Macromolecular synthesis during the sensitization of rat lymphocytes on mouse fibroblasts in vitro

lramunochemiary. Pergamon Press 1971. Vol.8, pp. 487-498. Printed in Great Britain

MACROMOLECULAR SYNTHESIS DURING THE SENSITIZATION OF RAT LYMPHOCYTES ON MOUSE FIBROBLASTS I N VITRO* W. R. CLARKt, G. BERKE*, M. FELDMAN and S. SARID Departments of Cell Biology and Biophysics, The Weizmann Institute of Science, Rehovot, Israel (Received 22 November 1970)

Abstract-The sensitization of rat lymphocytes against mouse cells in vitro and the effector phase of mouse cell lysis by the sensitized cells was found to proceed in the absence of soluble extracellular factors. The process of morphological transformation, and the acquisition of the capacity to lyse target cells, developed in the absence of detectable synthesis of new species of protein, including immunoglobulins. Hybridization competition experiments demonstrated that unlabeled small lymphocyte RNA was a successful competitor in the reaction between labeled RNA from sensitized lymphocytes and lymphocyte DNA. This suggests that resting small lymphocytes and sensitized lymphocytes possess similar species of RNA, and no gross changes in the various molecular species of RNA could be detected during sensitization. Our results do not, however, exclude the possibility that the synthesis of certain species of RNA present in the small lymphocyte is suppressed following transformation. INTRODUCTION The nature of at least the effector mechanism of humoral immune reactions is now fairly well understood: competent lymphocytes respond to antigen by synthesizing and exporting antibody molecules which interact with the antigen in a specific and well defined way. On the other hand, very little is known about the mechanism of the so-called cell-mediated immune reactions (Benacerraf, 1968; Perlmann and Holm, 1969). Heterograft reactions in vivo have been shown to be essentially cell-mediated. In previous publications, we have described a system for the in vitro induction of a heterograft reaction. This reaction appears to proceed without the involvement of antibody (Ginsburg et al., 1969). On the basis of the involvement of lymphoid cells, and the morphological transformation they undergo during sensitization (Ginsburg and Sachs, 1965; Tyler et al., 1969), the specificity of the reaction (Ginsburg, 1968; Berke et al., 1969), and the ability of lymphoid ceils sensitized in vitro to function in immune situations in viv0 (Berke a al., 1970), the in vitro reaction is a valid immune reaction. In order to gain some insight into the molecular basis of the graft reaction, we have undertaken a series of analyses of the synthesis of RNA and protein during the sensitization in vitro of rat lymphocytes on mouse fibroblasts. *This study was supported by the Max and Ida Hillson Foundation, New York and by the Freudenberg Foundation for Research on Multiple Sclerosis, Weinheim, Germany. ~'Supported by NIAID Postdoctoral Fellowship 2-FO2oAI-39111-02. Present address: Department of Zoology, University of California, Los Angeles, Calif. 90024, U.S.A. *Present address: Department of Surgery, Children's Hospital Medical Center, Boston, Mass. 02115, U.S.A. 487

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W . R . CLARK, G. BERKE, M. FELDMAN and S. SARID

MATERIALS AND METHODS

Cell culture Details of the preparation and culture of rat lymphocytes on mouse fibroblasts have been presented elsewhere (Berke et al., 1969; Berke et al., 1970). Lymphocytes were o f lymph node origin; medium (EM) was Eagle's medium supplemented with 4 × amino acids and vitamins, 20 per cent horse serum, and penicillin-streptomycin. Culture medium was changed on the third day. Protein synthesis At appropriate times of culture l~C-labeled amino acid mixture (Amersham CFB-104) was added to a final concentration of 2"5 tzc/ml. After the labeling period the cultures were placed on ice, and cells harvested by either gently pipetting the monolayer surface and collecting free-floating (lymphoid) cells, or by scraping the entire culture (lymphocytes plus monolayer) with a rubber policeman. Cells were transferred to a centrifuge tube and washed twice with cold phosphate buffered saline (PBS). The final cell pellet was stored in polyethylene microfuge tubes at--15 ° until assay. Uncentrifuged, freeze-thaw/sonicated suspensions of pulse-labeled cells were incubated 60 min at 37 ° in 10 vol. of a solution of 10 per cent acetic acid, 1 per cent sodium dodecyl sulfate (SDS) and 0"5 M urea. The suspensions were dialyzed overnight at room temperature vs. 1000 volumes phosphate buffer, 0.01M, pH 7.1, containing 0.1 per cent SDS and 0.5M urea (Shapiro et al., 1967). Five per cent polyacrylamide gels were prepared in the same buffer containing 0-1M phosphate; this buffer was also used for running of the electrophoresis at 5 ma. per gel for 3 hr. After running, the gels were either stained with Coomassie Blue stain, or frozen and sliced into 0.8 m m sections which were hydrolyzed with 0.4 ml 30 per cent H~O2 and counted in a liquid scintillation counter. Broken cell suspensions prepared as outlined above were also dialyzed against 1000 vol. saline overnight. Samples to be applied for immunoelectrophoresis were mixed with equal parts of whole rat serum. After electrophoresis the plates were developed with appropriate antisera, rinsed, stained and dried. Final plates were placed in contact with Kodak SB54 X-ray film for various lengths of time. Preparation of DNA DNA was extracted from rat lymphocytes by the method of Marmur (1961). For the preparation of (3H) thymidine-labeled lymphocyte DNA, lymphocyte cultures undergoing transformation were exposed for 72 hr to 1 ttCi of (methylall) thymidine per milliliter. DNA-RNA hybridization The technique used was essentially that reported by Gillespie and Spiegelman (1965). Nitrocellulose membrane filters were loaded with alkaline-denatured lymphocyte (3H)DNA. The DNA filters were then washed with 100 ml of 2 x SSC, dried at room temperature overnight, and baked at 80°C for 2 hr. Eighty to 100 per cent of the (3H)-labeled lymphocyte DNA deposited on the filters were

Macromolecular Synthesis

"

489

retained. DNA-RNA hybrids were formed by immersing the DNA filters in glass vials containing 1.7 ml of RNA in 2 x SSC at 67°C for 20 hr. After incubation the filters were removed, washed on each side with 50 ml 2 x SSC and incubated with 5 ml 2 x SSC containing 20/~g/ml of boiled RNase for 1 hr at room temperature, with occasional shaking. The filters were then washed again on each side with 50 ml 2 x SSC, dried, and assayed for radioactivity in a Packard Tri-Carb liquid scintillation spectrometer. RESULTS

Soluble factors in the transformation of lymphoid cells and lysis of target monolayers A number of systems have been described (Granger and Williams, 1968; Dumonde et al., 1969; Bennett and Bloom, 1968; Ruddle and Waksman, 1968) in which soluble factors are implicated as at least part of the effector mechanism in lymphocyte-mediated immune reactions. We have looked for the involvement of soluble factors in the transformation of rat lymphocytes in vitro on mouse fibroblast monolayers, and in the destruction of mouse target cells by in vitro sensitized rat lymphocytes. In the experiment shown in Table l, we tested the ability of media from mouse fibroblast cultures to cause rat lymphocytes to become sensitized against mouse antigens. Normal rat lymphocytes cultured for 5 days in the presence of such conditioned medium (CM) exhibited a slight increase in the ability to cause ~lCr release from labeled target monolayers; however, the values obtained were always small in comparison with lysis obtained when the lymphocytes were cultured for 5 days directly on fibroblast monolayers. In the experiments shown in Table 2, rat lymphocytes were cultured on mouse Table 1. Ability of medium alone from mouse fibroblast cultures to sensitize rat lymphocytes against mouse antigens* Group

Sensitizing agent

Counts/min in culture medium

Total counts/min per culture

% 51Cr released

A

C3H fibroblasts

10248 10008

18905 18683

54.2 53.5

B

20% CM~"

2764 2912

19458 20218

14-2 14.4

C

40% CM~"

3320 3556

19184 20239

17.3 17-5

D

--

2476 2560

19634 18974

12-6 13-4

*30 × 106 rat (Wistar) lymphocytes were seeded in 60 mm culture dishes in the following groups: A - C 3 H fibroblast monolayers+EM. B - N o fibroblasts; 20%CM+ 80%EM. C - No fibroblasts; 40%CM+ 60%EM. Medium was changed on the 4th day and cells were passed to ~Cr-labeled target monolayers for 48 hr on the fifth day. D - Spontaneous release of *XCrfrom target monolayers. ~'CM = medium collected from 4 day old derivative cultures of C3H fibroblasts.

I.M.M. V~/. 8 No. 6 - C

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W.R. CLARK, G. BERKE, M. FELDMAN and S. SARID

fibroblasts for 5 days, and then transferred to unlabeled mouse target monolayers for another 24 hr. When destruction of the target cells was visible, the culture medium only from these lytic cultures was transferred to a second set of 51Cr-labeled target monolayers. 51Cr release was determined 48 hr later. The SlCr release caused by such lytic medium was negligible, and not dose dependent. In another experiment, lyric medium was plated together with normal rat lymphocytes on labeled mouse target cells, and as shown in Table 3, the lyric medium was also unable to confer any lytic activity on normal lymphocytes. The foregoing experiments support our previous conclusion (Ginsburg, 1969) that the processes of sensitization and lysis in the in vitro heterograft reaction require direct cell-cell contact, and probably are not mediated to any significant extent by soluble, extracellular factors. It is likely, therefore, that these processes do not involve the synthesis of macromolecules for export, and if sensitization or lysis is accompanied by the synthesis of specific macromolecules, then these molecules are probably confined on or within the cell. Because of the fundamental implications of the synthesis of new molecular species during cellular immune reactions, for the understanding of the mechanism involved, a series of analyses of macromolecular synthesis within the reacdng cells themselves was undertaken. Table 2. Cytotoxicity of cells and medium taken from lytic cultures In vitro sensitized rat lymphoid cells plated*

Counts/min in culture medium

Total counts/min per culture

% 51Cr released~"

106

5796 7988

10214 12858

56"7 62"1

4 × 106

6000 6600

12890 13067

46"5 50"5

2 × 106

3964 4644

11402 12203

34-7 38.0

1

1828 2036

10885 10336

16"7 19"6

1:2

2048 2348

11722 11504

17-4 20"4

1:4

1632 2076

9857 10222

16"5 20.3

1676 1628 1200 1476 848

9743 9626 8522 9315 5502

15-9 (mean value)

8×

Lytic medium added*

*Lymphoid cells and lytic medium were diluted to the desired concentration with EM. "~51 Cr release was measured after 48 hr.

491

Macromolecular Synthesis Table 3. Lytic activity of medium and cells taken from lytic cultures Counts/min in culture medium

Total counts/min per culture

% 51Cr released*

C3H

765 741

1872 1859

40.8 39-8

EMC

C3H

283 222

2067 1909

13.6 11-6

EM

C3H

208 206

1806 1831

11.5 11.2

--

EM

C3H

225 194

1903 1954

10.5 9"0

Lew (anti-C3H)~" lymphoid cells

EM

C57BL

817 817

3675 3505

22"2 23.3

Normal Lew~ lymphocytes

EMC

C57BL

571 560

3333 3238

17.1 17"2

Normal Lew$ lymphocytes

EM

C57BL

523 554

3236 3365

16.1 16.4

EM

C57BL

545 532

3488 3570

15.6 14"9

Culture medium

51Cr-labeled fibroblasts

Lew (anti-C3H)~" lymphocyte cells

EM

Normal Lew:~ lymphocytes Normal Lewd: lymphocytes

Lymphoid cells

--

tLew (anti-C3H) lymphocytes were taken from cultures of Lew (anti-C3H) lymphocytes on C3H fibroblasts in which lysis of the fibroblasts was evident. $3 × 10 6 normal Lew lymphocytes were plated onto C3H target cells in either fresh EM, or EM from cultures of Lew (anti-C3H) lymphocytes on C3H fibroblasts, in which lysis of the fibroblasts was evident (EMC).

Synthetic patterns in rat lymphocytes undergoing transformation and sensitization on mousefibroblasts in vitro Rat l y m p h o i d cells were c u l t u r e d for sensitization o n m o u s e fibroblast monolayers as described previously (Berke et al., 1969; Berke et al., 1970). At various times d u r i n g sensitization, 14C-amino acids were a d d e d to the culture m e d i u m . At the e n d o f the labeling period the lymphocytes were collected, disrupted, and the cell contents p r e p a r e d for analysis by polyacrylamide gel electrophoresis (PAE) and r a d i o i m m u n o e l e c t r o p h o r e s i s (RIE) as described in the m e t h o d s section. As a test o f the ability o f these two analytical systems (PAE and RIE) to detect newly synthesized proteins in rat lymphoid cells, Lewis rats were h y p e r i m m u n i z ed with T-4 p h a g e a n d the l y m p h o i d cells f r o m the draining nodes were labeled in vitro with l~C-amino acids. As can be seen in Fig. 1, u n d e r the conditions described in the figure legend, a very distinct peak o f IgG was readily detected on PAE after electrophoresis o f the extracts o f cells stimulated in this m a n n e r . T h e p a t t e r n and distribution o f radioactivity o f such extracts, w h e n r u n as antigen in RIE, and d e v e l o p e d against goat (anti-rat) s e r u m is shown in Fig. 2. A

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Fig. ]. Polyacr~lamide gel clectrophero~ams of pulse-labeled T-4 stimulated rat lymph node cell extracts. Lew rats were injected in the footpad with 109 T-4 phage over a period of 15 days, and sacrificed on the 19th day. The draining lymph nodes were collected and lymphoid cell suspensions prepared. 25 × 106 lymphoid cells were incubated with 10 #c 1%-amino acids in 5 ml EM without horse serum. The cells were extracted and prepared for PAE as described in the Methods secdon. Upper panel. Cell extracts were preo incubated either with rabbit (anti-rat IgG) serum (dashed line) or normal rabbit serum (solid line) for 8 hr at room temperature. Lower panel. Cell extracts were dialyzed against 0.1 per cent #-mercaptoethanol as described by Shapiro et al. (1967). PAE was also carried out in the presence of ~mercaptoethanol. very sharply defined band o f radioactivity is seen to be congruent with the stained g a m m a globulin protein arc. These two techniques were then used to look for protein synthesized in rat lymphocytes in response to stimulation in vitro by mouse fibroblast monolayers. Lewis rat lymphocytes were cultured on mouse fibroblasts for 5 days. At 24 hr intervals, beginning at the time of plating, l~C-amino acids were a d d e d to the culture for 4 hr, after which the lymphocytes were harvested, extracted and analysed as described above and in the Methods section. T h e results o f the PAE analysis are shown in Fig. 3. At no time d u r i n g the 5-day culture period, d u r i n g which the rat lymphocyte population acquired the capacity to lyse the sensitizing monolayer a n d target cells of the same mouse strain, did we observe significant incorporation of labeled amino acids into any specific protein migrating into 5 per cent PAE columns. T h e lack of excess radioactivity at the top o f the columns (first fractions) f u r t h e r indicates that specific proteins larger than the exclusion limit of the gel (approximately 250,000 tool. wt.) were also not synthesized. Analysis of these samples on RIE gave variable results. T h e r e generally was a trace a m o u n t of radioactivity in the g a m m a globulin region of the serum

Fig. 2. Immunoelectropherogram and resultant autoradiogram of extracts of pulse-labeled T-4 stimulated rat lymph node cells. The extracts used for PAE in Fig. 1 were also run as antigen in RIE as described in the Methods section. Upper panel: original immunoelectrophoresis plate, stained with Amido Black. Lower panel: resultant autoradiogram. Top antigen well: immune lymphocytes were collected before labeling, and ‘“C-amino acid mixture added at beginning of extraction procedure; mixed in equal parts with whole rat serum. Bottom antigen well: immune lymphocytes labeled 1 hr with Wamino acids, collected and extracted as described in the text; mixed in equal parts with whole rat serum. Antibody trough: 0.2 ml goat (anti-rat serum) serum.

(Facing page 492)

4 5 6 7

Fig. 4. Upper panel: Immunoelectropherograms of cell extracts described in Fig. 3. Immunoelectrophoresis and autoradiography were performed as described in the Methods section. Antigen samples were all mixed 1: 1 with whole rat serum immediately prior to electrophoresis. All antiserum troughs contain 0.2 ml rabbit (anti-rat serum) serum. Antigen wells contain: 1 and 2whole rat serum only (stained with Amido Black); 3 -to labeled cell extract; 4-24 hr; 5-48 hr; 6-72 hr; 7-96 hr; S-120 hr. Lower panel. Radioimmunoelectropherogram from a separate experiment in which labeled precipitin lines were visible in the a-globulin region. Conditions of culturing and labeling were essentially the same as in Fig. 4a. In this experiment labeling in the o-globulin region occured only during the 48 hr pulse. Developed against rabbit (anti-rat serum) serum. Antigen wells: A-48 hr labeled cell extract; 72 hr extract (both mixed with equal parts whole rat serum).

493

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Fig. 3. Polyacrylamide gel electrophorograms of extracts of rat lymphocytes labeled at various times during sensitization on mouse fibroblast monolayers. 10 c~cof %-amino acids were added to duplicate cultures of Lew lymphocytes grown on C3H fibroblast monolayers at various times during sensitization. After 4 hr of incubation, the labeled cells were harvested, combined, and prepared for PAG as described in the Methods section. pattern (Fig. 4, upper panel), but it was not confined to the narrow gamma globulin precipitin line, nor did it appear to change in intensity as a function of sensitization time. Occasionally, there were faint but sharply defined traces of radioactivity in some of the alpha-globulin precipitin bands (Fig. 4, lower panel), but these were detected in only a small proportion of the experiments and again could not be correlated with any particular state of sensitization of the lymphocytes. Pick et al. (1969) have suggested that these may be related to hemoglobin. After one or two days of sensitization some of the lymphocytes begin to adhere firmly to the sensitizing monolayer. These lymphocytes cannot readily be separated from the fibroblasts. Since these lymphocytes might possibly represent those cells undergoing specific sensitization, experiments were also done in which the monolayer was collected with the lymphocytes after the labeling period, and the whole extracted and analyzed. Both the PAE and RIE profiles remained qualitatively unchanged from those in Figs. 3 and 4 in such experiments. These results suggest that no new detectable species of protein are synthesized in response to sensitization of rat lymphocytes on mouse fibroblasts in vitro, even though this sensitization process involves marked morphological changes, and results in the expression of a new function (cell lysis). We have subsequently examined the RNA patterns synthesized during sensitization using DNA-RNA hybridization and hybridization competition experiments. The competition experiments were based on challenging a fixed amount of pulse-labeled RNA from stimulated lymphocytes with lymphocyte DNA in the presence of increasing amounts of non-labeled RNA from various sources. The degree of saturation of the DNA with total RNA isolated from lymphocytes induced on mono-

494

W. R. CLARK,

G. BERKE,

M. FELDMAN

and S. SARID

layers is between 1 and 2 per cent of the DNA. On the other hand, saturation of the DNA with purified r-RNA and t-RNA gave no more than 0.2 per cent (Berke et al., 1971). The competition experiments were carried out at the level of maximum saturation which equals 100 per cent hybridization. As shown in Fig. 5, cold RNA taken from unstimulated rat lymphocytes competes as does cold RNA taken from fibroblast-stimulated lymphoid cells (LPC) in the hybridization of labeled RNA taken from rat LPC. Both the mitotic activity of transformed lymphoid cells, and the lytic activity of the lymphoid cell cultures, is maximal at day 5, and therefore, specific RNA molecules, if they are produced at all, might be expected to be synthesized maximally at this time. Hybridization competition was displayed also by RNA from PHA-stimulated rat lymphocytes (Berke et al., 1971). The differences obtained between unstimulated lymphocyte RNA and stimulated RNA do not exclude the possibility that RNA species present in the small lymphocyte are lost during induced transformation. Thus, within the limits of resolution of the DNA-RNA hybridization technique (Church and McCarthy, 1968; Melli and Bishop, 1969; Hansen et al., 1970) as employed here, it appears that these cell types (normal, PHA-stimulated, and mouse fibroblast-stimulated rat lymphocytes) contain similar major species of RNA. Hybridization competition experiments probably cannot detect minor differences in RNA species. Torelli et al. (1968) have shown that human peripheral blood lymphocytes stimulated with PHA produce no new species of RNA as revealed by DNA-RNA hybridization competition experiments. We also have shown (Berke et al., 1971)

ROtiO unlobelled

RNA

to LPC

P3*-

RNA

Fig. 5. The competition of unlabeled RNA from various sources, in the hybridization of labeled RNA from mouse fibroblast stimulated rat lymphocytes, with rat lymphocyte DNA. 32P-labeled RNA was prepared from C3H fibroblast stimulated Lew lymphocytes (LPC) and labeled with 250 /.x 32P for 24 hr. 12 w of LPC 32P-RNA (61760 cpm/pg) was challenged with 0 RNA from unstimulated rat lymphocytes; A LPC RNA; or 0 PHA stimulated (72 hr) rat lymphocyte RNA, as described in the Methods section. DNA content per filter was 0.99 pg.

495

Macromolecular Synthesis

that unlabeled RNA from unstimulated rat lymphocytes competes equally well as unlabeled RNA from PHA-stimulated rat lymphocytes, in the hybridization of labeled RNA from PHA-stimulated rat lymphocytes with rat DNA. In order to determine whether or not protein synthesis per se is required for sensitization of lymphoid cells or lysis of target cells, we tested the effect of cyclohexamide to block these processes. We found that cycloheximide was lethal to rat lymphoid cells undergoing sensitization on mouse fibroblast monolayers. The lymphoid cells degenerated and were all dead by the 3rd day of culture. On the other hand, lysis of mouse target monolayers by in vitro sensitized rat (antimouse) lymphocytes could be reversibly inhibited by cycloheximide (Table 4). This effect is specific for the lymphoid cells; preincubation of the target monolayer alone had little or no effect on subsequent lysis. Similar results were obtained by Brunner et al. (1968). Thus, while synthesis of specific proteins may not be required for the in vitro heterograft reaction, it would appear that general protein synthesis per se must be functioning for the reaction to occur. Table 4. Effect of cycloheximide treatment of C3H fibroblast target and I.ew anti-C3H effector cells, on target cell lysis* Experiment No.

1

2

Exposure to cycloheximide Target cells -6+Ohr -17+Ohr -6++17hr 0++17hr -

Lymphocytes -6+Ohr 0++17hr -6++17hr -17+0 -17+0

Period of interaction of lymphoid cells and ‘Cr-labeled target cells Ot--+ Ot + Ot+ Ot+ Ot+ Ot +

17hr 17 hr 17hr 17hr 17hr 17 hr

O-+ 18hr O-, 18hr 24 --* 41 hr

Net % released 83 79 74 56 20 0 58.6 11.1 32.6

*Lew lymphocytes were sensitized for 5 days on C3H fibroblast monolayers. On the 5th day they were harvested and resuspended in EM, plus or minus 0.5 pg/ml cycloheximide. 3.5 x lo6 cells were plated on SICr-labeled C3H target monolayers, some of which had been preincubated with cycloheximide. In some cases, the sensitized lymphoid cells were left suspended in cycloheximide at 37” for various times prior to plating. In experiment 2, the lymphoid cells were preincubated with cycloheximide for 17 hr, and either plated immediately on target cells (0 + 18 hr) or were washed and resuspended in fresh EM prior to plating. tZero time is the time of plating of the sensitized lymphocytes on the target cells. Negative times are pre-incubation periods prior to plating. DISCUSSION There are two possibilities with respect to the response of lymphocytes to foreign cell antigens. Either it is the case that those lymphocytes present in the normal population which recognize a particular cell as foreign already have all the necessary molecular apparatus for destroying the foreign cell; or else, it is the

496

W. R. CLARK,

G. BERKE, M. FELDMAN

and S. SARID

case that lymphocytes capable of recognizing the particular foreign cell cannot interact with it in a destructive way, but must undergo an additional differentiative event in which either new genes coding for proteins necessary for target cell destruction are expressed, or in which pre-existing information is translated into appropriate effector molecules. In the latter case, whatever the level of control, the result should be the appearance of new protein species in the stimulated lymphocyte. In the former case, mounting an effective immune response may consist simply of a selective proliferation of the specific recognition/effecter lymphocyte, without the synthesis of new protein molecules. In view of the evidence for the participation of other factors in the cellular immune response, such as antibody (Perper and Najarian, 1966; Lance et al., 1969) and other cells of the RES (Benacerraf, 1968), these two cases almost certainly represent an oversimplification of the cellular immune response as a whole, but nevertheless taken together present a valid boundary statement for the response of the lymphoid cells per se. The results presented here seem to favor the first case for lymphocyte response, namely, that no new macromolecules are required to mount an effective cellular immune response as expressed in the lysis of mouse target monolayers by rat lymphocytes sensitized against mouse fibroblasts in vitro. There are obvious limitations to the certainty with which such a conclusion may be drawn, the most apparent being the sensitivity of the methods employed. No quantitative conclusions can be drawn in this regard in the present study, for reasons discussed below, but it is certainly valid to say that the synthetic response of rat lymphoid cells to mouse cellular antigens in vitro, in terms of the synthesis of specific molecules, is at least well below the humoral response of rat lymphoid cells made hyperimmune to T-4 phage, which produce specifically IgG (Fig. 1). One of the major difficulties in trying to assess synthetic activity within a responding lymphocyte population is that the cells toward which the study is aimed are at least initially a very small proportion of the total lymphoid cell population (Tyler et at., 1969) and thus specific synthesis within the responding cells at early times in the response may be lost within the general turnover of synthetic activity of the population as a whole. This problem is further compounded in our system by the fact that after about 30 hr of culture, some of the lymphocytes begin to adhere firmly to the sensitizing monolayer, necessitating the collective removal and assay of both lymphocytes and monolayer, if these lymphocytes are to be assayed. Very little direct information relevant to protein synthesis in lymphoid cells undergoing sensitization to foreign grafts has been published. Prendergast et al. (1969) allogfafted fetal lamb in utero and pulse-labeled and analyzed the draining lymph nodes. They reported no significant synthesis of IgG or IgM, and only trace amounts of cu-globulins. Pick et al. (1969) immunized guinea pigs with tuberculin PPD, and subsequently cultured lymphoid cells from the draining lymph nodes in the presence or absence of PPD. They observed a general increase in protein synthesis in the lymphocytes cultured in the presence of PPD, and suggested specific incorporation of label into CY-and y-globulins. Wilson and Turk have reported the synthesis of specific proteins, including a- and ‘yglobulins, in the lymph nodes of guinea pigs contact-sensitized with oxazalone

Macromolecular Synthesis

497

(Wilson and Turk, 1968). They could not conclude whether the y-globulin synthesized were part of a cellular or humoral response to the oxazalone. Greaves and Roitt found no significant IgG production in response to PHA stimulation of human peripheral lymphocytes (Greaves and Roitt, 1968). Our failure to find at least IgG synthesis during heterograft sensitization in vitro was somewhat surprising, in view of the rather strong experimental evidence for the participation of antibody in heterograft reaction in vivo (Perper and Najarian, 1966; Lance et al., 1969). It may be that in our in vitro sensitization system those cells of the lymphoid line, i.e. plasma cells, which might have produced antibody for export, do not survive long in culture. Thus, while certain experimental and technical limitations should be placed on the quantitative interpretation of the studies presented here, one can at least set an upper limit on specific protein synthesis in the system; e.g. if new protein species are being produced by rat lymphocytes during sensitization to mouse fibroblasts in vitro, then they are being produced to a lesser extent than is the case for a humoral response demonstrated in lymphocytes from the same source as the cells undergoing such sensitization (Figs. 1 and 2), and in too minute quantities to be detected with the methods and conditions described in this paper. Acknowledgements- The authors wish to express their appreciation for the expert technical assistance of Miss Shoshana Levi and Mrs. Rivka Karakash. REFERENCES (1968) (1966) (1968) (1969) (1970) (1971) (1968) (1968) (1969) (1965) (1965) (L968) (1969) (1968) (1968) (1970) (1969) (1961) (1969) (1969) (1966) (1969) (1969) (1968) (1967)

Benacerraf B., Cancer Res. 48,1392. Benjamin T. L.,J. m&c. Biol. 16,359. Bennett B. and Bloom B., PTOC.Natl. Acad. Sci. 59,756. Berke G., Ax W., Ginsburg H. and Feldman M., Immunology 16,643. Berke G., Clark W. R. and Feldman M., submitted to Transplantation. Berke G., Sarid S. and Feldman M., manuscript in preparation. Brunner K. T., Mauel J., Cerottini J. C. and Chapuis B., Immunology 14,181. Church R. B. and McCarthy B. J., Biochem. Genet. 2,55. Dumonde D. C., Wolstencroft R. A., Panayi G. S., Mathew M., Morley J. and Howson W. T., Nature, Lond. 424,38. Gillespie D. and Spiegelman S.,J. molec.BioZ. 12,829. Ginsburg H. and Sachs L.,J. Cell. Camp. Physiol. 66, 199. Ginsburg H., ZmmunoZogy 14,62 1. Ginsburg H., Ax W. and Berke G., Excerpta Medica Intern. Cong. series 197. Pharmucobgical Treatment in Organ and Tissue Transplantation, p. 85. Granger G. A. and Williams T. W., Nature, Land. 2181253. Greaves M. F. and Roitt I. M., Clin. exp. Zmmunol. 3,393. Hansen J. N., Spiegelman G. and Halvorson H. O., Science X8,1291. Lance E. M., Levey H. R., Medawar P. B. and Ruszkiewicz M., Proc. Natl. Acad. Sci. 64,1356. Marmur J.,J_ m&c. Btil. 3,208. Melli M. and Bishop J. O.,]. mobc. Biol. 40, 117. Perlmann P. and Holm G., Adv. Immunol. 11,117. Perper R. J. and Najarian J. S., Transplantation 4, 700. Pick E., Krejeci J., Cech K. and Turk J. L., Immunology 17,741. Prendergast R. A., Silverstein A. M. and Parshall C. J., Transplantation 8,540. Ruddle N. H. and Waksman B. K.,J. exp. Med. 1281267. Shapiro A. L., Vinuela E. and Maize1 J. V., BBRC 28,815.

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and S. SARID

(1962) Sueoka N. and Cheng T.,J. molec. Biol. 4,161. (1968) Torelli K. L., Henry P. H. and Weissman S. M.,J. din. Invest. 47, 1083. (1969) Tyler R., Ginsburg H. and Everett N. B., Proc. of& Third AnnualLeucocyte Culture ConJ, p. 451. Appleton, New York. (1968) Wilson A. B. and Turk J. %., Immunochemistry5,33.