Int. J. Cancer: 16, 539-549 (1975)

SPECIFIC ANTI-TUMOR RESPONSES OF CULTURED IMMUNE SPLEEN CELLS

111. FURTHER CHARACTERIZATION OF CELLS WHICH SYNTHESIZE FACTORS WITH BLOCKING

(ADC) ACTIVITIES

by

AND ANTISERU M-DEPENDENT CELLULAR CYTOTOXIC

Karen NELSON l, Sylvia B. POLLACK, and Karl Erik HELLSTROM Departments of Pathology and Microbiology and Immunology, Seattle, Washington 9819.5;

and Division of Immunology, Fred Hutchinson Cancer Research Center, Seattle, USA

Spleen cells from BALBIc mice bearing syngeneic, methylcholanthrene-induced sarcomas were cultured in vitro. In agreement with previous observations, two tumor- specific activities could be demonstrated in the culture supernatants: the supernatants suppressed (blocked) specific lymph-node cell-mediated cytotoxicity directed against the respective neoplasm and they induced spec$c antibody-dependent cellular cytotoxicity (ADC) mediated by lymph-node cells fvom non-immune mice. In the present study, the eflects of specific depletion or enrichment of diferent cell types on the ability to synthesize the blocking and ADC activities was explored. Spleen cells passed through columns of Sephadex G-10, which removed plasma cells and macrophages, no longer synthesized factors with A DC activity though synthesis of blocking factors continued. However, lysis of Thy-l-positive cells with antiserum and complement did abolish the synthesis of blocking factors. Spleen cells from tumor-bearing mice were then enriched for Thy-I-positive cells by selective removal of macrophages and plasma cells on G-I0 columns and of immunoglobulin-bearing cells on anti-mouse immunoglobulin afinity columns. This T-cell-enriched population synthesized blocking factors and restored blocking factor synthesis in cultures depleted of Thy-1-positive cells. Similarly enriched spleen cells from normal control mice did not synthesize tumor-specific blocking factors but partially restored synthesis of blocking factor in tumor-bearer spleen cultures depleted of Thy-l-positive cells.

Spleen cells from mice bearing syngeneic cytotoxicity, ADC (Nelson et al., 19756). tumors, when cultivated in vitro, release factors Supernatants of spleen cultures from mice whose which specifically suppress (" block ") destruction tumors have been excised mediate ADC, but they of neoplastic cells by lymphocytes sensitized do not have blocking activity (Nelson et al., to their respective tumor-specific antigens (Nelson 19756). et al., 1975a). They also release factors which Both the blocking factors and the factors mediate antibody-dependent cell-mediated mediating ADC are retained on anti-mouse-

Received: July 22, 1975. Present address: Wallenberg Laboratory, University of Lund, Fack, 5-22007 Lund, Sweden.

539

NELSON ET AL.

immunoglobulin columns, and they can be re- covered by elution of such columns (Nelson et al., 1975a, b, c). When 14C-labelled leucine was added to the medium in which tumor-bearer spleen cells were cultivated, factors in the culture supernatants with blocking and ADC activities and with affinity for anti-immunoglobulin col- umns, were found to have incorporated the 14C label; they were found to bind preferentially to tumors having the " right " antigenic specificity (Nelson et af., 1975~). Synthesis of these factors could be inhibited by cycloheximide; this in- hibition was reversible (Nelson et al., 1975~). Altogether, these findings indicate that the factors responsible for the blocking and the ADC phenomena were synthesized, and not merely released in vitro.

The purpose of the present study was further to define the cells required for synthesis of factors mediating blocking and ADC activities in culture supernatants of spleen cells from tumor-bearing mice. We chose to separate spleen cells on Sephadex G-10 columns (Ly and Mishell, 1974) for two reasons. First, we wanted to identify which cells were responsible for the synthesis of factors in culture supernatants which mediate ADC. Previous work had shown that ADC activity of sera from mice bearing syngeneic sarcomas is associated with IgG, (Pollack and Nelson, 1975), and that the ADC activity of spleen culture supernatants from these mice is also associated with immunoglobulin (Nelson et af., 197%). This suggested that plasma cells may be responsible for the synthesis of ADC activity. Because plasma cells, as well as macro- phages and their precursors, are retained on Sephadex G-10 columns (Ly and Mishell, 1974), passage of spleen cells through such columns was chosen. Second, we have previously shown that removal of Thy-1-positive cells by treatment with antiserum and complement halts the synthesis of blocking activity in the cultures (Nelson et al., 1975~). It was, however, not clear at that time whether the effect of the anti-Thy-1 antiserum and complement was specific or was due to antigen-antibody complexes formed during lysis of the Thy-I-positive cells (Basten et al., 1975). Because the emuent of the G-10 columns is enriched for Thy-I-positive cells, cells from such effluents were cultured and the supernatants assayed for blocking activity. Cell populations enriched for Thy-I-positive cells were also tested

for their ability to restore the formation blocking factors in spleen cultures depleted Thy- 1 -positive cells.

of of

MATERIAL AND METHODS

Mice

Mice of the BALB/c strain were bred by brother- sister mating in this laboratory. In addition, female BALB/c mice were obtained from Jackson Laboratories, Bar Harbor, Maine, USA. Mice were sex- and age-matched in each experiment.

Tumor lines

Sarcomas were induced in adult BALB/c mice by subcutaneous injection of 3-methyl- cholanthrene. They were maintained by serial transplantation in vivo; tumors were used when in the fourth to eighth transplant generation.

Mice were inoculated subcutaneously with lo5 sarcoma cells. Spleens were taken for culture either when the tumors measured approximately 10 mm in diameter (" tumor-bearers ") or 3 to 4 weeks after surgical excision of tumors (" tumor-excised "). Each sarcoma was explanted and maintained in vitro as a monolayer culture to provide a source of target cells for the micro- cytotoxicity assays.

Spleen cultures

Spleens were removed from tumor-bearing, tumor-excised and non-immunized mice im- mediately following cervical dislocation. Prep- aration of the spleen-cell suspensions was done at 4" C in RPMI-1640. The cells were gently teased free of the capsule and the fragments sedimented at 1 xg. Erythrocytes were removed from single-cell suspensions by hypotonic shock, after which the remaining cells were washed twice. Aliquots of the suspensions were incubated with antiserum and complement or passed through columns as described below. Each spleen-cell suspension was cultured at 5 x lo8 cells per ml of medium in Erlenmeyer flasks at 37" C in a moist, 5 % CO, in air atmosphere. The culture medium was RPMI-1640 (GIBCO) buffered with NaHCO, and supplemented with 20% heat-inactivated (30 min at 56" C) fetal calf serum, 1 % sodium pyruvate, 2 % L-glutamine, 1001U/ml of penicillin, and 100pg/ml of streptomycin.

540

ADC AND BLOCKING FACTORS PRODUCED IN VITRO

Cells were spun free of the culture supernatants at 2- to 3-day intervals, washed twice and recultured at the original concentration in fresh medium. Cell viability was assessed by trypan blue dye exclusion. The cultures were discarded if the total number of viable cells was less than 80% of the original number. Culture supernatants were passed through a 0 . 4 4 ~ filter (Millipore Corporation, Bedford, Maine, USA) and stored at -70" C until assayed.

In vitro assay of the activity of culture super- natants

Supernatants were assayed for their ability to inhibit (block) the anti-tumor cytotoxicity of immune LNC and for their ability to mediate antiserum-dependent cellular cytotoxicity (ADC) by non-immune LNC in a modified microcyto- toxicity assay (Hellstrom and Hellstrom, 1971). They were heat-inactivated and assayed undiluted unless otherwise indicated. Target cells plated in the wells of Falcon 3040 Microtest plates received 0.1 ml supernatant, which was decanted 30 min later, when lo5 effector cells were added in 0.1 ml of medium. Immune lymph-node cells (LNC) were obtained from the lymph nodes of mice 5 days after the second of two intraperitoneal injections of lethally irradiated tumor cells given 10 days apart. Control LNC were obtained from the lymph nodes of non-immunized mice. The wells received 0.05 ml of medium containing 30 % fetal calf serum 45 min after the effector cells had been added. The plates were incubated for 36 to 40 h, stained with crystal violet, and the number of surviving target cells determined visually; all plates were coded and read " blind ". In all of four experiments, treatment of immune LNC anti-Thy-1 antiserum and complement (see below) reduced their percentage CMC (from 28*2 to 5&3). Thus, the immunization procedure used generated cytotoxic T cells, confirming earlier observations (Shiku et al., 1975).

Specificity of supernatant effects was deter- mined by testing each supernatant on a non- cross-reacting target cell line. As a control, tumor cells were treated with supernatants alone.

Calculations The calculations of percentage cell-mediated

cytotoxicity (% CMC), percentage blocking activity and percentage ADC activity have been

described (Nelson et al., 1975~). In this study, wells receiving LNC and culture supernatant of spleens from non-immunized mice served as controls. Each treatment group included 8 to 12 wells. The significance of the differences between the means of the treatment groups was determined by Student's t-test.

Separation of spleen cells prior to culture

Aliquots of spleen-cell suspensions received one of three treatments: (1) sequential treatment with an anti-Thy-1 antiserum and complement, (2) passage through Sephadex G-10 columns, or (3) sequential passage through Sephadex G-10 columns and affinity columns charged with anti- mouse immunoglobulin antiserum.

Preparation of the rabbit anti-Thy-I antiserum has been described (Pollack et al., 1975~). This antiserum was made specific for Thy-1 by ex- tensive adsorption with Thy-1 -negative lymphoma cells. Treatment of BALB spleen cells with anti- serum and complement abolished their response to PHA but did not reduce the number of direct plaque-forming cells detected in a Jerne assay. Normal rabbit serum served as the source of complement in these experiments. Treatment of spleen cells was essentially as before (Nelson et al., 1975~). One volume of spleen cells at 5 x lo7 cells per ml of 0.05 M phosphate-buffered saline (PBS) containing 2 % heat-inactivated fetal calf serum was mixed with one-half volume of diluted antiserum. The suspension was kept at room temperature for 15 min and on ice for 15 min longer. The cells were washed once, brought up to the original volume and mixed with one-half volume of a 1:15 dilution of complement. After 30min in a 37" C water bath, the remaining cells were washed twice and counted prior to culture.

The preparation of the Sephadex G-10 columns was as described (Ly and Mishell, 1974; Pollack et al., 1975b). The Sephadex was swelled, washed and autoclaved in PBS. A 15-ml bed volume was poured into a plastic syringe fitted with a cushion of glass wool and a stopcock. The gel was washed extensively with Hanks' balanced salt solution supplemented with 5 % heat-inactivated fetal calf serum and heated to 45" C. When the column was thoroughly washed and had attained a temperature of 37" C, 2 x lo8 spleen cells were passed through it. The column was flushed with warmed medium until the effluent was clear.

54 1

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ADC AND BLOCKING FACTORS PRODUCED IN VITRO

Cell losses averaged 52% (51 % to 53 %). The cells were washed once and counted prior to culture.

In pilot experiments to determine the efficiency of the columns, spleen cells were cultured for 48 h at 37" C in plastic Petri dishes with culture medium and 1.1 p latex beads (Difco Laboratories, Detroit, Michigan, USA). In contrast to the unfractionated spleen cells which were both adherent and phagocytic, effluent cells from the G-10 columns neither adhered to plastic nor in- gested latex beads.

In three experiments, the G-10 passed cells were also passed through affinity columns pre- pared by coupling the 7s fraction of a rabbit anti- mouse immunoglobulin antiserum to Sephadex G-100 (Pharmacia, Pisctaway, N. J., USA) (Mannik and Stage, 1971). The preparation and specificity of this antiserum has been described (Pollack and Nelson, 1975~). as has the prepara- tion of the columns (Pollack et al., 1975~). Spleen cells were passed through the columns at 4" C in Eagle's minimum essential medium supplemen- ted with 5 % fetal calf serum, 5 mM EDTA, and buffered with NaHCO,. The effluent of the columns contained less than 2% immuno- globulin-bearing cells by immunofluorescence (Pollack ef al., 1975~). The cells were washed twice and counted prior to culture.

RESULTS

Separation of spleen cells on Sephadex G-I0 columns

Spleens were obtained from mice bearing syngeneic sarcomas and from non-immune mice.

Plasma cells and macrophages and their pre- cursors were removed from a portion of the spleen-cell suspensions by passage through columns of Sephadex G-10 (Ly and Mishell, 1974). Supernatants were obtained from four series of cultures of G-10 passed and untreated spleen cells and assayed for blocking and ADC activities. Data from two experiments which were specificity controls for each other are shown in Table 1. Culture supernatants of untreated spleen cells from tumor-bearing mice had blocking and ADC activities specific for the tumor borne by the spleen donor. ADC activity was not de- tected in supernatants from cultures of G-10 passed cells; however, these supernatants were blocking. Similar results were obtained in all of four experiments of this type (Table 11). Because ADC activity is often more clearly detected at dilutions beyond (Nelson et al., 1975b), the supernatants were also assayed at a dilution of Supernatants of cultures of G-10 passed spleen cells did not have ADC activity at the high dilution either.

Restoration of the synthesis of blocking activity in T-depleted cultures by spleen-cell populations enriched for T cells

Although the effluent of the Sephadex G-10 columns was enriched for Thy-1 bearing cells (70-80 %), immunoglobulin-bearing cells (10 %- 15 %) also remained. Rapid passage of spleen cells through Sephadex G-100 columns charged with an anti-mouse immunoglobulin antiserum (RAMIG) decreases the number of immuno- globulin-bearing cells (Pollack et al., 1975~).

TABLE I1

SUMMARY OF FOUR EXPERIMENTS TESTING THE EFFECT OF PASSING SPLEEN CELLS FROM TUMOR-BEARING MICE THROUGH SEPHADEX G-10 COLUMNS ON THEIR

ABILITY TO SYNTHESIZE FACTORS MEDIATING BLOCKING AND ADC ACTIVITIES 1

Mean % blocking

activityfsr,

~

Mean % Mean ./,ADC ADC activity activity

f S D ( I o - ' ~ ) i S D

Untreated spleen cells G-10 passed spleen cells

12k29

83f18

3 8 1 8

3*2

3 0 k 4

- 2 1 6

Supernatants were assayed undiluted for blocking activity. The mean percent blocking activities were not significantly different (p>O.OS). Culturesupernatants were assayed both undiluted and at a dilution of 10 - ' ADC activity. At both dilutions the mean percent ADC activities produced by unseparated and G-10 passed spleen cells were significantly different at the p<O.001 level.

543

NELSON ET AL.

* * * * * * * N N P - Wl b b l N

+

Accordingly, in three experiments, aliquots of the G-10 passed spleen cells were passed through RAMIG columns. The effluent consisted of 88 %- 92 % Thy-I-positive cells. These cells were termed T-enriched; the ADC and blocking activities of their culture supernatants are shown in Figure 1. Detailed data from one experiment, A, are shown in Table 111. Removal of immunoglobulin- bearing cells from the (3-10 passed spleen cell population did not remove its ability to synthe- size blocking factors.

In each of the experiments shown in Figure 1, aliquots of the original spleen-cell suspension were treated with an anti-Thy-1 antiserum and complement. The supernatants of these T- depleted cultures did not have blocking activity, confirming our earlier observations (Nelson et af., 1975~). However, the synthesis of blocking activity could be restored in these cultures by addition of the T-enriched spleen cells (Fig. 1). Thus, the effect of the antiserum treatment was most likely the result of specific destruction of cells having the Thy-1 marker.

We then explored the effects of reconstituting T-depleted cultures with T-enriched spleen cells from non-immune mice. The results from one experiment are shown in detail in Table IV. Reconstituting a T-depleted culture with non- immune T cells partially restored the synthesis of factors with blocking activity. A11 of three additional experiments yielded similar results (Table V). In each case, the blocking activity of supernatants from cultures reconstituted with tumor-bearer T cells was about twice that of the supernatant from cultures reconstituted with non-immune T cells.

DISCUSSION

The present findings strongly implicate lympho- cytes carrying the Thy-1 marker (Reif and Allen, 1964; Shiku et ul., 1975) in the synthesis of factors which suppress (" block ") specifically immune lymphoid cells. Our data also indicate that cells which can be retained by G-10 columns are in- volved in the in vitro production of factors responsible for antibody-dependent cell-mediated cytotoxicity, ADC. According to our findings, Thy-1-positive lymphocytes do not synthesize factors responsible for ADC, and cells retained on G-10 columns do not synthesize blocking

ADC AND BLOCKING FACTORS PRODUCED IN VITRO

factors. These findings support and extend pre- indirect plaque-forming cells detected by a Jerne vious observations suggesting that different assay (Ly and Mishell, 1974) removed spleen cells are involved in the production of factors cells which were responsible for the ADC activity responsible for the blocking and the ADC in our culture supernatants. A role for macro- phenomena, respectively (Nelson et al., 19756). phages in this synthesis cannot be ruled out,

A B

I

1

Culture supernatants of spleen cells 0 Untreated T-enriched

T-depleted p T-depleted + T-enriched

The effect of adding spleen cells enriched for Thy-1-positive cells to spleen cultures depleted of T cells. In three experiments, A-C, culture supernatants of untreated and separated spleen cells from tumor-bearing mice were assayed for specific blocking and ADC activities. Separated spleen cells were: (A) treated anti-Thy-1 antiserum and complement (T-depleted); (B) passed se- quentially through columns of Sephadex (3-10 and Sephadex G-100 charged with RAMIG (T-enriched), or, (C) mixed in a ratio of 60% T-depletedf40x T-enriched spleen cells prior to culture. Culture supernatants of similarly treated spleen cells from non-immune mice served as controls. In each experi- ment, the T-enriched spleen cells restored the synthesis of blocking factors in the T-depleted cultures.

Our initial hypothesis was that factors mediat- ing ADC in supernatants of cultures of spleen cells from tumor-bearing mice are synthesized by immunoglobulin-forming cells. This hypothesis was supported by the results obtained in this study. Columns which remove the direct and

however, since the G-10 columns remove both plasma cells and macrophages and their pre- cursors (Ly and Mishell, 1974). This question can be directly tested using anti-plasma cell serum to specifically remove the plasma cells (Harris et a[., 1972).

545

NELSON ET AL.

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The identity of the cells which synthesize blocking factors remains enigmatic. Treatment of spleen cells from tumor-bearing mice with an anti-Thy-I antiserum and complement abolished their synthesis of blocking factors. Since this synthesis could be restored by a population of the same spleen cells which had been enriched for Thy-1-positive cells, it is likely that the effect of the antiserum and complement was specific for a population of cells essential for the synthesis of blocking factors and not due to inhibitory effects of complexes formed during the treatment (Basten et al., 1975). Our data thus indicate that Thy-I-positive cells are critical for this synthesis. However, the mechanism is not clear. Any hypothesis must account for the observed syn- thesis of a factor which (1) is produced by a population of spleen cells depleted of macro- phages, plasma cells and immunoglobulin- bearing cells ; (2) binds to an anti-immunoglobulin affinity column; and (3) exerts its immuno- suppressive effect on cytotoxic T cells. The evi- dence cited under (2) suggests synthesis of a T-dependent antibody; however, this conflicts with the evidence in (1) unless plasma-cell pre- cursors with little surface immunoglobulin are involved. The evidence cited under (1) and (3) suggests that what we are measuring is a T-cell control of another T-cell response. Similar interactions have been reported in other systems (Gershon, 1974). However, to account for the binding of blocking factors to an anti-immuno- globulin column (2), one would have to invoke an immunoglobulin-like factor synthesized either by the Thy-1-positive cells themselves or by the 10% of " undefined " cells present in the T-enriched populations. To mediate blocking activity, factors synthesized by either of these mechanisms may act alone or in concert with tumor antigens carried into the cultures on some cells present in tumor-bearer spleens.

The partial restoration of blocking factor synthesis by non-immune T-cells suggests that those cells may undergo sensitization to tumor antigens in vitro, resulting in de novo synthesis of blocking factors. Whether synthesis of blocking factors also occurs during generation of cyto- toxic lymphocytes reacting to antigens present on syngeneic tumors (Warnatz and Scheiffarth, 1974; Kall and Hellstrom, 1975; Treves et al., 1975) deserves investigation.

We wish to emphasize that all analyses of blocking and ADC activities made in this study

546

ADC AND BLOCKING FACTORS PRODUCED IN VITRO

were performed by incubating target tumor cells with culture supernatants and removing the supernatants before addition of the immune or control lymphocytes. The protocol favors the detection of blocking factors which are either antigen-antibody complexes (Sjogren et al., 1971 ; Baldwin et al., 1973; Tamerius et al., 1975) or free antibodies (Robins and Baldwin, 1974) rather than those which are free antigens (Sjogren et al., 1971; Currie and Basham, 1972; Thompson et al., 1973; Baldwin et al., 1973).

and McKhann, 1972). Removal of plasma cells decreased production of factors responsible for ADC without influencing that of blocking factors in our studies; improved ability to destroy tumor cells would not be expected under these circumstances. Our findings do suggest, however, that thymectomy might depress the formation of blocking serum factors. The depressed level of blocking factors in mice which had undergone neonatal thymectomy and the decreased fre- quency of spontaneous mammary carcinomas

TABLE V

EXPERIMENTS COMPARING THE ABILITIES OF PRIMED AND NON-IMMUNE, T-ENRICHED SPLEEN CELLS TO RESTORE SYNTHESIS OF FACTORS WITH BLOCKING ACTIVITY IN T-DEPLETED CULTURES.

Supernatant from cultures of

spleen cells spleen cells from from

Experiment T-depleted T-enriched

A Mice bearing MCA-1358 Mice bearing MCA-1358

B Mice b-aring MCA-1388 Mice bearing MCA-1388

Mice bearing MCA-1358 Non-immune mice

Mice bearing MCA-1388 Non-immune mice

C Mice bearing MCA-1386 Mice bearing MCA-I386 Mice bearing MCA-1386 Non-immune mice

% blocking * activity

125** 60*

133** 61* 17* 31

1 See footnote 1, Table 111. Probability that the observed differences were due to chance * p<O.O5, ** pi0.01.

Further insight into the nature of the T-cell- dependent blocking factors produced by the immune spleen-cell cultures may be obtained by assaying the supernatants at the lymphocyte level. In unpublished studies, 10 out of 13 super- natants blocked cell-mediated immunity at both the lymphocyte and target-cell levels (Nelson, unpublished data); it has not yet been studied whether the factors blocking at the lymphocyte and target cell levels, respectively, differed.

Concerning the possible in vivo implications of these studies, our data do not support the sugges- tion that removal of plasma cells from cell populations might decrease the formation of blocking factors, thereby allowing increased expression of cellular immunity (Jagarlamoody

in those mice (Heppner, 1970), is provocative in this context, although it cannot be used as a basis for conclusions as to the mechanisms involved.

ACKNOWLEDGEMENTS

This work was supported by grants IM-7 and IM 43F from the American Cancer Society and grant CA-14697 from the National Institutes of Health. We thank Caroline Hoelscher, Kathy Chorey, Linda Katzenberger and M. Craig Bailey for excellent technical assistance and DarIene Cheever for assistance in preparing the manuscript.

547

NELSON ET AL.

RBPONSES ANTITUMORALES SPECIFIQUES DES CELLULES SPLENIQUES IMMUNES EN CULTURE. 111. CARACTERISATION PLUS POUSSGE DES CELLULES

QUI SYNTHETISENT DES FACTEURS BLOQUANTS OU AYANT UNE ACTIVITE CYTOTOXIQUE DGPENDANT DE L'ANTISfiRUM (ADC)

Des cellules spliniques de souris BALBIc porteuses de sarcomes syngtniques induitspar le mkthylcholanthrtne ont k t i cultivies in vitro. Comme lors des observations preckdentes, deux activitis tumorispicifiques ont pu Ptre mises en ividence dans les surnageants: ceux-ci supprimaient (bloquaient) en effet la cytotoxiciti spici5que rnkdiie par les cellules de ganglions lymphatiques et dirigie contre le ntoplasme corres- pondant, et induisaient une cytotoxicite spicifique dipendant de I'anticorps ( A DC) mediie par les cellules de ganglions lymphatiques de souris non immunes. Dans la prtsente etude, les auteurs ont cherche' h diterminer les effets de la diplition ou de l'enrichissement spicifique en divers types de cellules sur l'aptitude h synthitiser l'activitd de blocage ou I'ADC. Les cellules splPniques passees dans des colonnes de Sephadex G-10, qui permettent de ricupirer les plasmocytes et les macrophages, ne synthetisent plus de facteurs induisant une ADC mais continuent de synthgtiser des facteurs bloquants. Toutefois, la lyse des cellules Thy I-positives avec l'antiskrum et le complkment supprime la synthbe des facteurs bloquants. Les cellules sple'niques de souris cancireuses ant ensuite e'tP enrichies en cellules Thy I-positives par suppression selective des macrophages e f des plasmocytes sur des colonnes G-I0 et des cellules porteuses d'immunoglobulines sur des colonnes d'afinite' contenant de l'antiskum anti-immunoglobuiine de souris. Cette population riche en cellules T synthitisait IIes facteurs bloquants et rttablissait leur synthtse dans les cultures privies de cellules Thy I-positives. Des cellules spliniques pareillement enrichies, provenant de souris- timoins normales, ne synthttisaient pas de facteurs bloquants spicifiques de la tumeur, mais ritablissaient partiellement leur synthkse dans les cultures de cellules splkniques d'animaux cancgreux privies de cellules Thy I-positives.

REFERENCES

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