Cell-mediated inhibition of proliferation and activation of alloreactive cytotoxic lymphocytes:...

CELLULARIMMUNOLOGY 101,105-121 (1986)

Cell-Mediated Inhibition of Proliferation and Activation of Alloreactive Cytotoxic Lymphocytes: Maintenance of Response Potential of Precursors and Dissociation between Proliferation and Effector

Function of Activated Cytotoxic Lymphocytes’

ROBERT D. STOUT,*‘~ JILL SUTTLES,~,~,~ DENISE M. PERsIANI,t’3

AND ODDMUND BAKKE?~

*Department of Microbiology, College ofMedicine, East Tennessee State University, Johnson City, Tennessee 37614, and tProgram in Biophysics and Department of Biology, Rosenstiel Research

Center, Brandeis University, Waltham, Massachusetts 02254

Received February 27, 1986; accepted March 21, 1986

Adherent layers of macrophages (M&c) generated in vitro from splenic precursors inhibit lymphoproliferative responses to mitogen and to alloantigen without inhibiting the production of interleukin-2 (IL-2). Analysis of spleen cells stimulated for 48 hr in the presence of M&c indicated that both blastogenesis (increased cell mass) and expression of IL-2 receptors (7D4 determinants) were reduced. Analysis of BrdU incorporation (frequency of S-phase cells) and total cellular DNA revealed that the M&c inhibited the progression from G, to S phase of cell cycle. The Mb-c not only inhibited the proliferative response to alloantigen but also prevented the generation of alloreactive cytotoxic T cells. The M&c were shown not to inhibit CTL responses by eliminating the stimulators or by inactivating precursors or inducing suppressors. The M$-c were affecting the induction of CTL activity since the M&c did not affect the expression of cytolytic activity by activated CTL. The M&-c did inhibit the proliferation of the activated CTL, suggesting that although cytolytic activity can be expressed in G, phase of cell cycle, the activation of cytolytic activity in CTL-P may require a G, to S phase transition. The cells recovered from 5&y MLC incubated in the presence of MI&-C were fully capable of generating a subsequent CTL response. This is in contrast to cells recovered from unstimulated cultures (no Mb-c) which have lost the ability to generate CTL responses. The M&c therefore prevent the generation of CTL responses in a totally reversible fashion, so as to allow activation and proliferation of CTL-P which have been removed from the influence of the M&c. These observations are discussed in the context of the currently hypothesized role of tissue macrophages in mtcroenvironmental regulation. 0 1986 Academic Press, IX.

’ This research was supported by Grant CA-38408 from the National Cancer Institute, National Insti- tutes of Health.

’ To whom correspondence should be addressed. 3 J.S. was supported in part by National Institutes of Health Training Grant AI-07069. D.M.P. was

supported by National Institutes of Health Training Grant GM-08596. 4 Present address: Department of Microbiology, College of Medicine, East Tennessee State University,

Johnson City, Tenn. 376 14. ’ O.B. is a fellow of the Norwegian Cancer Society. Present address: Institute for Biophysics, University

of Trondheim, 7034 Trondheim NTH, Norway.

105

0008-8749186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any farm reserved.

106 STOUT ET AL.

INTRODUCTION

Although tissue macrophages are usually studied in the context of their immuno- stimulatory role (1, 2), resident splenic macrophages have been shown to express strong cytostatic or immunosuppressive activity during a variety of chronic perturba- tions of the hematopoietic system, such as cancer (3-9), muscular dystrophy (lo), and some chronic microbial infections ( 1 l- 14). In contrast to the suppressive effector function of peritoneal macrophages, the effector mechanism of the resident splenic macrophages does not appear to be dominated by the secretion of toxic and nontoxic products (4, 10, 12, 13, 15-21). The resident splenic macrophages are apparently capable of inhibiting cell proliferation by mechanisms that do not involve secretion of significant (detectable) quantities of proteases, peroxides, prostaglandins, or thymidine ( 12, 13, 16, 2 1) and therefore are more likely to be involved in microenvironmental regulation than in systemic regulation. Resident splenic and bone marrow macrophages are known to play a key role in hematopoietic microenvironmental regulation (22-24). However, the influence of such a microenvironmental regulatory function on immune responses would not be expected to be easily detectable in cultures of dispersed spleen cells unless, perhaps, excessive numbers of the resident macrophages had accumulated in the spleen, such as occurs in the hyperplastic spleens of some tumor-bearing animals (6-9). In such cases, or in experimental situations wherein the macrophage content is artificially increased, the immune responsiveness of the spleen cell culture is dramatically reduced (9, 19, 25-27). Despite the obvious biomedical relevance, the nature of the immunoinhibitory effect of these resident splenic macrophages is still unresolved.

Adherent layers of macrophages can be generated in vitro from either bone marrow or spleen cell populations (9,22-25,28-32). These macrophages can be induced to express Ia and to produce interleukin- 1 (IL- 1)6 and they can be activated to express a strong cytostatic effect on the proliferation of normal and neoplastic cells (9,25,28- 32). This inhibitory activity has been shown to be independent of the secretion of prostaglandins, peroxides, or thymidine by the macrophages, to be nontoxic, and to be completely reversible (32). The in vitro generated splenic macrophages have been demonstrated to inhibit lectin-induced lymphocyte proliferation without inhibiting the production and secretion of IL-2 lymphokines (3 1). In the present report, data are presented which demonstrate that although the macrophages do not inhibit IL-2 production by stimulated lymphocytes, they do inhibit the cell cycle progression of the lymphocytes from Gi to S phase, the expression of IL-2 receptors, and the differentiation or activation of cytotoxic T lymphocytes (CTL) in allogeneic mixed lymphocyte cultures.

MATERIALS AND METHODS

Mice. Inbred female C57B1/6J, DBA/2J, and CBA/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine). Only mice between the ages of 7 and 14 weeks were used for experimentation.

6 Abbreviations used: BrdU, bromodeoxyuridine; CAS-CM, conditioned medium from 24-hr cultures of Con A-stimulated spleen cells; Con A, concanavalin A; CTL, cytotoxic T lymphocyte; D’PBS, Dulbec~o’s phosphate-buffered saline; PBS, fetal bovine serum; IL, interleukin; LU, lytic unit; MLC, mixed lymphocyte culture; m&c, adherent macrophages generated from nonadherent precursors in culture.

REVERSIBLE INHIBITION OF T-CELL CYTOTOXIC RESPONSES 107

Cell preparation. The methods for generating monolayers of splenic macrophages have been recently described in detail (32). Briefly, spleen cells were suspended to 5 X 1 O6 cells/ml in RPM1 1640 containing L-glutamine (GIBCO, Grand Island, N.Y .) and supplemented to 5% v/v with heat-inactivated fetal bovine serum (FBS; GIBCO), 10 mM Hepes (GIBCO), and 50 pg/ml gentamicin sulfate (Sigma Chemical Co., St. Louis, MO.) and dispensed in 2-ml aliquots into Costar 24-well cluster plates (Cam- bridge, Mass.). The cultures were incubated for 5 days at 37°C and washed 3X with warm Dulbecco’s phosphate-buffered saline (D’PBS) to remove nonadherent cells. Based on counts of representative areas, the wells contained approximately l-4 X 1 O5 adherent cells which were predominantly Thy 1 -, MAC- 1 +, FcR’ macrophages (32). The macrophages were treated with 250 ~1 of 25 pg/ml mitomycin C (Sigma Chemi- cal Co.) for 30 min at 37°C after which they were washed 3X prior to use. Concana- valin A (Con A; Miles Laboratories, Elkhart, Ind.)-stimulated lymphoblasts were obtained by culturing 4 X lo6 spleen cells/ml for 48 hr with 2 pg/ml Con A. In some experiments the macrophages were preactivated (32) by coculture with 4 X lo6 spleen cells plus 2 pg/ml Con A for 18-24 hr. The culture wells were then washed thoroughly with D’PBS containing 0.05 Mcu-methylmannoside prior to mitomycin C treatment. Interleukin-2 (IL-2) levels in 24-hr culture supernates were assayed using the IL-2- dependent CTLL-2 cell line (33) and the growth assay which has been described previously (32, 34).

Flow cytometric analyses. Cells expressing receptors for IL-2 or cells expressing the L3T4 glycoprotein were detected by incubating 2 X IO6 cells for 30 min at room temperature with 50 ~1 of a 1:5 dilution of culture supernates of the 7D4 hybridoma (35) or the GK 1.5 hybridoma (36), respectively. The cells were washed and incubated for another 30 min in 50 ~1 of a 1:20 dilution of directly fluoresceinated mouse anti- rat K chain monoclonal antibody (Becton-Dickinson Immunocytometry Systems, Mountain View, Calif.). The cells were washed through a cushion of FBS and analyzed on a Becton-Dickinson FACS 420 with a Consort 30 data analysis system. The flow cytometric system, filtration, laser, and data handling have been described in detail recently (32). Cells expressing Thy 1.2 or Lyt 2 determinants were detected by labeling with directly fluoresceinated monoclonal anti-Thy 1.2 (32, 37) or directly fluoresceinated monoclonal anti-Lyt 2 (Becton-Dickinson Immunocytometry Sys- tems), respectively.

Cell cycle analysis using mithramycin and BrdU (both from Sigma Chemical Co.) have been described previously (32). Briefly, cells were pulsed for 30 min at 37°C with lo-’ M BrdU, washed 3X, and fixed in 70% ethanol. For BrdU analysis, the fixed cells were washed, incubated for 30 min at room temperature in 2 NHCl, and washed with PBS until a neutral pH was obtained (three-five washes). The cells were then incubated with a 1: 10 dilution of fluoresceinated anti-BrdU monoclonal antibody (Becton-Dickinson Immunocytometry Systems) for 30 min, washed, and analyzed. For total DNA content, fixed cells were incubated with 100 pg/ml mithramycin for 2-3 hr at 4°C in the dark after which they were analyzed as previously described (32). To quantitate total protein content of the cells, the fixed cells were incubated for 1 hr with 20 pg FITC (Sigma Chemical Co.) at 4°C. The cells were washed at least 2~

through a cushion of FBS prior to analysis. Mixed lymphocyte cultures. Spleen cells to be used as stimulators for mixed lym-

phocyte cultures (MLC) were incubated at 5 X 10’ cells/ml in 25 pg/ml mitomycin C for 30 min at 37°C and washed 3X prior to use. Responder and stimulator spleen

108 STOUT ET AL.

TABLE 1

Normal IL-2 Production in the Absence of Proliferation in Lymphocyte Cultures Held Static by Mg-c

Culture cpm [‘H]Thymidine’ IL-2 production* Incorporation (X 10-j) (Units/ml)

Con A-stimulated 148 f 12 780 Con A + M&c-stimulated 8f 3 970 1” MLC 41+ 4 422 l”MLC+M&c 5a 3 393

a DBA/2 spleen cells (4 X 10’ per microwell) were stimulated with Con A or with mitomycin C-treated C57B1/6 spleen cells in the presence or absence of M+-c. After 3 days the cultures were pulsed for 4 hr with [3H]thymidine. Values represent arithmetic means of triplicate cultures + SD.

* DBA/2 spleen cells were stimulated with Con A or with mitomycin C-treated C57B1/6 spleen cells in the presence or absence of M&c for 24 hr after which supernates were harvested and assayed for IL-2 activity.

cells were cultured in 2-ml vol at 4 X 1 O6 cells/ml RPM1 1640 supplemented to 10% v/v with heat-inactivated FBS, 10 mM Hepes, and 5 X 1 Op5 2-mercaptoethanol. After 5 days, the cells were harvested and centrifuged over Lympholyte-M (Accurate Chemical and Scientific Co., Westbury, N.Y.) to reduce dead cells and debris. The cells were assayed for cytolytic activity and/or used as responders in a second MLC. The 2” cultures were supplemented with 10% supernate from 24-hr cultures of Con A-stimulated spleen cells (CAS-CM) (3 1,34) as a source of growth factors. a-Methyl- mannoside was added to 0.05 M to the CAS-CM to block residual Con A activity.

The El-4 (H-2b), P815 (H-2d), YAC-1 (H-2”) and BW 5 147.3 (H-2k) tumor cell lines were used as targets in cytolysis assays. The tumor cells were incubated for 30 min at 37°C with 10 &i 5’Cr (NaCr04; Amersham, Arlington Heights, Ill.) per lo6 cells in 100 ~1 culture medium. The cells were washed twice through FBS, resus- pended to lo5 cells/ml, and dispensed in loo-p1 aliquots to round-bottom microwell plates (Falcon Plastics, Oxnard, Calif.) containing 0.6-20 X lo4 MLC-derived lymphocytes. The plates were centrifuged lightly (5Og, 5 min), and incubated 3 hr at 37°C. The plates were then centrifuged at 5OOg for 10 min and 100 ~1 of supernate was mixed in 3 ml of Aquasol (New England Nuclear Corp., Boston, Mass.) and counted in a Beckman LS 7000. Specific 51Cr release was determined by the formula

% specific lysis = experimental cpm - spontaneous cpm

maximum cpm - spontaneous cpm x 100.

Maximum releasable cpm was determined by freezing and thawing the targets in a dry ice/ethanol bath 3X. For studies on response kinetics, the cytolysis titration data were converted into lytic units per lo7 cells as previously described (38). A lytic unit is defined as the number of cells required to effect 30% lysis of 1 O4 target cells in 3 hr. In those experiments in which cytolytic assays were run in the presence of cytostatic macrophages, the spontaneous release control was also run in the presence of the macrophages. Spontaneous release values were lower (20-30% reduction) in the presence of the macrophages.

RESULTS Inhibition of cellproliferation. Macrophages generated in vitro (M+c) from splenic

monocyte precursors can inhibit lymphocyte proliferation responses to lectin and to


alloantigen without inhibiting the production of IL-2 T-cell growth factors (29-3 1) (Table 1). To determine if the inhibitory effect of the cytostatic macrophages involved an arrest of the stimulated lymphocytes at a particular stage of cell cycle, spleen cells were stimulated with Con A for 48 hr in the presence or absence of the M&-c after which the lymphocytes were fixed and labeled with mithramycin. Flow cytometric analysis indicated that the majority of spleen cells stimulated in the presence of the M&c remained in Gi or early S phase of cell cycle (Fig. 1). The actual frequency of cells in S phase was determined by analyzing BrdU incorporation per cell. The cultures stimulated for 48 hr with Con A in the presence of M&c contained a reduced frequency of cells incorporating BrdU (7%) as compared to cultures stimulated in the absence of M&c (27%) (Fig. 2, Table 2).

Although in the presence of M&c the majority of spleen cells appear to remain in G, phase of cell cycle, a small increase in apparent size can be detected by forward angle light scatter. To determine if this change in light scattering characteristic is correlated with an actual change in cell mass, the cells were fixed and their intracellu- lar protein was labeled with FITC. The cells stimulated in the absence of M&c displayed two size populations, one with a mean fluorescence of 49 units and one with

FLUORESCENCE INTENSITY

(Mithramycid

FIG. 1. Cell cycle distributions of spleen cells stimulated in the presence of M$-c. Spleen cells were stimulated for 48 hr with Con A in the presence or absence of M+c. The cells were fixed and labeled with mithramycin. Profiles represent analyses of 1 x lo4 cells in each group using linear amplification.

110 STOUT ET AL.

LIl” Cl CO” A 0 jx_,

100 10’ lo2 103 104

FLUORESCENCE INTENSITY (SRdU)

FIG. 2. BrdU incorporation rates of spleen cells stimulated in the presence of M@-c. Spleen cells were stimulated for 48 hr with Con A in the presence or absence of Mg-c. The cells were pulsed for 30 min with BrdU before being fixed and labeled with fluoresceinated anti-BrdU. Profiles represent analyses of 1 X lo4 cells in each group using logarithmic fluorescence amplification.

a mean fluorescence intensity of 2 18 units (Fig. 3). In the presence of M&c, only one major size population could be resolved. This population had a mean fluorescence intensity of 34 units, which was twice the mean of the unstimulated spleen cells ( 15 units) (Fig. 3).

The expression of IL-2 receptors on those cells was examined by labeling the cells with the 7D4 rat anti-IL-2 receptor antibody. Stimulation of spleen cells for 48 hr in the presence of M&c resulted in a fourfold reduction in the frequency of 7D4+ cells (11%) compared to the frequency of 7D4+ cells (42%) detected after stimulation in the absence of M&c (Fig. 4, Table 2). This reduction in expression of IL-2 receptors was also indicated by a reduced ability of the cells to absorb IL-2 activity from a supernate containing IL-2 (3 1).

Inhibition of CTL generation. When spleen cells were stimulated with allogeneic cells in the presence of M+c, not only was the proliferative response inhibited (Table l), but no allospecific cytotoxic ,activity was generated (0% lysis at a 20: 1 E:T) (Fig. 5). This inhibition of CTL generation, like the M+c-mediated inhibition of cell proliferation (3 l), could not be overcome by the addition of lymphokines in CAS-CM


or in supemates of 4%hr 1” MLC. The presence of preactivated M&c during the 3- hr lytic assay of alloreactive CTL derived from a normal 5-day MLC did not cause a significant decrease in target cell lysis (Fig. 6). The macrophages themselves did not mediate significant lysis of the target cells during the 3-hr assay.

To determine if the failure to induce CTL activity was the result of rapid M&c- mediated elimination of the stimulators, the nonadherent cells harvested from a 5- day M&c-inhibited DBA/2J anti-C57B1/6J MLC were used as the sole source of H-2b stimulators in an MLC set with fresh DBA/2J spleen cells. The mitomycin C-treated nonadherent cells were capable of stimulating an anti-H-2b CTL response (Fig. 7).

Flow cytometric analysis indicated that there was only a small difference in the frequency of Thy l+ cells in populations harvested from 5-day MLC stimulated in the presence of M&c compared to control MLC (Table 3). When size (forward light scatter) was included in the analysis, the large cells (any cell greater than 32 units, which would include lymphoblasts but not exclusively) from M&c-inhibited MLC were found to contain only half as many Thy If cells as control MLC (Fig. 8, Table 3). More notably, the large cell fraction of the M&c-inhibited MLC population was nearly devoid of Lyt 2+ cells (Fig. 8). In contrast, the small cell fraction of M+c- inhibited MLC and of control MLC populations contained nearly identical frequencies of both Thy l+ and Lyt 2+ cells (Table 3).

Since a significant frequency of Lyt 2+ cells could be detected in the M&c-inhibited MLC populations even though no CTL effector activity was evident, the question was raised as to whether the Lyt 2+ population could express CTL-p activity. Cells harvested after a 5-day MLC in the presence of M&c were washed and restimulated with the same allogeneic cells in the absence of M&c. Lytic activity became detectable 3 days after restimulation and increased thereafter (Fig. 9). The response kinetics and intensity were similar to those of the primary MLC of freshly prepared spleen cells (Fig. 9). In all cases, the observed cytolytic responses were allospecific in that neither P8 15 nor BW5 147.3 targets were lysed by C57B1/6-stimulated effecters. The EL-4 and P8 15 tumor cell lines carried in our laboratory (R.D.S.) have been shown to be insensitive to NK-mediated lysis (39).

The above experiments indicated that M&c were capable of inhibiting the generation of CTL activity but did not address the question of whether M&c could inhibit cytolytic activity after the CTL had been activated. To address this question, the responding lymphocytes from a 5-day 1” MLC were restimulated in the presence or

TABLE 2

Inhibitory Effect of M&c on Frequency of S-Phase and 7D4-Positive Cells in Con A-Stimulated Cultures

Cell population” 70 7D4+ % S-Phase

Spleen, unstimulated 2-4 2-3 Spleen + Con A 36-42 27-34 Spleen + Con A + M&c 8-13 6-9

’ Spleen cells were stimulated with Con A for 48 hr, washed, and analyzed for 7D4 reactivity or for BrdU incorporation (S-phase cells). Values are based on analysis of lo4 cells and represent range of values obtained in three experiments.

112 STOUT ET AL.

250 Bl Con A+hW

250 a con A

0 .-i 100 10’ 102 103 10’

FLUORESCENCE INTENSITY (FITC)

FIG. 3. Analysis of cellular protein content of spleen cells stimulated in the presence of M$-c. Spleen cells were stimulated for 48 hr with Con A in the presence or absence of M&-c. The cells were fixed and labeled with FITC. Profiles represent analyses of 1 X lo4 cells in each group using logarithmic fluorescence amplification.

absence of M+c. After l-3 days of culture, the cells were harvested, washed, and assayed for [3H]thymidine incorporation and cytolytic activity. The M&c caused a 7080% reduction in the proliferative activity of the 2” MLC population within 24 hr of culture onset and maintained that level of cytostasis during the subsequent 48- hr culture period (Fig. 10). Analysis of forward angle light scatter intensity (cell size) of the Lyt 2-positive cells in the populations revealed that the Lyt 2-positive cells had decreased in size in the presence of M&c (Fig. 11). In contrast, the M+c did not cause a similar decrease in cytolytic activity during the first 48 hr of culture (Fig. 12). Analysis of BrdU uptake and DNA content (mithramycin labeling) of the 2” MLC populations 48 hr after restimulation indicated that the M&c had caused an arrest of the cycling population in G1 (Table 4). However, a similar reduction was not observed in expression of IL-2 receptor determinants recognized by the 7D4 monoclonal antibody (Table 4).

DISCUSSION

We have previously demonstrated that adherent macrophages generated from nonadherent precursors in spleen cell cultures can be activated to express potent cyto-


static activity against tumor cells and continuous cell lines (29-32). In contrast to peritoneal macrophages (15- 19), the cytostatic mechanism of the M&c does not involve prostaglandins, peroxides, or thymidine secretion (2 1, 29, 32). Maximum tu- morostatic activity can be expressed by these macrophages within 9 to 18 hr after addition of activating agents such as mitogen-stimulated lymphocytes (32). The activating lymphocytes themselves are subject to the cytostatic effect of the macrophages. The presence of the M@-c during stimulation of lymphocytes with mitogen or alloantigen results in inhibition of the lymphoproliferative response (29-3 1). The cytostatic effect of the M&c involves an inhibition of progression of the cells from G, to S phase of the cell cycle as determined by analysis of BrdU uptake and cellular DNA content. This cell cycle arrest has been shown to be totally reversible when tested on cell lines (32) and when tested on CTL-P (Fig. 9). Classical blastogenesis also seems to be inhibited as assessed by total protein content and by forward light scatter analysis (e.g., Fig. 8). Some early G,-associated events in lymphocyte activation are not inhibited by the M&c. For example, the M&c do not inhibit the production of IL-2 lympho-

200 A) Unslimulated

200 C) Con A

FLUORESCENCE INTENSITY (7D4)

FIG. 4. Expression of 7D4 determinants on spleen cells stimulated in the presence of M&c. Spleen cells were stimulated for 48 hr with Con A in the presence or absence of M&c. The cells were labeled with 7D4 and fluoresceinated anti-rat kappa. Profiles represent analyses of 1 X lo4 cells in each group using logarithmic fluorescence amplification.

114 STOUT ET AL.

+M0 0 0 Q 7

2.5 5 10 20

EFFECTOR : TARGET RATIO

FIG. 5. Inhibition ofCTL generation by M&c. DBA/2J spleen cells were cultured with C57B1/6J stimulators for 5 days in the presence (0) or absence (0) of M&c prior to assay against EL-4 targets. Each point represents the average of triplicate assays.

kines (31). Therefore, the lymphocytes are receiving and responding to all signals required for IL-2 production (34).

The lymphocytes stimulated in the presence of M&c do display a sharp reduction in activation-associated expression of IL-2 receptors, as detected by 7D4 monoclonal antibody (35). This reduced reactivity with 7D4 antibody appears to reflect a reduction in functional IL-2 receptors since lymphocytes activated in the presence of M&c have been shown to be deficient in their ability to absorb IL-2 activity from a standard lymphokine preparation (3 1). The reduction of both 7D4 binding sites and IL-2 binding sites, which are not sterically incompatible (35), makes it unlikely that the M&c

40

v) 73 2 30

2 G L 20. fn d?

10.

2.5 5 10 20


FIG. 6. Lack ofM+-c effect on CTL lytic activity. DBA/ZJ anti-C57B1/6J CTL activity, generated during a S-day MLC, was assayed against EL4 targets in the presence (0) or absence (0) of M&c. The M&c were preactivated by 20-hr coculture with spleen cells plus Con A prior to the lytic assay. Activated M&c, without added DBA/2J effecters, did not cause EL-4 lysis at approximately a 1:l ET (A). Each point represents a mean of triplicate assays. Vertical lines indicate SD.


40-

0 2.5 5 10 20


FIG. 7. Presence of H-2b stimulator activity in cell population harvested from 5-day MLC incubated in the presence of M$-c. Cells harvested from a 5day DBA/2J anti-C57Bl/6J MLC, incubated in the presence of DBA/ZJ M&J-C, were treated with mitomycin C (0) or treated with anti-Thy 1 + C’ prior to mitomycin C (0) and used as the stimulator population with fresh DBA/2J spleen cells in a 1” MLC. After 5 days, the cells were assayed for lytic activity against EL-4 targets. Each point represents the average of triplicate assays.

are producing a factor that blocks the IL-2 receptor. The existence of a receptor block- ing factor is essentially precluded by the observations that the M&c can inhibit the proliferation of activated lymphocytes in 2” MLC without effecting a proportionate decrease in IL-2 receptor expression (Table 4), that the M+c do not produce detectable factors which inhibit lymphocyte proliferation (2 1, 29, 32), and that the supernates of lymphocytes stimulated in the presence of M&c contain normal levels of IL-

TABLE 3

Surface Phenotype of 5-Day 1” MLC Populations Generated in the Presence of M&c”

Frequency (%) in population

Phenotype 1” MLC control 1” MLC + M&c

Small Thy 1’ 25 29 Large Thy I+ 36 19

Total Thy+ 61 48

Small L3T4+ 15 16 Large L3T4+ 21 16

Total L3T4+ 36 32

Small Lyt 2+ Large Lyt 2+

Total Lyt 2+

13 17 30

14 3

17

a Cells were harvested from 5day 1’ MLC generated in the presence or absence of M&c. The cells were labeled with anti-Thy 1, anti-L3T4, or anti-Lyt-2 and analyzed for frequency of positive cells within two size populations. The criteria for size and fluorescence windows are displayed in Fig. 8. Frequencies are derived from analyses of IO4 cells in a typical experiment.

STOUT ET AL. 116

30 60

I ‘0 L “0

0 30 60 FORWARD LIGHT SCATTER

(CELL SIZE)

FIG. 8. Thy 1, Lyt 2, and cell size analysis of S-Day MLC populations. DBA/ZJ anti-CS7B1/6J MLC, cultured for 5 days in the presence (c, d) or absence (a, b) of DBA/ZJ M&c, were harvested and labeled with anti-Thy 1 (a, c) or anti-Lyt 2 (b, d). The profiles of fluorescence versus cell size of IO4 cells are displayed. The criteria for small (sector 1) and large (sector 2) fluorescent cells used as the basis for the percentages in Table 3 are also displayed.

2 activity, as assayed on IL-Zdependent cell lines (3 1)-i.e., do not contain factors which inhibit IL-2 binding to the IL-Zdependent cell lines. In fact, the action of the M&c does not appear to be directly related to IL-2 or IL-2 receptors. After being activated, the M&c can inhibit the proliferation of a variety of cell lines, including monocytic tumor cell lines (e.g., P8 15) which do not require T-cell growth factor and do not express IL-2 receptors (32).

The common event in the cytostatic effect of the M&c on all primary and continuous cell lines tested is an inhibition of cell cycle progression (32). It is therefore proba- ble that the inhibition of cell cycle progression is the primary effect of the M&c on stimulated lymphocytes and that the reduced expression of IL-2 receptors on cells activated in the presence of M&c is a result of cell cycle arrest. It has been demonstrated that the activation of IL-2 production involves a Go to G1, progression (40, 4 1) and that IL-2 augments the expression of IL-2 receptors (42,43). The expression


DAYS OF CULTURE

FIG. 9. CTL-P activity in S-day MLC populations cultured in the presence of M&c. DBA/2J spleen cells were cultured with C57Bl/6J stimulators in the presence (0---) or absence (a-) of DBA/2J M&c for 5 days. The cells were harvested and cultured for an additional l-5 days in the presence of 10% CAS-CM and mitomycin C-treated C57Bl/6J stimulators. As a control for 1” MLC reactivity, fresh DBA/ZJ spleen cells were cultured under identical conditions (O--). The titration of lytic activity against EL-4 for each day was converted into lytic units. No lytic activity (~10 LU/lO’ cells) was detected using P815 or BW5 147.3 targets.

of critical levels of IL-2 receptors must therefore be an event which occurs after the onset of IL-2 production and after the G,, to G,, transition (40,41). In this context, it would appear that the M&c may inhibit cell cycle progression at a stage subsequent to the onset of IL-2 production and prior to the expression of IL-2 receptors. (One alternative explanation might be that IL-2 production begins prior to the expression of cytostatic activity by the macrophages. However, the M&c do not alter, or some- times augment, the production of IL-2 over a 4%hr period, even when the M&c are “preactivated” (3 1,32).

Since the M&c are known to inhibit cell proliferation, the question was raised as to whether the cytostatic effect, in itself, could prevent the generation of CTL effecters. Several investigators have reported that cell proliferation is not required for the generation of allo-CTL responses (44-46). However, those experiments were designed to test whether inhibitors of mitosis or DNA synthesis (S-phase or mitotic poisons) prevented CTL activation. The question as to whether a G1 to S phase transition (initia- tion of cell cycling) was required for CTL activation was not addressed. As discussed above, the expression of IL-2 receptors is a transitional event in lymphocyte activation, occurring in late G, or early S phase (40-43). Although there is some contro- versy over how many of what type of factors are required for generation of CTL responses, all investigators seem to agree that IL-2 is required for the generation of CTL responses (47-50). Inhibition of IL-2 production or availability (5 1) or inhibition of IL-2 receptor expression (52, 53) results in an inhibition of CTL responses. The G, arrest which the M&c have been shown to effect, with the associated reduc-

118 STOUT ET AL.

103- 0 1 2 3

DAYS IN CULTURE

FIG. 10. Inhibition ofcell proliferation in established MLC by M&c. Cells harvested from a 5day DBA/ 25 anti-C57Bl/6J MLC were restimulated with C57B1/6J splenocytes in the presence of 10% CAS-CM and in the presence (0) or absence (0) of DBA/ZJ M&c. The cells were harvested l-3 days later, passed through Lympholyte-M, and 1 X 10’ viable cells were pulsed for 3-4 hr with [‘Hlthymidine. Averages of triplicate samples and standard deviation are displayed.

tion in IL-2 receptor expression, could therefore result in an inhibition of CTL generation. This also implies that contrary to earlier reports (44-46), at least one round of cell cycle activity may normally occur in the induction of CTL activity.

Whether the G, arrest is the only effect exerted by the M&c that is relevant to inhibition of CTL generation is not yet known with certainty. However, the M&c do not have a demonstrable effect on cytolytic activity per se nor do they have an effect on the continued expression of cytolytic activity of activated CTL during a 48-hr

B ii 1 i 8

0

0 100 200 LIGHT SCATTER

(CELL SIZE)

FIG. I 1. Size distribution of Lyt 2-positive MLC lymphocytes after 48 hr culture with M&c. Cells harvested from a 5-day DBA/2J anti-C57Bl/6J MLC were restimulated with C57Bl/6J splenocytes plus 10% CAS-CM in the presence (light line) or absence (dark line) of M&-c. After 48 hr culture, the cells were harvested and labeled with fluoresceinated anti-Lyt 2. The forward light scatter histograms of 8000 Lyt 2-positive lymphocytes are displayed. Dead cells and debris (scatter below channel 58) were excluded from the cell count and display.


a) 2’MLC CONTROL

70 -

60 -

cn 5o 5

cl 40-

0

4

!J

30-

ae 20-

10 -

I 1.2 2.5 5 10 20

70 -

60 -

50 -

40 -

30 -

20 -

10 -

b) PMLC on M’&c


FIG. 12. Effect of M&c on activated CTL. Cells harvested from a 5-day DBA/2J anti-C57B1/6J MLC were restimulated with C57B1/6 splenocytes in the presence of 10% CAS-CM and in the presence (b) or absence (a) of DBA/ZJ M&-c. The cells were harvested l-3 days later, passed through lympholyte M and assayed for cytolytic activity against EL-4 targets. The heavy line (do) represents the lytic activity at the time of 2” stimulation. The averages of triplicate assays for Day 1 (dl; 0), Day 2 (d2; A), and Day 3 (d3,O) are displayed. No lytic activity against P8 15 was observed at 20: I effector to target ratio.

period, even though the proliferation of the activated CTL is inhibited within the first 24 hr. The M&c do not appear to be inducing suppressor cells, inactivating or delet- ing CTL-P, or otherwise instilling a tolerant state since the recovered cells, once removed from the M&c, can respond to alloantigen with an allospecific CTL response. The M+c do not prematurely terminate the response by eliminating the stimulators since the stimulators can be demonstrated to be actively present in MLC cultures incubated 5 days with M&c.

The maintenance of the potential to respond in the absence of a response during in vitro culture is itself of interest. ZH vitro culture of lymphocytes in the absence of allostimulation results in the loss of the potential to respond. This loss of responsiveness has been hypothesized to be due to a disruption of regulatory equilibrium of T helper, inducer, and suppressor activities (54). Macrophages are known to be a key

TABLE 4

Effect of M&x on Frequency of S-Phase and 7DCPositive Cells in Activated MLC

% 7D4+ % S-Phase

Cell population’ I II III I II III

2” MLC 17 17 20 15 18 19 2” MLC + M+c 12 18 17 5 4 7

’ Lymphocytes were harvested from a 5day DBA/2J anti-C57Bl/bJ MLC and were restimulated with mitomycin-C-treated C57B1/6J spleen cells plus 10% CAS-CM for 48 hr in the presence or absence of activated M@-c. Values obtained in three trials are presented and are based on analysis of l-2 X lo4 cells.

120 STOUT ET AL.

component of the hematopoietic microenvironment that regulates homing, proliferation, and differentiation of hematopoietic cells in bone marrow (22-24). Macro- phages are also a component of bone marrow adherent layers generated in vitro which are considered to be an in vitro model of the hematopoietic microenvironment (22- 24). Those adherent layers have been shown to maintain hematopoietic activity in bone marrow cultures over long culture intervals and to exert a cytostatic effect that appears functionally similar to that of M&c (23, 32). Although the in vivo cytostatic effects of resident splenic macrophages have been obvious only during chronic dis- eases (3- 14), there is indirect evidence that a cytostatic influence is exerted in the spleen during in vivo allograft responses. The CTL isolated from spleen cells after in vivo allostimulation are not lymphoblasts and are not proliferating (46). These characteristics are in sharp contrast to the large proliferating CTL generated during in vitro responses (46) but are very similar to the smaller nonproliferating CTL obtained by culturing in vitro activated CTL on M&c. Whether resident splenic macrophages are responsible for the differences in T-cell blastogenesis observed in spleen cells after in vivo versus in vitro stimulation remains to be resolved. These studies do raise the interesting possibility that splenic macrophages may provide intraorgan (microenvironmental) immunoregulation which limits clonal expansion in select areas of the spleen and favors activation of emigrating T cells.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of Mary Carrier and Barbara Carter-Hamm and the secretarial assistance of Janette Taylor and Barbara Stokes.

REFERENCES

1. Unanue, E. R., Annu. Rev. Zmmunol. 2,395, 1984. 2. Mizel, S. B., Zmmunol. Rev. 63,5 1, 1982. 3. Elgert, K. D., and Farrar, W. L., J. Zmmunol. 120, 1345, 1978. 4. Kruisbeek, A. M., Zijlstra, J., and Zurcher, C., Eur. J. Zmmunol. 8,200, 1978. 5. Kennard, J., and Zolla-Pazner, S., J. Zmmunol. 124,268, 1980. 6. Berman, J. E., and Zolla-Pazner, S., Cell. Zmmunol. 87, 137, 1984. 7. Nutter, R. L., Gridley, D. S., Slater, J. M., and McMillan, P. J., Anat. Rec. 197,363, 1980. 8. Yamagishi, H., Pellis, N. R., Macek, C., and Kahan, B. D., Eur. J. Cancer 16, 1417, 1980. 9. Shibata, Y., Shibuya, E., and Ishida, N., Cell. Zmmunol. 85,45, 1984.

IO. Kline, K., andsanders, B.G., J. Zmmunol. 132,2813, 1984. 1 I. Ottesen, E. A. J. Zmmunol. 123,1639, 1979. 12. Klimpel, G. R., Okada, M., and Henney, C. S., J. Zmmunol. 123,350,1979. 13. Suzuki, Y., and Kobayashi, A., Cell. Zmmunol. 85,4 17, 1984. 14. Brett, S. J. Cell. Zmmunol. 89, 132, 1984. 15. Hume, D. A., and Weidemann, M. J., “Mitogenic Lymphocyte Transformation,” Chap. 2. Elsevier/

North-Holland, Amsterdam/New York, 1980. 16. Gery, I., and Davies, P., In “Biology of the Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim,

Eds.), p. 347. Academic Press, New York, 1979. 17. Allison, A. C., Zmmunol. Rev. 40,3, 1978. 18. Stadecker, M. J., Calderon, J., Kamovsky, M. L., and Unanue, E. R., J. Zmmunol. 119, 1738, 1977. 19. Adams, D. O., and Hamilton, T. A., Anna Rev. Zmmunol. 2,283, 1984. 20. Bixler, J. S., Jr., and Booss, J., J. Zmmunol. 127, 1294, 198 1. 21. Liu, Y-N., Uchida, T., Ju, S-T., and Dorf, M. E., Cell. Zmmunol. 94,49, 1985. 22. Rich, I. N., and Kubanek, B., In “Hematopoietic Stem Cell Physiology” (E. P. Cronkite, N. Dainiak,

R. 0. McCatTrey, J. Palek, and P. J. Quesenberry, Eds.), p. 283, Liss, New York, 1985.


23. Dexter, T. M., Spooncer, E., Toksoz, D., and Lajtha, L. G., In “Control of Cellular Division and Development,” Part B (D. Cunningham, E. Goldwasser, J. Watson, and C. F. Fox, Eds.), p. 67. Liss, New York, 1980.

24. Wang, S-Y., Castro-Malaspina, H., and Moore, M. A. S., J. Zmmunol. 135, 1186, 1985. 25. Unanue, E. R., Adv. Zmmunol. 31, 1, 1981. 26. Gershon, R. K., Contemp. Top. Zmmunobiol. 3, 1, 1974. 27. Folch, H., and Waksman, B. H., Cell. Zmmunol. 9, 12, 1973. 28. Warren, M. K., and Vogel, S. N., J. Zmmunol. 134,982, 1985. 29. Stout, R. D., and Fisher, M., J. Zmmunol. 130, 1573, 1983. 30. Stout, R. D., and Fisher, M., J. Zmmunol. 130,1580, 1983. 3 1. Stout, R. D., Cell. Zmmunol. 85, 168, 1984. 32. Stout, R. D., Cell. Zmmunol. 96,83, 1985. 33. Baker, P. E., Gillis, S., and Smith, K. A., J. Exp. Med. 149,273, 1979. 34. Bykowsky, M. J., and Stout, R. D., J. Zmmunol. 130,2093, 1983. 35. Malek, T. R., Robb, R. J., and Shevach, E. M., Proc. Natl. Acad. Sci. USA 80,5694, 1983. 36. Dialynas, D. P., Quan, Z. S., Wall, K. A., Pierres, A., Quintas, J., Loken, M. R., Pierres, M., and Fitch,

F. W., J. Zmmunol. 131,2445, 1983. 37. Marshak-Rothstein, A., Fink, P., Gridley, T., Raulet, D. H., Bevan, M. J., and Gefter, M. L., J. Zmmu-

nol. 122,2491, 1979. 38. Herberman, R. G., Ortaldo, J. R., and Timonen, T., Methods Enzymol. 79,477, 198 1. 39. Suttles, J., Schwarting, G. A., and Stout, R. D., J. Zmmunol. 136, 1986. 40. Kristensen, F., Walker, C., Bettens, F., Joncourt, F., and DeWeck, A. L., Cell. Zmmunol. 74, 140,

1982. 41. Bettens, F., Kristensen, F., Walker, C., and DeWeck, A. L., Eur. J. Zmmunol. 12,948, 1982. 42. Welte, K., Andreelf, M., Platzer, E., Holloway, K., Rubin, B. Y., Moore, M. A. S., and Mertelsmann,

R., J. Exp. Med. 160, 1390, 1984. 43. Reem, G. H., and Yeh, N.-H., J. Zmmunol. 134,953,1985. 44. Hengst, J. C. D., Chan, K. K., and Mitchell, M. S., Cell. Zmmunol. 90,281, 1985. 45. MacDonald, H. R., and Lees, R. K., J. Exp. Med. 150, 196, 1979. 46. Kimura, A. K., and Wigzell, H., J. Zmmunol. 130,2056, 1983. 47. Erard, F., Corthesy, P., Nabholz, M., Lowenthal, J. W., Zaech, P., Plaetinck, G., and MacDonald,

H. R., J. Zmmunol. 134,1644,1985. 48. Erard, F., Corthesy, P., Smith, K. A., Fiers, W., Conzelmann, A., and Nabholz, M., J. Exp. Med. 160,

584, 1984. 49. Mannel, D. N., Falk, W., and Droge, W., J. Zmmunol. 130,2508, 1983. 50. Garman, R. D., and Fan, D. P., J. Zmmunol. 130,756, 1983. 5 1. Gunther, J., Haas, W., and von Boehmer, H., Eur. J. Zmmunof. 12,247, 1982. 52. Glasebrook, A. L., and MacDonald, H. R., J. Zmmunol. 130, 1552, 1983. 53. Malek, T. R., Ortega, G., Jakway, J. P., Chan, C., and Shevach, E. M., J. Zmmunol. 133, 1976, 1984. 54. Weksler, M. E., Moody, C. E., Jr., and Kozak, R. W., Adv. Zmmunol. 31,271, 1981.

Cell-mediated inhibition of proliferation and activation of alloreactive cytotoxic lymphocytes:...

Documents

Transcript of Cell-mediated inhibition of proliferation and activation of alloreactive cytotoxic lymphocytes:...