Blocking of the induction and expression of immunologically functional T lymphocytes by rat antiactivated T-cell serum

CELLULAR

IMMUNOLOGY

73, 3 I l-323 (1982)

Blocking of the Induction and Expression of Immunologically Functional T Lymphocytes by Rat Antiactivated T-Cell Serum RITA EFFROS,*JOHNFAN,* JOHNHISERODT,*STEVEKESSLER,? IRWIN ScHER,t AND BENJAMINBONAVIDA* *Department

of Microbiology and Immunology, UCLA School of Medicine, University Los Angeles, California 90024, and fDepartment of Immunology, Naval Medical Research Institute, Bethesda, Maryland 20014

of California,

Received May 21, 1982; accepted August 9, 1982 The preparation of a xenogeneic rat anti-mouse CTL serum, RAT*, which potently inhibits CTL function in the absence of complement, has recently been described. In the present study, this serum is further characterized. The membrane antigens recognized by RAT* were shown by FACS analysis to be detected most intensely on CTL, but are also present in lesser amounts on splenic T cells, thymocytes, and B cells. The T-cell proliferative response induced by Con A, PHA, TCGF, and modified self were all inhibited by RAT*, but no effect was seen on LPSinduced B-cell proliferation. In addition, the in vitro induction of secondary CTL was not observed in the RAT&-treated cultures. Finally, the effect of RAT* pretreatment is shown to be irreversible. It is proposed that RAT* serum reacts with receptors on T cells which are intimately involved in the “activation” step during the induction or expression phases of the response.

INTRODUCTION Inhibition by serological reagents of T cell-mediated cytotoxicity at the effector cell level has been difficult to demonstrate (1-6, 1I), and therefore such inhibitory sera should serve as valuable probes to dissect the mechanism of cytotoxicity. These reagents can be used to analyze the surface structures involved in cellular recognition and/or function. We have, therefore, recently generated a xenoantiserum, RAT*’ serum, which inhibits the cytotoxic T-cell function in the absence of complement. The blocking activity was shown to be at the level of the effector T cells. Further characterization of RAT* serum revealed that blocking was at the postbinding level (11). In addition, RAT* serum was not idiotype specific and inhibited allosensitized CTL and H-2-restricted influenza-specific CTL of various mouse strains (1). These data suggestedthat the cell-surface determinants recognized by RAT* serum were probably not directly involved in antigen recognition, but rather in the induction ’ Abbreviations used: ADCC, antibody-dependent cellular cytotoxicity; C, complement; CMC, cellmediated cytotoxicity; CML, cell-mediated lympholysis; Con A, concanavalin A; CTL, cytotoxic T lymphocyte; FACS, fluorescence-activated cell sorter; MASH, multiple automated sample harvester; MLC. mixed lymphocyte culture; MLR, mixed lymphocyte reaction; NRS, normal rat serum; PEL, peritoneal exudate lymphocytes; PHA, phytohemagglutinin; RAT*, rat antiactivated T cell; TCGF, T-cell growth factor. 311

312

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ET AL.

or effector phase of cytotoxic function. Accordingly, it would be predicted that RAT* serum could interfere with the activation of T cells by mitogens and antigens. The present study was undertaken to examine the effect of RAT* serum on mitogen and TCGF-induced proliferation, and the induction of secondary CTL. Furthermore, the reversibility of the inhibition of the functional activity of CTL following exposure to RAT* serum was examined. MATERIALS

AND METHODS

Injluenza Virus Immunization The Influenza A viruses used in the present study were A/PR8 [A/PR/8/34 (HONl)] and [A/Hong Kong/g/68 (H3N2)]. They were grown from stocks generously provided by Dr. Peter C. Doherty (Wistar Institute, Philadelphia, Pa.) in Day 10 embryonated hens’ eggs, from which the allantoic fluid yields 1200-3000 hemaglutinating units/ml (HAU). Immunization of mice was done intraperitoneally, using a 1:6 dilution of allantoic fluid containing virus. “Memory” spleens were derived from mice any time after 21 days following immunization.

Antibodies and Complement Monoclonal anti-Lyt-1 (No. 53-7313) and anti-Lyt-2 (No. 53-672) were obtained from the Salk Cell Distribution Center, and were used as 10X concentrated culture supematants, as previously described (12). The anti-Thy- 1.2 hybridoma, No. HO3 14 (Salk Cell Distribution Center), was grown as ascites,and used at a 2 X 1Oe4dilution. The complement was lyophilized guinea pig complement (Flow Laboratories, Inc., McLean, Va.), diluted 1:10 in RPMI.

In Vitro Induction of SecondaryAnti-Influenza CTL Splenic lymphocytes from Balb/c mice (Jackson Laboratories, Bar Harbor, Maine) primed to Influenza A/Hong Kong 1 month previously were cultured with A/Hong Kong-infected anti-Thy- 1.2 + C-treated syngeneic spleen cells (stimulator cells). Each culture consisted of 8 X lo6 memory and 6 X lo6 stimulator cells in 12 ml of RPM1 1640 supplemented with 10% FCS, antibiotics, sodium pyruvate, glutamine, nonessential amino acids, and 2-mercaptoethanol (“complete RPMI”). After culturing for 5 days at 37°C cells were harvested, counted, and tested for specific cytotoxicity in a S’Cr-releaseassay. Secondary in vitro stimulation was performed as described above, but cells were not harvested on Day 5. Instead, they were restimulated on Day 12, and then 8 days later washed and cultured in TCGF-containing medium. The TCGF was prepared as previously described (7). Briefly, Wistar/Furth rat spleen cells were cultured at 5 X 106/ml in complete RPM1 containing 5 fig/ml Con A (Miles-Yeda, Rehovot, Israel) for 45 hr. The cells were then pelleted (20 min at 2000 x-pm)and the supernatant was filtered through an 0.2~pm Nalgene filter, and stored at -20°C.

“Cr-Release Assay The influenza-infected target cells were prepared as described previously (8). Briefly, targets (either P8 15Y or L cells) were labeled with Na5’Cr04 at a concen-

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tration of 250 &i/10’ cells, and after 30 min, virus was added at a concentration of 50 HAU/106 cells. After an additional hour at 37°C targets were washed twice and utilized in an 8- to lo-hr “Cr-release assay. Alternatively, target cells were maintained at 37°C for an additional 4 hr to allow for expression of viral antigens, then washed again, and used in a 3- to 4-hr assay. Both forms of the assay yielded similar results and were used interchangeably. The percentage specific “Cr-release was calculated as: cpm experimental - cpm spontaneous x 100. cpm maximal - cpm spontaneous All results are the average of at least three replicates. The standard error of the mean for these experiments averaged & 2. In general, assay wells consisted of 100 ~1 each of targets and effector cells. For inhibition assays, 50 ~1 of effecters was first plated with 50 ~1 of the appropriate dilution of antibody and then 100 ~1of targets was added to each well. The percentage inhibition of killing was determined as:

100 x

(% specific release in the presence of RPM1 or NRS) - (% specific release in the presence of specific antibody) (% specific release in the presence of RPM1 or NRS)

Mitogen Stimulation Splenic lymphocytes from normal C57B1/6 mice (Jackson Laboratories), 6-8 weeks of age, were stimulated with either Con A (Miles-Yeda, at 1 pg/ml), PHA (Burroughs Wellcome, at 0.5%) or LPS (Sigma, at 10 pg/ml) in microtiter wells, and then pulsed with tritiated thymidine for the final 24 hr of culture. Proliferation was expressed in counts per minute of samples harvested on a “MASH” apparatus and the radioactivity was counted in a Beta scintillation counter. TCGF Production (1) Rat. Cell-free supernatant from a 45-hr culture of rat splenocytes was used as a source of TCGF. Cells were cultured at a density of 5 X 106/ml in RPM1 containing 10% FCS and 5 Fg/ml of Con A (Miles-Yeda). Culture supernatants were filtered and stored at -70°C. (2) Human. Peripheral blood lymphocytes from normal donors were separated over Ficoll-Hypaque and cultured at a concentration of 5 X 106/ml in RPM1 containing 0.1% PHA-P (Sigma), 0.25% BSA, antibiotics, and glutamine. Culture supernatants were harvested after 18 hr. TCGF Assay The procedure used was modified from that originally described by Gillis et al. (9). Briefly, CTLL-2 were plated in loo-p1 aliquots containing 4 X lo3 cells in flatbottom microtiter wells. Following the addition of 100 ~1 of RAT* or medium, 50 ~1 of either rat or human TCGF was added to each well. After 20 hr at 37°C 0.5 &i/well of tritiated thymidine (Amersham) was added, and 4 hr later, plates were harvested on a multiple automated sample harvester, and incorporated radioactivity

314

EFFROS ET AL. TABLE 1 Comparison of Preincubation with RAT* and Anti-Lyt-2 Antibodies Percentage specific 5’Cr-release Serum

4’C Pretreatment

Anti-Lyt-2 RAT* NRS Anti-Thy- 1.2

5

7

15 16 16

5 NT

37°C Pretreatment

14

Note. Splenic lymphocytes from CBA/J mice, primed 4 days previously with A/PRI were treated in duplicate aliquots with each serum, one portion being maintained at 4”C, and the other at 37’C for I hr. Cells were then washed twice and tesed for specific cytotoxicity in a “Cr-release assay at an E:T of 100:1 on PRI-infected L cells.

was measured as counts per minute on a beta scintillation counter. The entire assay was performed in Click’s medium (Altick Associates) containing 2% FCS antibiotics and glutamine.

Preparation of IgG The IgG fractions of RAT* serum and NRS were prepared by precipitation with 42% ammonium sulfate at 4°C for 1 hr. The precipitate was pelleted by centrifugation at 15,OOOg for 30 min, resuspended in phosphate-buffered saline, pH 7.2, and chromatographed on a Sephacryl S-200 (Pharmacia, Piscataway, N.J.). The IgM and IgG peaks were collected; the IgG peak was further purified by chromatography through DEAE cellulose and was used at a concentration of 3 mg/ml for the FACS analysis.

FACS Analysis of Fluorescent Cells Single-cell suspensions of lymphoid cells were prepared and red blood cells were removed by treatment with ACK lysing buffer (Microbiological Associates, Walkersville, Md.). For immunofluorescence, l-2 X lo6 cells in 100 ~1 of RPM1 1640 plus 10% FCS in 0.1% Na azide were incubated with lo- 15 ~1 of RAT* IgG (3 mg/ ml) for 30 min at 4°C. The cells were washed two times and then incubated with 5 ~1 of undiluted fluorescent conjugated Ig fractions of goat anti-rat IgG (Cappel Laboratories, Cochranville, Pa.) for 30 min at 4°C. After washing the cells two times, the fluorescence was analyzed on a FACS II flow cytometer (Becton-Dickenson FACS Systems, Mountain View, Calif.). A total of 1 X lo4 stained cells were analyzed for each cell population studied, and the fluorescence profiles were depicted as increasing fluorescence intensity on the abscissaand relative number of cells on the ordinate.

Production of RA P Serum This antiserum was prepared as described previously (1). Briefly, Sprague-Dawley rats weighing approximately 300 g were immunized with preparations of activated alloimmune (C57BL/6, H-2b anti-P8 15, H-2d) peritoneal exudate T lymphocytes.

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The immunizing cells were prepared by collecting Day 9 or 10 alloimmune peritoneal exudate cells, passaging them over nylon wool columns, and incubating the effluent cells (nylon wool-nonadherent T lymphocytes) on PHA-P-coated L929 fibroblasts. Each PHA-P/L929 monolayer consisted of 5 X lo6 L929 cells coated with PHA-P (Difco, Detroit, Mich.) at 100 pg/75-cm* T flask (Falcon, Oxnard, Calif.) to which 50 X lo6 PEL were added in 25 ml serum-free RPM1 1640. After a 2- to 4-hr incubation at 37°C the lymphocytes were collected and washed twice in serumfree medium, and 20 X lo6 cells were injected ip into each rat. Immunizations were repeated biweekly for 8 weeks, then reduced to monthly intervals. Animals were bled from the tail, and the serum was collected and heat inactivated (56°C 45 min) before experimental use. All serum was routinely absorbed on P815-X2 cells, using two absorptions, each with 1.5 X lOa cells/ml at 4°C for 1 hr. This procedure removed all antibody + complement activity against P8 15 cells. RESULTS Blocking of CTL Activity by RAP Serum Is Not Due to Anti-Lyt-2 Antibody Activity Both anti-Lyt-2 antibody and RAT* serum have been shown to inhibit CTL function in the absence of complement (1, 3, 10, 11). However, anti-Lyt-2 has been shown to block the binding of effector lymphocytes and target cells (12), whereas blocking by RAT* serum is at the postbinding phase of the cytolytic process (11). Our observation (unpublished) of a Lyt-2 negative CTL clone which was inhibitable by RAT* and not by anti-Lyt-2 prompted us to seek other distinguishing characteristics between the two reagents. The effect of preincubation of in viva-sensitized CTL with anti-Lyt-2 antibody or RAT* serum on the cytotoxic activity is shown in Table 1. Both antibodies are inhibitory following pretreatment of effecters at 37°C for 1 hr. However, blocking of CMC is seen only with anti-Lyt-2 antibodies following pretreatment of CTL at 4°C. Using an in vitro-maintained CTL line, it was established that a 1:24 dilution of anti-Lyt-2 antibody and 1:36 of RAT* serum resulted in identical inhibition of

IO

20

30

40

% INVIBITION

50

60

70

80

90

OF 5’ CR RELEASE

FIG. 1. Comparison of RAT* and anti-Lyt-2 antibodies. Preliminary titration of RAT* and anti-lyt2 antibodies was performed to determine the concentration of each which resulted in equivalent inhibition when present during the 5’Cr-rekase assay. These.concentrations were then used for a 37” pretreatment of fresh aliquots of the same effector cell population. Following washing, these effecters were then assayed for cytotoxicity. Both 5’Cr-release assayswere performed on the same day, using the same target cells. Effecters were secondary in vitro stimulated CBA/J spleens, and targets were PR8-infected L cells. Effector:target was 5: 1.

EFFROS ET AL.

-

FIG. 2. FACS profiles of four different lymphocyte populations obtained from C57B1/6 mice. (A) Day 10 alloimmune PEL (C57BL/6, H-2b anti P815, H-2d); (B) splenic T lymphocytes; (C) thymocytes; and (D) splenic B lymphocytes. The dotted line represents binding with NRS, and the solid line represents profile with RAT* serum.

cytotoxicity when present throughout the assay. These functionally equivalent concentrations were then compared during the 37°C pretreatment step, using the same CTL line. From the data in Fig. 1, it can be seen that inhibition by RAT* serum was 2.5 times greater than that induced by anti-Lyt-2 antibody. Furthermore, RAT* serum recognizes cell-surface molecules (30) of sizes distinct from Lyt-2 antigens ( 13). Thus, we suggestthat blocking by RAT* and anti-Lyt-2 antibodies are mediated through distinct antigenic sites on the membrane of CTL. Assessment of the Binding of RA P Serum to Lymphocytes by Immunofluorescence In view of the ability of RAT* serum to inhibit the functional activity of CTL, we examined the expression of cell-surface antigens recognized by RAT* serum on various subpopulations of lymphocytes by indirect immunofluorescence and analyzed on a fluorescence-activated cell sorter (FACS). The FACS profiles shown in Fig. 2 indicate that RAT* serum binding is well above the background level seen with NRS. The highest intensity of fluorescence is associated with alloimmune PEL. Moderate fluorescence is associated with thymocyte and splenic T cells and, some-

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what unexpectedly, for B cells as well. These results showed that RAT* serum contains antibodies detecting antigens present on both T and B cells.

lnhibition by RAT* Serum of PHA- and Con A-Induced Proliferation and Failure to Inhibit LPS Response Since RAT* serum binds to both T and B cells, we proceeded to examine its effect on the antigen-nonspecific proliferative responses induced by T- and B-cell mitogens. The results in Fig. 3 show that the PHA- (Fig. 3A) and Con A- (Fig. 3B)

I

5 a 0

IO4

r /

A

NRS

0

RAT*

3

NOSERUM

A

NRS

0

RAT*

0

NOSERUM

A NRS 0

RAT*

0

NO SERUM

II-i-.

IQ0 DILUTIONS

-1

l/80

~~

I/320

OF ANTISERUM

FIG. 3. Effect of RAT* serum on mitogen stimulation. Splenic lymphocytes from C57BL/6 mice were cultured in the presence of RAT* serum, NRS, or no additional serum, and tritiated thymidine uptake was measured for each mitogen. (A) Con A, (B) PHA, and (C) LPS.

318

EFFROS ET AL. TABLE 2 Inhibition of TCGF-Dependent Proliferation Source of TCGF

RAT*

Rat Rat Human Human -

+ + -

cm 23,400 11,028 17,923 466 903

Note. TCGF assay was performed as described under Materials and Methods. Rat and human TCGF were utilized at final dilutions of 1:16 and 1:2, respectively. The RAT* serum was used at a 1:6 final dilution.

induced proliferative responses were markedly inhibited by RAT* serum and the inhibition was dependent on the concentration of serum used. In contrast, it was found that the response to a B-cell mitogen, LPS, was not affected by RAT* serum and, in fact, appeared to be slightly enhanced (Fig. 3C). Thus, the presence of RAT* serum throughout the culture period does not result in some nonspecific toxic effect, but appeared to be selectively inhibitory for the T-cell subpopulations.

Inhibition of TCGF-DependentProliferation We next examined the effect of RAT* on the proliferation of the Interleukin-2dependent cell line CTLL-2 (graciously provided by Dr. Steven Gillis). These cells respond to both human and rat TCGF, and it can be seen in Table 2 that RAT* is inhibitory in both situations. No inhibitory effect was seen with NRS or several rat anti-mouse T-cell hybridomas including anti-Thy-l (data not shown). In addition, the inhibitory effect of RAT* was maintained following absorption with normal C57BL/6 spleen cells (3 X lO*/ml of serum). This is reminiscent of our previous reports showing that CTL inhibitory activity was retained after absorption of RAT* with immune T cells (1) and thymocytes (11).

Inhibition by RAT* Serum of the In Vitro Induction of Secondary CTL We have previously reported that RAT* serum blocks the cytotoxic activity of both alloimmune and H-2 restricted CTL ( 1, 10). It was therefore of interest to now extend these studies to the induction phase in the generation of CTL. It can be seen from Table 3 that pretreatment of the memory spleen cells with RAT* serum at 37°C followed by extensive washing, results in the inhibition of the in vitro induction of 2” CTL directed against influenza-infected P815 target cells. Control cultures treated with NRS or RPM1 have no inhibitory effect. Since the in vitro anti-influenza cultures utilized in the present study are dependent on the presence of primed responder cells, the observed effect of RAT* serum is primarily directed at the antigen-specific expansion of a memory-T-cell population. In other experiments, we have observed that RAT* serum also inhibits the in vitro sensitization of lo and 2” CTL directed against ahogeneic cells (data not shown). Thus, the induction of both lo and 2” CTL is inhibited by RAT* serum, suggesting that brief exposure to this serum prevented these cells from responding to alloantigen or virally modified syngeneic cells.

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TABLE 3 RAT* Pretreatment of Influenza-Primed Spleens Inhibits Induction of Secondary in Vitro Culture Percentage specific 5’Cr release Cell yield after culture

Pretreatment None RAT* 2 hr at 37°C wash NRS 2 hr at 37°C wash RPM1 2 hr at 37°C. wash

12.1 x 2.7 X 9.5 x 10.3 x

lo6 lo6 lo6 10”

5:l

1O:l

20: I

16 3 58 78

78 7 67 80

91 NT 78 84

Note. Splenic lymphocytes from Balb/c mice which had been primed 2 months previously with A/ PR8 pretreated as indicated, then washed twice with RPMI. These responder cells were cultured with syngeneic anti-Thy-l.2 + complement-treated spleen cells which were infected for 1 hr with A/PR8 (stimular cells). Cultures were harvested after 5 days and assayed for specific cytotoxicity.

Irreversibility of the Inhibition by RAF Serum It should be emphasized that in the in vitro influenza cultures discussed above, the only contact that the spleen cells had with RAT* serum was during the pretreatment process, and that no additional RAT* serum was added to the culture. Thus, it was somewhat surprising to observe that the cells did not recover from the inhibitory effect during the course of the 5-day culture period. A similar effect was previously observed for CTL in the “Cr-release assay,whereby pretreatment of CTL with RAT* serum did not lead to the recovery of cytotoxic function (unpublished). To further identify the target cell inhibited by RAT* serum, a TCGF-dependent cultured CTL line specific for influenza virus-infected cells was used as a tool to further explore the inhibitory characteristic of RAT* serum. This anti-influenza line has maintained its virus-specific function for over 8 months and parallels the in

I 6

12

24

(rot

a

48

60

Ly I ) DILUTION

6

24

48

(rat

12

a

Ly 2)

OF

REAGENT

63

6

12 (RAT*

X

24

48 1

IO-’

FIG. 4. Inhibition of TCGF-dependent CTL. Various dilutions of monoclonal rat anti-Lyt- 1 and Lyt2 culture supernatants, RAT* and NRS were added in IOO-~1amounts to CTL effector cells in microtiter wells. 5’Cr-Labeled targets were added to each well and plates were incubated at 37°C for 2 hr.

320

EFFROS ET AL. TABLE 4 Recovery from RAT* Inhibition Percentage specific Wr release Serum

Tested immediately

Recultured for 24 hr

NRS RAT* ( 1:12 dilution)

33 15

38 4

Note. Cultured CTL were washed free of TCGF medium, then divided into four aliquots. All were preincubated for 1 hr with serum at 37”C, then washed twice. Two samples were used immediately as effecters in a 3-hour s’Cr-release assay, and two others were placed in TCGF-containing medium for 24 hr, at which time they were similarly tested.

v&-primed CTL in its H-2 restriction and cross-reactivity for serologically distinct influenza A viruses. In addition, its cytotoxic function is inhibited by RAT* serum and anti-Lyt-2 antibodies, but not by anti-Lyt-1 antibody (Fig. 4). Thus, by several criteria, the cultured line was similar to the in viva-derived CTL. The data presented in Table 4 demonstrate that preincubation of the cultured CTL with RAT* serum resulted in the inhibition of cytotoxicity when these cells were tested in a 5’Cr release assay.Furthermore, aliquots of CTL that were preincubated in parallel, then washed extensively and recultured in TCGF medium, did not recover their cytotoxic function, even after 24 hr. These results showed that the blocking of the CTL line by RAT* serum persisted and was not overcome by TCGF. DISCUSSION The present study demonstrates that RAT* serum, previously shown to- block CTL activity, selectively inhibits other T-cell-dependent functions, namely, mitogen response, CML induction, and TCGF-dependent CTL activity. The inhibition obtained was not reversible for 24 hr, even though lymphocytes were only exposed to RAT* serum for 60 min. These results, therefore, suggest that RAT* serum recognizes membrane-associated molecules of T lymphocytes which are vital to the expression of its functional activity. Although studies from a number of laboratories have shown that antisera directed against target cell antigens are capable of inhibiting cytotoxicity ( 14-2 l), it has been extremely difficult to demonstrate blocking with antisera directed against cell-surface determinants of the effector lymphocytes. Since anti-Lyt-2 is one of the few antibodies capable of inhibiting CTL function, we sought to demonstrate possible differences between it and RAT* serum. The distinction was initially suggestedby our observation that one of our TCGF-dependent CTL clones was Lyt-2 negative and yet possessedmembrane antigens which could be immunoprecipitated with RAT* serum (30). The findings reported in the present study offer additional evidence that different cell-surface determinants are recognized by anti-Lyt-2 antibody and RAT* serum. Since RAT* serum was shown to bind to both T and B lymphocytes, it is not clear whether this interaction was with the same or distinct cell surface molecules on the two cell types. However, there are precedents in the literature for the notion that a common cell-surface antigen may exist on functionally diverse populations.

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For example, a low-molecular-weight protein recently isolated from T cells stimulated via their antigen receptor was found on Con A-activated T cells as well (22). Also, the LFA- 1 polypeptide described by Davignon et al. (23) appears to be involved in a variety of T-cell functions, such as MLR, CML, and T-dependent antibody production. Furthermore, this antigen, defined by a monoclonal antibody, was shown to be present on B cells, in greater amounts on T cells, and in still increased quantity on activated T cells (24). Our own experiments also suggest a common cell-surface antigen, since immunoprecipitation with RAT* using B- and T-cell fractions resulted in identical patterns (J. Fan, personal observation). Finally, an “activated lymphocyte antigen” reported by Feeney and Hammerling was present on both T and B cells (25). One might speculate that some sort of “activation molecule” may be expressed by all lymphocytes. Thus, for example, the CTL at the peak of their functional activity may display the same surface antigen as both a memory T cell and a T cell that is in the process of being activated by a mitogen or TCGF. The fact that this molecule may be present at a lesser density on normal T cells (Fig. 2) is not necessarily contradictory to this hypothesis, since the “activated state” may simply be an exaggeratedform of the normal cell. Furthermore, “normal” spleen cells may continually be exposed to antigens, even without our intentional immunization, so that we may be detecting a subpopulation of cells which is actually an “activated” state. Although fluorescence studies indicate that the molecule recognized by RAT* serum may be expressed by B cells, the failure of RAT* to inhibit the LPS response suggeststhat B-cell activation may operate via a different pathway than the T-cell induction step. This is consistent with the studies of Davignon et al. using monoclonal antibodies (23). The interaction of RAT* serum with its antigen(s) seemsto inactivate the T cell in an irreversible manner. We have examined the recovery from RAT* seruminduced inhibition in two different situations. From the data in Table 3, it can be seen that incubation of memory spleens with RAT* serum at 37°C (followed by washing) induced a modification that was not reversed during the entire 5-day culture period, despite the absence of RAT* serum in the culture medium. From previous experiments, one would expect to see evidence of proliferation by Day 2, so that even if full recovery took several days, there should have been some evidence of stimulation by Day 5. Indeed, the inhibited cultures showed no blast-like cells. and the cell yield indicated that no proliferation has occurred. The second situation in which the T cells failed to recover from RAT* seruminduced inhibition involved the TCGF-dependent CTL line (Table 2). Since such cultures contain only CTL, the lack of recovery cannot be due to the elimination of effector lymphocytes by an ADCC or opsonization mechanism. Furthermore, NRS has never been inhibitory in any situation, so that nonspecific toxic effects of xenogeneic serum can also be ruled out. Finally, the IgG fraction of RAT* serum causes inhibition which is comparable to that of the whole serum (unpublished observation), so that inhibition is indeed mediated by antibody. It appears that the binding of antibody to a functionally relevant membrane molecule causes an irreversible inhibition of both memory T cells and TCGF-dependent CTL. It is not clear why the T cells cannot recover from the inhibitory effect of RAT* serum. Studies on patching and capping of both T- and B-lymphocyte surface molecules indicate that within 4 to 12 hr after antibody treatment, the antigens reappear on the membrane (26). Interestingly, all our attempts at dem-

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onstrating capping and patching of CTL membrane antigens with RAT* serum were unsuccessful (unpublished observation). Possibly the loss of lateral mobility, analogous to the effect of high levels of Con A described by Edelman (27) could account both for the absence of capping and the lack of recovery following incubation with RAT* serum. A second explanation for the irreversible inhibition caused by RAT* serum relates to the molecular signals that may be requisite for activation. The cell-surface molecules recognized by RAT* serum might be blocked at a critical stage in a precisely coordinated series of events whose order and timing cannot be modified. This situation is somewhat reminiscent of the irreversible modulation of antigen receptors on immature B cells described by Sidman and Unanue (28). They first demonstrated that immature B cells have the ability to reexpress their Ig as completely and as rapidly as adult B cells when the Ig is removed with Pronase. However, treatment with antibody to the Ig resulted in the irreversible removal of Ig from the surface of immature B cells with the subsequent loss of all functional potential. A third possible explanation is that RAT* serum may be recognizing structures involved in cellular homeostatic mechanisms. Such molecules might be regulators of activation which when blocked cause a state of suppression even in the presence of activation signals. The situation may not be unlike the prolonged inhibition of CTL function reported by Chang and Eisen (29), where the programming for the lysis step was impaired for l-4 days by the protease inhibitor iVu-tosyl-chloromethylketone. In conclusion, the results of the present study suggest that RAT* serum may be a valuable probe for analyzing functionally relevant surface molecules found on T cells. RAT* serum has been shown to produce dramatic effects on various T-cell functions, but not on the response of B cells to mitogens. In addition, the inhibitory effect of RAT* serum appears to be at least temporarily irreversible. It is not clear whether the inhibitory activity of various T-cell functions is mediated by the same or distinct antibodies present in RAT* serum. However, continued analysis of RAT* serum, together with preparation of monoclonal reagents, may yield important information on the mechanism of T-cell function. ACKNOWLEDGMENTS This research was supported by CA 12800 from the National Cancer Institute, DHEW, and in part by a grant from the Cancer Research Coordinating Committee. We wish to thank David Anisman for technical assistance and Susan Stehn for typing the manuscript.

REFERENCES 1. 2. 3. 4. 5.

Ethos, R. B., Hiserodt, J. C., and Bonavida, B., J. Immunol. 125, 1879, 1980. Redelman, D., and Trefts, P. E., J. Immunol. 121, 1532, 1978. Sachs, D. H., and Shinohara, N., J. Exp. Med. 150,433, 1979. Kimura, A. K., J. Exp. Med. 139, 888, 1974. Nakayama, E., Shiku, H., Stockert, E., Oettgen, H. F., and Old, L. J., Proc. Nut. Acad. Sci. USA 76, 1971, 1979. 6. Rabinowitz, R., Laskov, R., and Schlesinger, H., In “Macrophages and Lymphocytes, Nature, Functions and Interactions, Part B.” (M. Escobar and H. Friedman, Eds.), p. 247. Plenum, New York, 1980. 7. Nabholz, M., Engers, H. D., Collavo, D., and North, M., Curr. Top. Microbial. Immunol. 81, 176, 1978.

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