Immunology 1979 36 619


BENEDITA ROCHA*, A. FREITAS* & MARIA DE SOUSAt Department ofBacteriology and Immunology, Western Infirmary, Glasgow

Received 30 January 1978; acceptedforpublication 17 October 1978

Summary. Spleen cells from BN rats were separated in a discontinuous density gradient. Cells from different fractions were shown to be functionally diverse, light cells enhancing DNA synthesis and cell division of the dense cell fractions, while dense cells suppressed DNA synthesis and cell division of light cells. Both these effects were also present in the absence of cell stimulation, were proportional to the number of modulating cells added to the cultures, and totally independent of the magnitude of response of controls. In Con Astimulated cultures, mitogen dose also influenced the intensity of help and suppression. Both these effects are blocked by cell treatment with cycloheximide and can be mediated by cell supernatants. A T cell seems to be responsible for both helper and suppressor effects.

antigens and can also exert suppressive effects to the same antigens. Non-specific stimulation of T lymphocytes with polyclonal activators (mitogens) can also generate helper or suppressive activities (Dutton, 1975; Rich & Pierce, 1973). It has been attempted to correlate these different effector functions to independent subsets of T cells. For such purpose, lymphocytes have been studied according to different cell properties, e.g. irradiation, sensitivity, sedimentation rates, density and cell surface markers (Dutton, 1975; Tse & Dutton, 1976; Whisler & Stobo 1976; Cantor & Boyse, 1976). Although most of these studies have been interpreted to indicate the existence of at least two subsets of T cells (helper and suppressive) the factors that condition the stimulation of one or another cell population and the functional interaction between different cell types have not yet been clarified. (Eardley & Gershon, 1975; Cantor, McVay-Bondreau, Hugenberger, Naidorf, Shen & Gershon, 1978; Eardley, Hugenberger, McVay-Boundreau, Shen, Gershon & Cantor, 1978). The data presented here suggest that amplification and suppression of the in vitro response to Con A reflects the interplay of different T-cell populations, separable by their buoyant density.


Thymus dependent lymphocytes can function as helper cells in the induction of immune responses to * Present address: Faculdade de Ciencias Medicas de Lisboa, Departmento de Imunologia, Campo Santana, 130, Lisboa, Portugal. t Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York NY 10021, U.S.A. Correspondence: Dr B. Rocha, Department of Immunology, Faculdade de Ciencias Medicas de Lisboa, Campo Santana 130, Lisboa, Portugal. 0019-2805/79/0400-0619$02.00 © 1979 Blackwell Scientific Publications


Normal, inbred BN rats, bred in the Department of Bacteriology and Immunology or purchased from 619


Benedita Rocha, A. Freitas & Maria de Sousa

Microbiological Associates, Bethesda, MD, between 5 and 12 weeks old, were used. For each experiment, animals of the same age and sex were selected. Cell suspensions and cell separations on a discontinuous BSA gradient Spleen cells were harvested by wrapping the spleen in a sterile cotton gauze and teasing it with two forceps in cold Eagle's medium. The resulting cell suspensions were freed of tissue clumps by decantation, washed twice and resuspended in 17% BSA (Albumin bovine fraction V from Sigma Chemical Co., St. Louis). The cells were then layered on top of a 2% step discontinuous BSA gradient, ranging from 17 to 31O% and separated by differential flotation according to the method of Dicke, Hooft & Van Bekkum (1968). The different BSA concentrations were made from a stock solution of 35% BSA in Tris-buffer with a final osmolarity of 330 mOsms and a pH of 5-2. The successive dilutions were made in PBS, 300 mOsms, pH 7-2 and the BSA concentration of the different gradient layers was checked with the help of a refractometer. Several stocks of BSA powder were used. In order to obtain reproducibility of results from batch to batch, the pH, osmolarity and refractive index of the stock solution and the different fractions were kept constant. The gradient was centrifuged at 1000g for 40 min at 40 in a PR-2 international centrifuge, using a horizontal head. The cells were collected from the interphases with a Pasteur pipette and washed three times in large volumes of ice-cold Eagle's medium. After the final wash, cells were counted and their viability assessed by exclusion of 0-2% Trypan blue. The cells from the 17/23, 19/21 and 21/23 interphases were pooled and are referred to hereafter as the 1 7/23Traction. Preparation of enriched T-cell suspensions Cells from the different fractions were depleted of Ig-bearing cells by passage through nylon wool columns (Leuko-Pack Leukocyte Filters, Fenwal Laboratory, Morton Grove, IL) equilibrated with 10% foetal calf serum (FCS) in Eagle's medium at 370 as described by Julius et al. (Julius, Simpson & Herzenberg, 1973). The frequency of Ig-bearing cells in the effluent populations, was found always to be less than 4%.

Mitomycin-C treatment Cells were incubated in Eagle's medium for 30 min at 370 with 80 jg/ml per 107 cells of Mitomycin-C (Mito-

mycin-C, Sigma Chemical Co., St. Louis, MO) and subsequently washed three times in fresh medium. At this concentration of M-C, [14C]-thymidine incorporation of 72 h cultures of cells stimulated with an optimal Con A concentration was eliminated, but the ability of the cells to stimulate the mixed lymphocyte reaction was unimpaired.

Cycloheximide treatment Cells were incubated in Eagle's medium for 30 min at 370 with 400 ig/ml per 107 cells of CHX (cycloheximide, Sigma Chemical Co., St. Louis, MO), followed by three washes in fresh medium. This treatment was found to reduce protein synthesis of 72 h spleen-cell cultures stimulated with optimal doses of Con A (measured by the incorporation of [3H]-leucine) to about 2% of the synthetic rate of the untreated cells.

Cell cultures Cell fractions were cultured in triplicate in RPMI 1640 medium (Flow Lab), supplemented with 10% foetal calf serum, 2% L-glutamine (200 mm,, Flow Lab.), 2% penicillin-streptomycin (100 iu/ml each, Flow Lab.), and HEPES buffer (0 02 M). The cells at a concentration of 2 x 106 cells/ml were cultured in glass tubes in I ml volume per culture. Cultures were incubated at 370 in a 10% CO2 atmosphere for 72 h. Except for controls, different concentrations of Con A in 50 p1 of saline were added before the incubation was started. For the determination of DNA synthesis, a 24 h pulse of ['4C]-thymidine (specific activity 57 mCi/mmol) diluted to 0 1 Ci/culture was given. DNA was precipitated with 10% trichloroacetic acid, washed with methanol, and incorporation of the isotope was evaluated in a Tri-carb Spectrometer (Packard). Results are expressed as counts per minute per 106 cells planted in culture (c.p.m./106 cells). Cell supernatants Cell fractions were incubated in RPMI + 10% FCS for 3 h at 370 at the concentration of 5 x 107/ml per culture. Cells were spun down and supernatants kept at -20° until used. Statistical analysis For each experiment, the significance of the differences observed was estimated by Student's t test. RESULTS Response to Con A of cells from various density fractions Cells with different densities responded differently to Con A (Fig. 1). Cells lighter than 23% BSA (light cells)

Separation of helper and suppressor cells were more responsive to Con A than any other fraction or the infractionated spleen cells. Medium density cells (23/25 and 25/27 cell fractions) responded similarly to unfractionated spleen cells. Cells denser than 27% BSA (dense cells) responded poorly to Con A. The response of light cells also differed from that of other fractions in that maximum response was obtained with Con A concentrations ranging from I to 15 ug/ml of Con A (Fig. 1). The differences in the mitogen response of the collected fractions were not due to different concentrations of Con A receptors, since in the range of mitogen concentrations we studied (5-100 ,ug/ml of Con A), various fractions were found to have identical ability to bind ['25I]-Con A (not shown). The low response of dense fractions was not due to contamination with red blood cells or to an inhibitory effect of high BSA concentrations since culturing of 17/23 fraction (that had no red blood cell contamination) with as much as 600% red blood cells or previous incubation with 31O% BSA for 40 min at 40, did not alter the response (not shown). Also, it cannot be attributed to a marked enrichment of B cells in these fractions since BN rat spleen B lymphocytes were


found to distribute homogeneously throughout the gradient (Rocha, 1978). Cells from light fractions exert an enhancing effect To investigate if light cells could influence the response to Con A of cells from the denser fractions mixed cultures of cells from these fractions were studied. Different numbers of light cells (ranging from 5 x I05 to 1 5 x 106) treated with mitomycin-C were added to different numbers of dense reacting cells, in order that the total number of cells per culture was kept constant (2 x 106/ml per culture). The DNA synthesis of mixed cultures was compared with that of control cultures of cells from the dense fractions with the same number of reacting cells, mitomycin C-treated cells and total number of cells per culture. At concentrations equal or higher than 1 x 106, light cells enhanced the incorporation of ['4C]-thymidine


E 0


0 0 0'







0 0.


1 2-5 5 Con A dose (gg/mL)


Figure 1. Response to Con A of rat spleen cell density fractions. Results are expressed as c.p.m./106 planted cells, each point representing the mean of quadriplicate cultures. *, 17/23; &, 23/25; A, unseparated cells; o, 25/27; o, 27/29; a, 29/31.

pg/ml 5 pghnl 10 pg/mL Figure 2. Enhancing effect of various numbers of light cells (treated with mitomycin-C) on the DNA synthesis of dense cells, in the presence and absence of Con A, expressed according to the formula: M x 100 Percentage enhancement = -100,

where M, [I4C]-thymidine incorporation of mixed cultures of dense and light cells; D, ['4C]-thymidine incorporation of dense cell controls. Hatched columns, 1 x 106 light cells/culture; open columns, 1 5 x 106 light cells/culture.

Benedita Rocha, A. Freitas & Maria de Sousa


by the dense cell fraction (Fig. 2) in the presence or absence of Con A. The enhancing effect was proportional to the number of light cells added to the cultures and totally independent of the intensity of the response of control cultures. In Con A-stimulated cultures, mitogen concentration also influenced the intensity of the enhancing effect: optimum helper effect was obtained in the presence of suboptimal Con A concentrations (I pg/ml) while high Con A doses induced a less intense enhancing effect. With the lowest dose of cells tested (5 x 105 light cells), no significant effects were observed either in the presence or absence of Con A.

100 I




80 _








T 60

a 6n

I l

0 c 0


40 _





5 Con A (g±g/mt)

Figure 3. Suppressive effect of various numbers of mitomycin-C treated dense cells on the DNA synthesis of cells from the light fractions, in the presence and absence of Con A. Each point represents the mean+ SD of five to six experiMx 100 ments. Percentage suppression =100where M, ['4C]-thymidine incorporation of mixed cultures of light and dense cells; L, ['4C]-thymidine incorporation of light cell controls. *, 1 x 106 dense cells/culture; 1.5 x 106 dense cells/culture; *, 1-75 x 106 dense cells/culture. .,

Dense cells are suppressive When the situation was reversed, i.e. mitomycin Ctreated dense cells were added to light cells, then the presence of dense cells suppressed incorporation of [I4C]-thymidine by light cells both in the presence and absence of Con A (Fig. 3). The intensity of the suppressive effect was independent of the intensity of the response of control cultures and directly proportional to the number ofdense cells added to the cultures, until a maximum suppressive effect of 75-90% was reached. In Con A-stimulated cultures, once maximum suppression was obtained, a further increase in the number of dense cells did not alter the intensity of the suppressive effect (Fig. 3). In non-stimulated cultures, however, higher doses of cells induced a less intense suppressive effect (Fig. 3).

Table 1. Blast transformation and cell division in mixed cultures

Con A dose

(pg/ml) 0

Mitomycin-C Reacting cells treated cells Dense Light Dense


Dense Dense Dense Dense Light


Light Light

Dense Light Dense



Light Light

Total no. of cells per culture Blast cells (%) (x 10-6) 0-68 164 0 34 08 17 0-7 2-5 12

23 23 24 54 35 21 93 85

Mixed cultures were done by incubating 1 x 106 reacting cells with I x 106 mitomycin-C-treated cells. After 72 h the number of cells present in each culture was evaluated in a haemocytometer and the percentage of large pyroninophilic blast cells evaluated in methyl-green pyronin-stained slides.

Separation ofhelper and suppressor cells


Mitogen concentration also influenced the intensity of suppression. When higher Con A concentrations were used, a lower number of cells were needed to induce maximum suppressive effect (Fig. 3). Conversely, when a low dose of Con A was used, a higher number of cells was required. Blast transformation and cell division in mixed cultures Incorporation of labelled thymidine is not always proportional to the degree of blast transformation in culture (Piguet, Dewey & Vassalli, 1975; Piguet, Dewey & Vassalli, 1976). Therefore we evaluated the number of blast cells and total number of cells in both control and mixed cultures. As shown in Table 1, changes in ['4C]-thymidine i05-


CL 0.

E Q.










5 ConA (pg/mL)



Figure 5. Influence of cycloheximide treatment (protein synthesis during the culture period

Characterization of rat spleen-cell populations. I. Cell interactions in the regulation of in vitro response to concanavalin A.

Immunology 1979 36 619 Characterization of rat spleen-cell populations 1. CELL INTERACTIONS IN THE REGULATION OF IN VITRO RESPONSE TO CONCANAVALIN A...
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