogen and a subpopulation of lymphocytes responding to the mitogen. The above result might simply indicate that Con A is more efficient in stimulation of T lymphocytes than LPS for B lymphocytes. It is evident that non-T lymphocytes, when appropriately stimulated, gave rise to V-PFC, since significantly V-PFC were observed in 0-positive lymphocyte (and macrophage) depleted population of nude spleen. It will be disputable to conclude that splenic lymphocytes from nude activated by LPS are defined to be B lymphocytes. Four main types of lymphocytes (Ig6O-, Ig-O+, Ig+O- and Ig+O+) were detected and
882
T. Kasahara, K. Shioiri-Nakano & A. Sugiura
classified in nude mice (Roelants, Loor von Boehmer, Sprent, Hagg, Mayor & Ryden, 1975; Roelants, et al., 1976). We could exclude completely the participation of 09-positive lymphocytes in the formation of V-PFC, but not that of Ig-negative lymphocytes. The amount of Ig-negative lymphocytes in nude spleen was estimated to be 50-55%Y (see legend for Table 2). Ig-0--lymphocytes were determined to be 25 Y. in nude spleen (Roelants et al., 1975). It is quite interesting to know which types of lymphocytes (Ig+9- or Ig-O9) were activated by LPS and became V-PFC. Blastoid lymphocytes from nude spleen stimulated with LPS were found to have distinct surface Ig by immunofluorescent antibody technique (unpublished data), suggesting that V-PFC resulted from Ig+0--lymphocytes. Non-T lymphocytes so-called in this paper may therefore be said to be B lymphocytes, although the possibility that Ig-0-- lymphocytes are stimulated and become V-PFC is not ruled out. The capability to support the replication of VSV is thought to be one of the events that follow the activation of both T and B lymphocytes. Measles virus has also been shown to be capable of replicating in both T and B lymphocytes of human blood. The virus replication was more efficient in lymphocytes stimulated with mitogens than in unstimulated lymphocytes (Joseph et al., 1975). An analogous situation is the recent finding that the migration inhibitory factor, formerly considered to be a specific product of activated T lymphocytes, was also released from B lymphocytes activated by a mitogen or an antigen (Yoshida, Sonozaki & Cohen, 1973; Sugane, Kasahara & Shioiri-Nakano, 1975). Most cell lines derived from B lymphocytes, unlike LPS-activated non-T lymphocytes described above, did not support the growth of VSV (Nowakowski et al., 1973; Bloom et al., 1974a). It is not surprising that cells directly taken from the animal differ in many respects from the cells in continuous culture in vitro for a long time. In this study, the cells smaller and less active in DNA synthesis than blastoid cells, were also detected as V-PFC. This was particularly evident in the activated non-T lymphocytes and unstimulated lymphocytes. It has been shown that lymphoid cells in continuous culture gradually increased in size during the transition from the G1 to G2 phase in the cell cycle (Shall, 1973). If this finding also holds true for lymphocytes taken from the animal, our results may indicate that the acquisition of virus susceptibility precedes the active DNA synthesis. In this
respect, the finding by Bloom et al. (1974b) with WI-L2, human lymphoid cells in continuous culture, might be relevant. They showed that synchronously growing cells gave rise to the maximum number of V-PFC when infected at the stage of late G1 to S phase. It was feasible to separate lymphoid cells and fibroblast cells in continuous culture into G1, S, and G2 stages by use of unit gravity sedimentation and to start synchronously growing cultures (Everson, Buell & Rogentine, 1973; MacDonald & Miller, 1970; Shall, 1973). The technique of collecting activated lymphocytes in well synchronized states is of great significance in order to know how many of the activated lymphocytes were enumerated as V-PFC. While 10-15% of cells were seen labelled by autoradiography in normal lymphocytes incubated with Con A for 2 h pulse labelling of [3H]Tdr, the proportion of V-PFC was not more than 2% of the infected cells. Furthermore, when Con A-activated cells were more precisely separated into more than 10 fractions, over 25 % of cells in the fraction most active in DNA synthesis were labelled by autoradiography; in contrast, the proportion of V-PFC was only 1-2% of the infected cells (unpublished data). This suggests that all the activated lymphocytes followed by DNA synthesis do not always permit virus-replication. This might be explained by the following two notions; one is that only lymphocytes in some confined stage of cell cycle before DNA synthesis permit virus-replication and the other is that only some subsets of lymphocyte subpopulations may permit virus-replication. The former might be resolved by the use of a more refined technique of separation than that used by us or another method of synchronization to separate activated lymphocytes in different cell stages. The latter is partially evidenced by the work of Kano et a. (1973) that the proliferative T lymphocytes in a certain combination of mixed lymphocyte culture is not consistent to those possessing cytotoxic effector function and V-PFC. The results of further investigations clarifying the relationship between V-PFC detected by VPA and the functional lymphocytes in cell-mediated immunity will be of interest. REFEREN CES BLOOM B.R., JIMENEz L. & MARCUS P.1. (1970) A plaque assay for enumerating antigen-sensitive cells in delayedtype hypersensitivity. J. exp. Med. 132, 16. BLOOM B.R., STONER G., FISCHETTI V., NOWAKOWSKI M.,
Virus plaque assay on T and non-T cells MUSCHEL R. & RUBINSTEIN A. (1974a) Products of activated lymphocytes and the virus plaque assay. Progress in Immunology II (ed. by L. Brent and J. Holborow), volume 3, p. 113. North-Holland Publishing Co., Amsterdam. BLOOM B.R., NOWAKOWSKI M. & KANO S. (1974b) An approach to the mechanism of viral interaction with lymphoid cells. Mechanism of virus disease (ed. by W.S. Robinson and C.F. Fox), p. 25. W.A. Benjamin Inc., New York. EDELMAN R. & WHEELOCK E.F. (1966) Vesicular stomatitis virus replication in human leukocyte cultures: enhancement by phytohemagglutinin. Science, 154, 1053. EDELMAN R. & WHEELOCK E.F. (1967) Specific role of each human leukocyte types in viral infection. 1. Monocyte as host cell for VSV replication in vitro. J. Virol. 1, 1139. EVERSON L.K., BUELL D.N. & ROGENTINE G.N. (1973) Separation of human lymphoid cells into GI, S, and G2 cell cycle populations by use of a velocity sedimentation technique. J. exp. Med. 137, 343. JERNE N.K. & NORDIN A.A. (1963) Plaque formation in agar by single antibody producing cells. Science, 140, 405. JIMENEZ L. & BLOOM B.R. (1970) Virus plaque assay for antigen sensitive cells in delayed-hypersensitivity. In vitro methods in cell-mediated immunity (ed. by B.R. Bloom and P.R. Glade), p. 553. Academic Press, New York. JOSEPH B.S., LAMPERT P.W. & OLDSTONE M.B.A. (1975) Replication and persistence of measles virus in defined subpopulations of human leukocytes. J. Virol. 16, 1638. KANO S., BLOOM B.R. & HOWE M.L. (1973) Enumeration of activated thymus-derived lymphocytes by the virus plaque assay. Proc. nat. Acad. Sci. (Wash.), 70, 2299. KASAHARA T., SHIOIRI-NAKANo K. & OSAWA T. (1975) Correlation betwcen mature and immature thymocytes. Int. Arch. Allergy, 48, 452. KREEFTENBERG J.G., LEERLING M.F. & LOGGEN H.G. (1975)
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B- and T-cell markers on human lymphoblasts after stimulation with mitogen or antigens. Clin. exp. Immunol. 22, 121. MACDONALD II.R. & MILLER R.G. (1970) Synchronization of mouse L cells by a velocity sedimentation technique. Biophys. J. 10, 834. MILLER R.G. & PHILLIPS R.A. (1969) Separation of cells by velocity sedimentation. J. Cell Physiol. 73, 191. NOWAKOWSKI M., FELDMAN J.D., KANO S. & BLOOM B.R. (1973) The production of vesicular stomatitis virus by antigen- or mitogen-stimulated lymphocytes and continuous lymphoblastoid lines. J. exp. Med. 137, 1042. ROELANTs G.E., LOOR F., VON BOEHMER H., SPRENT J., HXGG L.B., MAYOR K.S. & RYDEN A. (1975) Five types of lymphocytes (Ig-0-, Ig-O+weak, Ig-O+strong, Ig+0and Ig+t+) characterized by double immunofluorescence and electrophoretic mobility. Organ distribution in normal and nude mice. Europ. J. Immunol. 5, 127. ROELANTs G.E., KATHLEEN S.M., HAGG L.B. & LOOR F. (1976) Immature T lineage lymphocytes in athymic mice: presence of TL, life span and homeostatic regulation. Europ. J. Immunol. 6, 75. SHALL S. (1973) Selection synchronization by velocity sedimentation separation of mouse fibroblast cells grown in suspension culture. Methods in cell biology. VII (ed. by D.M. Prescott), p. 269. Academic Press, New York. SUGANE K., KASAHARA T. & SHIoIRI-NAKANo K. (1975) Release of migration inhibitory factor from mouse T and B cells activated by insoluble phytomitogens. Jap. J. exp. Med. 45, 19. WILLEMs F.T.C., MELNICK J.L. & RAWLS W.E. (1969) Replication of poliovirus in phytohemagglutinin-stimulated human lymphocytes. J. Virol. 3, 451. YOSHIDA T., SONOZAKI H. & COHEN S. (1973) The production of migration inhibitory factor by B and T cells of the guinea pig. J. exp. Med. 138, 784.
Detection of mitogen-activated T and non-T lymphocytes by virus plaque assay VIRUS PLAQUE ASSAY ON THE CELLS FRACTIONATED BY UNIT GRAVITY SEDIMENTATION
T. KASAHARA, K. SHIOIRI-NAKANO & A. SUGIURA Department of Medical Biology and Parasitology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken and Department of Microbiology, the Institute of Public Health, Minato-ku, Tokyo, Japan
Received 27 August 1976; accepted for publication
November 1976
considered 'to be general properties panying lymphocyte activation.
Summary. Virus plaque assay (VPA) was utilized for the quantitative evaluation of activated lymphocytes. We examined what types of cells, especially which of activated T and non-T lymphocytes, were detected as infective centres after infection with vesicular stomatitis virus. Marked increases in DNA synthesis and in virus-plaque forming cells (V-PFC) were observed not only during the activation of T lymphocytes with Con A, but also, though to a lesser extent, during the activation with lipopolysaccharide (LPS) of non-T lymphocyte preparations of nude spleen from which 0-positive lymphocytes and macrophages were completely depleted. The latter observation was further confirmed by the VPA on the populations enriched in LPS-activated non-T lymphocytes fractionated by the unit gravity sedimentation method. Fast sedimenting cells were found to be more active in DNA synthesis and contained more infective centres after infection than those sedimenting slowly and original unfractionated cells. Both the capacity for DNA synthesis and virus-replication
were
accom-
INTRODUCTION Lymphocytes, when transformed by appropriate stimulation, have been known to become permissive to the replication of certain viruses (Edelman & Wheelock, 1966; Willems, Melnick & Rawls, 1969; Bloom, Jimenez & Marcus, 1970; Joseph, Lampert & Oldstone, 1975). Based on this phenomenon, Bloom and co-workers (Bloom et al., 1970; Jimenez & Bloom, 1970) developed a method, comparable to Jerne's haemolytic plaque assay for antibodyforming cells (Jerne & Nordin, 1963), to enumerate sensitized T lymphocytes by virus plaque assay (VPA). It was reported that VPA detected activated T lymphocytes but not activated B lymphocytes (Kano, Bloom & Howe, 1973; Nowakowski, Feldman, Kano & Bloom, 1973). It was also found that continuous lymphoblastoid cell lines derived from T lymphocytes supported the replication of vesicular stomatitis virus (VSV), while cell lines derived from B lymphocytes were non-permissive except MOPC 104, a plasmacytoma presumed to be
* This work was supported in part by research grants from the Ministry of Education of Japan (010703, 087155). Correspondence: Dr T. Kasahara, Department of Medical Biology and Parasitology, Jichi Medical School, Minamikawachi-machi Tochigi-ken, 329-04, Japan.
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11
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T. Kasahara, K. Shioiri-Nakano & A. Sugiura
of B-cell origin (Nowakowski et al., 1973). We found, however, that lymphocytes from mice with thymic deficiency became permissive to the replication of VSV, when appropriately activated. This was evident particularly when activated lymphocytes were separated according to their size by unit gravity sedimentation. The results indicate that non-T splenic cells, which are considered mainly to be B lymphocytes, as well as T lymphocytes are detected by VPA.
by Dr M. Nakano, Jichi Medical School) were used at the concentration of 3 pg/ml. Cells incubated in the presence of Con A were washed twice with 0.1 M a-methyl-D-mannoside to remove Con A. Depletion of adherent cells from splenic cell suspensions The method is described elsewhere (Kasahara et al., 1975). Briefly, nonadherent cells were obtained after spleen cells suspended in MEM containing 2% FCS were adhered to plastic dishes (Lux, 5220) at 370 twice for 30 min.
MATERIALS AND METHODS
Animals Male and female BALB/c, C3H/He and AKR mice, maintained in our laboratory, were used at 6-12 weeks of age. Congenitally athymic nude (nu/nu) mice derived from BALB/c were purchased from the Center of Laboratory Experimental Animals, Tokyo, Japan, and used at 4-6 weeks of age. Neonatally thymectomized (NTX) mice were also used and the mice with visible thymus remnants were not employed.
Cell cultures L cells were propagated in Eagle's MEM supplemented with 5 % heat-inactivated calf serum. Monolayer cultures for VPA were prepared in 60-mm LUX petri dishes (no. 5220, LUX Scientific Corp. Thousand Oaks, California) in humidified atmosphere containing 5% CO2 (CO2-incubator).
Lymphocytes Spleens were removed aseptically and teased. Viable lymphocytes were separated from granulocytes, erythrocytes, dead cells and cell debris by velocity sedimentation through a Ficoll-Urografin gradient (Kasahara, Shioiri-Nakano & Osawa, 1975). Cells were washed, resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS, Flow Laboratories, Rockville, Maryland) at the concentration of 2.0 x 106 viable cells/ml. In small scale culture, 1.0 ml of cell suspension was incubated in a sterile glass tube with or without mitogens in a C02-incubator at 370 for 2-3 days. In large scale culture, 15-20 ml of cell suspension was incubated in a glass bottle with a flat bottom. The mitogens concanavalin A (Con A, Pharmacia, Uppsala, Sweden) and lipopolysaccharide (LPS, from Salmonella typhimurium LT2, kindly provided
Unit gravity sedimentation through Ficoll gradient The methods described by Miller & Phillips (1969) and Everson, Buell & Rogentine (1973) were modified. By these methods, cells were separated mainly on the basis of their cell size, according to the equation of s = r2/4, in which s is sedimentation velocity (mm/h) and r is the radius of a cell (pm). Ficoll was dissolved in MEM and sterilized by autoclaving. Starting from the 20% stock solution, various concentrations of Ficoll solution were prepared by dilution in MEM. Discontinuous gradient was prepared in a 50 x 80-mm glass bottle by successively layering 15 ml of 12, 10, 8, 6 and 4% Ficoll. Lymphocytes that had been incubated with or without mitogens in large scale culture, usually 5060 ml of cell suspension, were washed, resuspended in 2 ml of 50% FCS, layered carefully on the top of a gradient and allowed to sediment at 40 for 5 h at the unit gravity. Fractions were collected from above through a glass capillary tubing with lower end upwardly curved in J shape. The tip of the glass tubing was placed about 1 mm below the meniscus and the fluid above was gently aspirated by a peristaltic micropump connected to the other end of the glass tubing. Usually six to ten fractions were collected. 60-90% of layered lymphocytes were recovered after sedimentation. Virus plaque assay The method developed by Jimenez & Bloom (1970) was followed. Stocks of VSV, Indiana strain, were prepared in L cells and stored at - 700. Lymphocytes, either unfractionated or fractionated as described above, were washed twice with MEM and resuspended in MEM supplemented with 2% FCS at the concentration of 2-0 x 106 viable cells/ml. Lymphocytes suspended in 0-2 ml were infected VSV at a dose with of 20 PFU/cell. Virus adsorption
877
Virus plaque assay on T and non-T cells carried out for 1.5-2 h in a C02-incubator at 370. Excess virus was removed by two washings with MEM, followed by neutralization with 0.15 ml of adequately diluted rabbit anti-VSV serum (final dilution (1:50) for 1 h at 4°. At this dilution, antiserum was not toxic to lymphocytes. Infected cells were washed once and diluted. From 5 x 104 to 5 x 103 viable cells suspended in 0.15 ml were plated onto a confluent monolayer culture of L cells. Immediately after the plating of lymphocytes, 0.5 ml of agar overlay medium (MEM with 0 7% Ionagar no. 2, 1 % calf serum, 10 ,4g/ml DEAE dextran, and 2 4ug/ml fungisone) was added and plated lymphocytes were thoroughly mixed with the overlay medium by pipetting. After this layer had solidified, a second 4 ml of agar overlay medium was added. Three monolayer cultures of L cells were used per dilution of lymphocytes. After incubation for 24 to 36 h in a CO2 incubator at 370, the agar overlay was removed, monolayers were stained with 0.1 % crystal violet in 20% ethanol and plaques were counted. was
Assay of DNA synthesis Lymphocytes incubated for 2-3 days were collected and 4 x 105 viable lymphocytes in a volume of 0.2 ml were reincubated for 2 h in the presence of 0.5 pCi 3H-labelled thymidine ([3H]Tdr, specific activity 5 Ci/mmol, Radiochemical Centre, Amersham). The radioactivity incorporated into trichloroacetic acidinsoluble materials was determined as described previously (Kasahara et al., 1975).
RESULTS Correlation between DNA synthesis and VPA in lymphocytes activated by mitogens The applicability of VPA for the quantitative study of lymphocyte activation was confirmed by simultaneous determination of DNA synthesis and virus plaque-forming cells (V-PFC) in various lymphocyte preparations (Table 1). When spleen cells from normal mice were incubated in the presence of Con
Table 1. DNA synthesis and virus plaque assay in lymphocyte preparations incubated with
mitogens*
Expt no. 1. 2.
Source of lymphocytes
Normal spleen Normal spleen treated with
anti-O+ complement 3.
NTX spleen§
4.
nu/nu spleen¶
5.
nu/nu spleen treated with anti-O+ complement
[3H]Tdr uptake
VPA
c.p.m./106 cells
V-PFC/106 cells
3363+ 157 35,952+2,724 9921+ 1,346 3349+ 289 6453+ 890 16,557+ 1,057 1396+60
2866+ 363 19,310+2,204 5190+ 1,125 4700+420 6050+450 9160+ 1,150 2180+380 3760+ 520 6720+ 1,780 1725+ 280 2280+ 350 6470+ 1,650 2600+390 3155+ 150 8550+ 1,850
Mitogenst None Con A LPS None Con A LPS None Con A LPS None Con A LPS None Con A LPS
3350+ 115 8732+650 815+ 75 1138+ 31 8906+ 242 1423+ 100 2032+ 116 7197+ 38
* Lymphocytes prepared as described in the Materials and Methods section were incubated in a glass tube (small scale culture) with or without mitogens. t The optimal dose of mitogens was added, which was 3 ,pg/ml for both Con A and LPS. I Spleen cells were treated with anti-0 serum (rabbit anti-serum to brain-associated 0 antigen of C3H mice, final dilution 1:20) and agar-absorbed normal guinea-pig complement. Viable lymphocytes were purified by Ficoll-Urografin and incubated. § 6-13Y4 of 0-positive lymphocytes existed at the initiation of incubation. T 2-9%. of 6-positive lymphocytes existed and Ig-positive lymphocytes were determined to be 45-50%. by FITC-conjugated rabbit anti-mouse IgG (MBL, Fujizoki, Tokyo, Japan).
T. Kasahara, K. Shioiri-Nakano & A. Sugiura
878
Table 2. Effects of depletion of adherent cells from splenic preparations
Expt no. 1.
Source of lymphocytes
BALB/c spleen, untreated BALB/c spleen, non-adherentt BALB/c spleen, non-adherent
2.
nu/nu spleen, untreated nu/nu spleen, non-adherentt
*3
[3H]Tdr uptake
VPA
c.p.m./106 cells
V-PFC/106 cells
880+ 34 1726+ 24 1326+ 12 2642+ 160 380+ 26 1998+ 184 16,967+ 806 415+30 447+ 21 4870+425 578+ 15 967+ 142 2331+218
960+ 169 1780+ 244 880+ 63 2387+ 390 813+ 162 2533+ 192 9660+ 260 914+ 106 951+ 201 1470+ 197 460+ 105 478+ 102 2011+ 137
Mitogen* None LPS None LPS
F-1t F-3 F-5
None Con A LPS None Con A LPS
4ug/ml for both LPS and Con A was used.
t See the Materials and Methods section. Cell recovery was 15-30%4 after plastic-adherent cells were extensively removed. t Non-adherent cells were incubated with LPS for 3 days and fractionated by Ficoll unit gravity sedimentation into five fractions. For other details, see legends for Figs 1 and 2.
A, both DNA synthesis and the number of V-PFC markedly increased. The increment after incubation with LPS was also appreciable but less pronounced. This could be either due to a low mitogenic activity of LPS compared with Con A, or to inherently low reactivity of B lymphocytes. Stimulation of normal spleen cells treated with anti-@ serum and complement, cells from NTX and nude mice, with Con A was marginal. Their reactivity to LPS, however, did not differ from that of normal lymphocytes. Since nude mouse spleen was considered to contain T-cell precursors in a negligible percentage (Roelants, Kathleen, Hagg & Loor, 1976), the cells from nude mice were treated with anti-l serum and complement. The response to LPS in DNA synthesis and the number of V-PFC was not decreased (Table 1, Expts 4 and 5). At least two different types of leucocytes can be mentioned in non-T splenic preparations which formed V-PFC, that is, macrophages and non-T lymphocytes. The former is known to be permissive for VSV (Edelman & Wheelock, 1967; Nowakowski et al., 1973). We examined therefore, the effect of depletion of adherent cells from nude splenic leucocytes. Cells adherent to plastic surface were removed extensively. As shown in Table 2, a nonadherent population free from monocytes and
macrophages remains to make V-PFC when stimulated with LPS. V-PFC derived, at least, from macrophages existing in non-T splenic leucocytes could be excluded (Table 2, Expt 2). It was concluded that lymphocyte preparations actively synthesizing DNA after incubation with mitogens contained a correspondingly greater number of V-PFC when infected with VSV, and that, when appropriately activated, non-T lymphocytes as well as T lymphocytes became permissive to the replication of VSV. The latter fact was further substantiated by the experiments described below. DNA synthesis and VPA in lymphocytes fractionated by unit gravity sedimentation In our experiments the number of V-PFC did not reach more than 2-3%. of the infected lymphocytes with VSV, while a considerable number of small lymphocytes from mouse spleen was observed to be transformed to large blastoid cells activated by Con A. If the activated lymphocytes allow the replication of VSV, more V-PFC must be detected in the preparations containing relatively more of activated lymphocytes. After 2 days' incubation with Con A, cells from normal mouse spleen were fractionated by unit gravity sedimentation (Fig. 1). In these large
879
Virus plaque assay on T and non-T cells
I 0
c'0 0()
0
6. E L
0D
H-
CL
I0
0. I3
>
BL CON A +) unfractionated
0
2
3
i- -F IF-F-i F-2 F-3 F-4 F
5
4
Sedimentation velocity -
F-5
6
(mm / h) -
F-F
F-I F-7
Figure 1. DNA synthesis and virus plaque assay in fractions of Con A-activated spleen cells. Spleen cells from normal mice were incubated with Con A in large scale culture for 46 h and fractionated by unit gravity sedimentation. Fraction number was named from upper of the gradient. The inset shows the distribution of viable lymphocytes among fractions. For other details, see the Materials and Methods section.
I
6.0
c
E
4.5 0
ci
C
-
30 0 "L--
n
-F
1.5 ..I-
I FL CON A LPS
crnrcct1oncated
4 3 5 2 Sed mentotion velocity (mm/h) - -i - i m I-i F-. F-2 F-3 F-4 F-5 8-6 F-7
Figure 2. DNA synthesis and virus plaque assay in fractions of LPS-activated non-T lymphocytes. Spleen cells from NTX mice were incubated with LPS in large scale culture for 42 h and fractionated by unit gravity sedimentation. The left ordinate is [3H]thymidine incorporated into 106 viable lymphocytes.
T. Kasahara, K. Shioiri-Nakano & A. Sugiura
880
Table 3. DNA synthesis and virus plaque assay in fractions of LPS-activated non-T lymphocytes from nu/nu mice*
Fraction number
Sedimentation velocity
Distribution of viable cells
[3H]Tdr uptake
VPA
(mm/hr)
(0)
c.p.m./106 cells
V-PFC/106 cells
Unfractionated None LPS (3,ug/ml) F-I F-2 F-3 F-4 F-5 F-6
0-80 1-3 2-1 2-9 3-8 52
230 3230
725 1680
Fractionated (LPS 3 ug/ml) 150 12-3 19-7 310 27-0 2180 16-8 6672 15-0 9980 90 9172
450 1800 5085 5220 3645 1485
* Spleen cells from athymic nu/nu mice were incubated with LPS in large scale culture for 46 h and fractionated by unit gravity sedimentation as described in the Materials and Methods section.
scale cultures, the activity of DNA synthesis and the number of V-PFC of the unfractionated cells were far less than those in small scale cultures. In microscopic examination, 80-90% of large blastoid cells were recovered in the bottom three fractions. The incorporation of [3H]Tdr was also marked in these fractions. The number of V-PFC per 106 viable cells inoculated was highest in F-6, the fraction most active in DNA synthesis, which was four to five times as high as that in the unfractionated preparation. There were, however, also V-PFC in the fractions sedimenting more slowly. The number of V-PFC relative to [3H]Tdr incorporation was always greater among cells sedimenting slowly (F-2-F-4) than among faster sedimenting cells. The disproportionality between the two parameters suggested that V-PFC comprised, in addition to large blastoid cells actively synthesizing DNA, cells smaller and relatively inactive in DNA synthesis. Non-T lymphocytes incubated with LPS for 2 days were fractionated in the same way (Fig. 2, Table 3). Again, faster sedimenting cells were more active in [3H]Tdr incorporation and contained a higher proportion of V-PFC. This was additional evidence that activated non-T lymphocytes could become V-PFC upon infection with VSV. The number of V-PFC relative to the rate of DNA synthesis was greater in the upper half of the gradient (Fig. 2, F-1-F-4). While the proportion of V-PFC was highest in the fraction of activated T lympho-
cytes in which DNA synthesis was also most active (Fig. 1, F-6), the V-PFC peak usually preceded the peak of DNA synthesis by one to two fractions in activated non-T lymphocytes (Fig. 2; Table 3). Activated T lymphocytes were fractionated more precisely into more than ten fractions (data not shown), but the peak of DNA synthesis and that of V-PFC were not completely separated from each other. It might be that non-T lymphocytes which supported the replication of VSV were generally smaller in size than the corresponding T lymphocytes.
V-PFC detected in unstimulated lymphocyte preparations Lymphocytes incubated in the absence of mitogens always contained a small number of V-PFC upon infection with VSV. The nature of these background V-PFC was examined by fractionation. Spleen cells from normal mice were incubated for 72 h in the absence of added mitogens and were separated by unit gravity sedimentation (Fig. 3). Being unstimulated, even the fraction with the highest rate of DNA synthesis (F-4) incorporated only twice as much [3H]Tdr as the whole population. On the other hand, V-PFC were found in ten-fold higher proportion in F-2 than in unfractionated preparation. The separation of V-PFC peak from the peak of DNA synthesis indicated that lymphocytes actively synthesizing
881
Virus plaque assay on T and non-T cells
I
0 C-) AD
C-
b C3a
I L
'O -!
LL.
-r >~
2
0
5
_
4
Sedimentation velocity (mm/h) F-I F-2 F-3
F-4
F-5
F-6
Figure 3. DNA synthesis and virus plaque assay in fractions of normal spleen cells incubated without mitogens. Spleen cells from normal mice were incubated for 72 h in the absence of added mitogens. For other details, see the legend for Figs 1 and 2. Note the scale in ordinate.
DNA were not necessarily susceptible to VSV. It has been reported that FCS was slightly mitogenic for B lymphocytes (Kreeftenberg, Leerling & Loggen, 1975). V-PFC found in a supposedly unstimulated preparation of lymphocytes might have resulted from B lymphocytes activated by FCS contained in the medium. An alternative possibility that cells other than lymphocytes, e.g. monocytes and macrophages, gave rise to V-PFC is also considered (Edelman & Wheelock, 1967; Nowakowski et al., 1973). The contribution of the latter to the background V-PFC can be estimated from Table 2. When adherent cells were completely removed and incubated without mitogens, there remained more than half of the original background V-PFC, indicating that the amount of V-PFC derived from monocytes and macrophages co-existing in our splenic preparation is not so large. It can be seen from Fig. 3 that the sum of V-PFC contained in fractions of lymphocytes exceeded the V-PFC present in the original unfractionated preparation. This phenomenon, though occasionally observed, is unexplicable at present. DISCUSSION The main purpose of this study was to determine what subtypes of lymphocytes were detected by VPA.
The incubation of normal spleen cells with Con A, i.e. activation of T lymphocytes, resulted in a sharp increase in the number of V-PFC, whereas the incubation with LPS generated fewer V-PFC. It is worth pointing out that the relative augmentation of VSV permissive cells after LPS stimulation in our experiment (Table 1, Expt 1: 2860-5190 V-PFC/106 cells) was of the same order of magnitude as that reported by Kano et al. (1973) and Bloom, Nowakowski & Kano (1974b) (1 800-3800 V-PFC/106 cells). The interpretation of this finding by earlier investigators was that VPA detected primarily activated T lymphocytes, but not activated B lymphocytes (Kano et al., 1973; Nowakowski et al., 1973; Bloom, Stoner, Fischetti, Nowakowski, Muschel & Rubinstein, 1974a). The extent of increase, however, would depend upon the particular combination of a mitogen and a subpopulation of lymphocytes responding to the mitogen. The above result might simply indicate that Con A is more efficient in stimulation of T lymphocytes than LPS for B lymphocytes. It is evident that non-T lymphocytes, when appropriately stimulated, gave rise to V-PFC, since significantly V-PFC were observed in 0-positive lymphocyte (and macrophage) depleted population of nude spleen. It will be disputable to conclude that splenic lymphocytes from nude activated by LPS are defined to be B lymphocytes. Four main types of lymphocytes (Ig6O-, Ig-O+, Ig+O- and Ig+O+) were detected and
882
T. Kasahara, K. Shioiri-Nakano & A. Sugiura
classified in nude mice (Roelants, Loor von Boehmer, Sprent, Hagg, Mayor & Ryden, 1975; Roelants, et al., 1976). We could exclude completely the participation of 09-positive lymphocytes in the formation of V-PFC, but not that of Ig-negative lymphocytes. The amount of Ig-negative lymphocytes in nude spleen was estimated to be 50-55%Y (see legend for Table 2). Ig-0--lymphocytes were determined to be 25 Y. in nude spleen (Roelants et al., 1975). It is quite interesting to know which types of lymphocytes (Ig+9- or Ig-O9) were activated by LPS and became V-PFC. Blastoid lymphocytes from nude spleen stimulated with LPS were found to have distinct surface Ig by immunofluorescent antibody technique (unpublished data), suggesting that V-PFC resulted from Ig+0--lymphocytes. Non-T lymphocytes so-called in this paper may therefore be said to be B lymphocytes, although the possibility that Ig-0-- lymphocytes are stimulated and become V-PFC is not ruled out. The capability to support the replication of VSV is thought to be one of the events that follow the activation of both T and B lymphocytes. Measles virus has also been shown to be capable of replicating in both T and B lymphocytes of human blood. The virus replication was more efficient in lymphocytes stimulated with mitogens than in unstimulated lymphocytes (Joseph et al., 1975). An analogous situation is the recent finding that the migration inhibitory factor, formerly considered to be a specific product of activated T lymphocytes, was also released from B lymphocytes activated by a mitogen or an antigen (Yoshida, Sonozaki & Cohen, 1973; Sugane, Kasahara & Shioiri-Nakano, 1975). Most cell lines derived from B lymphocytes, unlike LPS-activated non-T lymphocytes described above, did not support the growth of VSV (Nowakowski et al., 1973; Bloom et al., 1974a). It is not surprising that cells directly taken from the animal differ in many respects from the cells in continuous culture in vitro for a long time. In this study, the cells smaller and less active in DNA synthesis than blastoid cells, were also detected as V-PFC. This was particularly evident in the activated non-T lymphocytes and unstimulated lymphocytes. It has been shown that lymphoid cells in continuous culture gradually increased in size during the transition from the G1 to G2 phase in the cell cycle (Shall, 1973). If this finding also holds true for lymphocytes taken from the animal, our results may indicate that the acquisition of virus susceptibility precedes the active DNA synthesis. In this
respect, the finding by Bloom et al. (1974b) with WI-L2, human lymphoid cells in continuous culture, might be relevant. They showed that synchronously growing cells gave rise to the maximum number of V-PFC when infected at the stage of late G1 to S phase. It was feasible to separate lymphoid cells and fibroblast cells in continuous culture into G1, S, and G2 stages by use of unit gravity sedimentation and to start synchronously growing cultures (Everson, Buell & Rogentine, 1973; MacDonald & Miller, 1970; Shall, 1973). The technique of collecting activated lymphocytes in well synchronized states is of great significance in order to know how many of the activated lymphocytes were enumerated as V-PFC. While 10-15% of cells were seen labelled by autoradiography in normal lymphocytes incubated with Con A for 2 h pulse labelling of [3H]Tdr, the proportion of V-PFC was not more than 2% of the infected cells. Furthermore, when Con A-activated cells were more precisely separated into more than 10 fractions, over 25 % of cells in the fraction most active in DNA synthesis were labelled by autoradiography; in contrast, the proportion of V-PFC was only 1-2% of the infected cells (unpublished data). This suggests that all the activated lymphocytes followed by DNA synthesis do not always permit virus-replication. This might be explained by the following two notions; one is that only lymphocytes in some confined stage of cell cycle before DNA synthesis permit virus-replication and the other is that only some subsets of lymphocyte subpopulations may permit virus-replication. The former might be resolved by the use of a more refined technique of separation than that used by us or another method of synchronization to separate activated lymphocytes in different cell stages. The latter is partially evidenced by the work of Kano et a. (1973) that the proliferative T lymphocytes in a certain combination of mixed lymphocyte culture is not consistent to those possessing cytotoxic effector function and V-PFC. The results of further investigations clarifying the relationship between V-PFC detected by VPA and the functional lymphocytes in cell-mediated immunity will be of interest. REFEREN CES BLOOM B.R., JIMENEz L. & MARCUS P.1. (1970) A plaque assay for enumerating antigen-sensitive cells in delayedtype hypersensitivity. J. exp. Med. 132, 16. BLOOM B.R., STONER G., FISCHETTI V., NOWAKOWSKI M.,
Virus plaque assay on T and non-T cells MUSCHEL R. & RUBINSTEIN A. (1974a) Products of activated lymphocytes and the virus plaque assay. Progress in Immunology II (ed. by L. Brent and J. Holborow), volume 3, p. 113. North-Holland Publishing Co., Amsterdam. BLOOM B.R., NOWAKOWSKI M. & KANO S. (1974b) An approach to the mechanism of viral interaction with lymphoid cells. Mechanism of virus disease (ed. by W.S. Robinson and C.F. Fox), p. 25. W.A. Benjamin Inc., New York. EDELMAN R. & WHEELOCK E.F. (1966) Vesicular stomatitis virus replication in human leukocyte cultures: enhancement by phytohemagglutinin. Science, 154, 1053. EDELMAN R. & WHEELOCK E.F. (1967) Specific role of each human leukocyte types in viral infection. 1. Monocyte as host cell for VSV replication in vitro. J. Virol. 1, 1139. EVERSON L.K., BUELL D.N. & ROGENTINE G.N. (1973) Separation of human lymphoid cells into GI, S, and G2 cell cycle populations by use of a velocity sedimentation technique. J. exp. Med. 137, 343. JERNE N.K. & NORDIN A.A. (1963) Plaque formation in agar by single antibody producing cells. Science, 140, 405. JIMENEZ L. & BLOOM B.R. (1970) Virus plaque assay for antigen sensitive cells in delayed-hypersensitivity. In vitro methods in cell-mediated immunity (ed. by B.R. Bloom and P.R. Glade), p. 553. Academic Press, New York. JOSEPH B.S., LAMPERT P.W. & OLDSTONE M.B.A. (1975) Replication and persistence of measles virus in defined subpopulations of human leukocytes. J. Virol. 16, 1638. KANO S., BLOOM B.R. & HOWE M.L. (1973) Enumeration of activated thymus-derived lymphocytes by the virus plaque assay. Proc. nat. Acad. Sci. (Wash.), 70, 2299. KASAHARA T., SHIOIRI-NAKANo K. & OSAWA T. (1975) Correlation betwcen mature and immature thymocytes. Int. Arch. Allergy, 48, 452. KREEFTENBERG J.G., LEERLING M.F. & LOGGEN H.G. (1975)
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B- and T-cell markers on human lymphoblasts after stimulation with mitogen or antigens. Clin. exp. Immunol. 22, 121. MACDONALD II.R. & MILLER R.G. (1970) Synchronization of mouse L cells by a velocity sedimentation technique. Biophys. J. 10, 834. MILLER R.G. & PHILLIPS R.A. (1969) Separation of cells by velocity sedimentation. J. Cell Physiol. 73, 191. NOWAKOWSKI M., FELDMAN J.D., KANO S. & BLOOM B.R. (1973) The production of vesicular stomatitis virus by antigen- or mitogen-stimulated lymphocytes and continuous lymphoblastoid lines. J. exp. Med. 137, 1042. ROELANTs G.E., LOOR F., VON BOEHMER H., SPRENT J., HXGG L.B., MAYOR K.S. & RYDEN A. (1975) Five types of lymphocytes (Ig-0-, Ig-O+weak, Ig-O+strong, Ig+0and Ig+t+) characterized by double immunofluorescence and electrophoretic mobility. Organ distribution in normal and nude mice. Europ. J. Immunol. 5, 127. ROELANTs G.E., KATHLEEN S.M., HAGG L.B. & LOOR F. (1976) Immature T lineage lymphocytes in athymic mice: presence of TL, life span and homeostatic regulation. Europ. J. Immunol. 6, 75. SHALL S. (1973) Selection synchronization by velocity sedimentation separation of mouse fibroblast cells grown in suspension culture. Methods in cell biology. VII (ed. by D.M. Prescott), p. 269. Academic Press, New York. SUGANE K., KASAHARA T. & SHIoIRI-NAKANo K. (1975) Release of migration inhibitory factor from mouse T and B cells activated by insoluble phytomitogens. Jap. J. exp. Med. 45, 19. WILLEMs F.T.C., MELNICK J.L. & RAWLS W.E. (1969) Replication of poliovirus in phytohemagglutinin-stimulated human lymphocytes. J. Virol. 3, 451. YOSHIDA T., SONOZAKI H. & COHEN S. (1973) The production of migration inhibitory factor by B and T cells of the guinea pig. J. exp. Med. 138, 784.
