Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin

CELLULAR IMMUNOLOGY 1 lo, I - 13 ( 1987)

Suppression of the Cytotoxic T-Lymphocyte Response in Mice by Pertussis Toxin

PRINCE K. ARORA, RONALD D. SEKURA, AND EDGAR E. HANNA

Laboratory ofDevelopmental and Molecular Immunity, NICHD National Institutes ofHealth,

Bethesda, Maryland 20892

Received January 13,1987; accepted July 5, 1987

Pertussis toxin (PT), the major toxin produced by Bordeteliapertussis, has been reported both to enhance and to suppress immune responsiveness. These findings suggested that PT contrib- utes to the virulence of B. pertussis through mechanisms involving immune regulation. We report that PT suppressed both the primary and the secondary cytotoxic T-lymphocyte (CTL) responses of mouse spleen cells cultured against two different allogeneic stimulator spleen cells in vitro. This suppression was dependent on the dose of PT used. PT must be present during the initial stages (within the first 24 hr) of CTL generation. Soluble factor(s) obtained from spleen cells preexposed to PT did not suppress the CTL response. Suppression of the CTL response observed was not due to depletion of the antigen by PT. The cytotoxic activity of CTL clones could not be suppressed by PT. The analysis of responder spleen cells, fractionated by anti- immunoglobulin panning techniques, provided evidence that L3T4-, Lyt 2+ cells mediate the PT-induced immunosuppression. We propose that suppression of the CTL response by PT is generated through the activation of L3T4-, Lyt 2+ suppressor T lympohcytes. o 1987 Academic

Press, Inc.

INTRODUCTION

Microbial toxins have been shown to have immunomodulatory activities (l-7). Acellular components extracted from Bordetellu pertussis were shown to have some of these immunomodulatory activities (8-14). One of these components, pertussis toxin (PT),’ an oligomeric protein exotoxin, has been shown to both augment ( 15- 17) and suppress ( 18, 19) antibody responses. Pretreatment of mice with PT suppresses antibody responsiveness to sheep erythrocytes (SRBC), tetanus toxoid, or to influenza virus. Kumazaza and Mizuno (20) observed that PT suppressed both primary and secondary in vitro anti-SRRC responses of immune spleen cells. The suppression by PT was greatest during the inductive phase of the response and appeared to be related to a suppressor factor produced by T lymphocytes. These findings sug-

’ Abbreviations used: PT, pertussis toxin; SRBC, sheep red blood cells; CTL, cytotoxic T lymphocyte; HTL, helper T lymphocyte; STL, suppressor T lymphocyte; APC, antigen-presenting cell; Con A, concanavalin A; HBSS, Hanks’ balanced salt solution; SEM, standard error about the mean; FBS, fetal bovine serum; LPF, lymphocytosis-promoting factor; SEA, staphylococcal enterotoxin-A.

0008-8749187 $3.00 Copyright 0 1987 by Academic pnsS Inc. All ri8bt.s of reproduction in any form reserved.

2 ARORA, SEKURA, AND HANNA

gested that PT may contribute to the virulence of B. pertussis through immune suppression mechanisms.

While modulatory effects of several bacterial exotoxins upon the regulatory T lymphocytes have been investigated (l-7), reports relative to modulation of effector T lymphocytes by microbial toxins are rare. This report concerns our investigation of the role of PT in the modulation of cytotoxic T-lymphocyte (CTL) responses. We report that PT suppresses both primary and secondary CTL responses, and suggest that this suppression is mediated by L3T4-, Lyt 2+ cells.

MATERIALS AND METHODS

Animals. Mouse strains NFR/N, BALB/cN, AKR/N, and C57BL/6N were obtained from the Small Animal Section, Division of Research Services, NIH. All mice used were females and 8- 12 weeks of age.

Pertussis toxin preparation. PT was purified to homogeneity according to the method of Sekura et al. (2 1). The stock solution was diluted 1: 10 in Hanks’ balanced salt solution (HBSS, GIBCO, Grand Island, NY) and dialyzed for 4 hr in HBSS at room temperature. The dialysate was then sterilized by membrane filteration (0.22 pm), and used.

Sensitization of cultures. Cytotoxic effector cells were generated in 24-well, flat- bottom tissue culture plates (Costar, Cambridge, MA) as described (22). Briefly, re- sponding spleen cells (5 X 106, not treated with ACK), were cocultured with 2000- rad irradiated (‘37Cs source; Gammator Isomedix, Inc., Parsipany, NJ), allogeneic stimulator cells (2 X 106) that had been freed of erythrocytes by ACK treatment. The cultures were incubated at 37°C in a 95% air, 5% CO2 atmosphere for 5 days,

Cytotoxicity assay. Effector cells were tested for cytotoxicity by using a “Cr-release assay (23). Briefly, varying dilutions of cells in lOO-~1 aliquots were added to microtiter wells (Linbro Chemical Co.). Target cells used were 48-hr concanavalin A (Con A, Sigma Chemical Co.)-stimulated blast spleen cells, labeled with 200 PCi of 5’Cr (Naz CrO3 (New England Nuclear, Boston, MA). After three washings in medium containing 10% FBS, target cells were counted and added (loo-p1 aliquots) in triplicate to microtiter wells containing effector cells. The plates were centrifuged for 3 min at 400 rpm, and incubated at 37°C for 4 hr in a 95% air, 5% CO2 atmosphere. After the incubation, the plates were centrifuged for 5 min at 800 rpm, and the supernatant was collected with the Titertek Supernatant Collection System (Skatron Inc., Sterling, VA) and counted in a Beckman Auto Gamma scintillation spectrometer. The percentage of lysis and standard error of the mean (SEM) were calculated based on triplicate samples as described previously (24) using the equation

% specific lysis = Exp,, - Con,, x 1oo ~Q&pnl - Spent,, ’

where Expwm was the number of counts released by effector cells cultured against allogeneic stimulator cells, Con,, , the number of counts released by effector cells cultured against autologous cells, Max,,,,, , the number of counts released by 100 ~1 of target cells in the presence of a solution of 2.5% T&on-X detergent, and Spent,, was the number of counts released by 100 ~1 of target cells cultured with the medium alone.

MODULATION OF CTL RESPONSES BY PERTUSSIS TOXIN 3

Lymphocyte blastogenesis. Spleen cells were cultured in 96-well flat-bottom microtiter tissue culture plates (Falcon Plastics, Becton-Dickinson Co., Mountain View, CA). Quadruplicate wells each contained 2 X lo5 spleen cells in 200 ~1 of complete medium and PT. The plates were incubated for 72 hr at 37°C in humidified atmosphere containing 95% air and 5% COP. Each culture was pulsed with 1 &i of [3H]thymidine (sp act 6.7 Ci/mmol, New England Nuclear) for 18 hr. The cells were harvested with a MASH II cell harvester and [3H]thymidine uptake was measured in a liquid scintillation counter.

Separation of T and B lymphocytes by anti-imrnunoglobulin panning. Cells bearing surface immunoglobulin (sIg+) were separated from whole spleen cells by the anti- immunoglobulin panning technique of Mage et al. (25). Affinity-purified goat anti- mouse immunoglobulins (Cappel Laboratories, Malvern, PA) were diluted to concentrations of 1 mg/ml in sterile phosphate-buffered saline (PBS). Portions (6 ml) of the anti-mouse immunoglobulin solution were added to Falcon 100 X 15-mm tissue culture dishes and the dishes were incubated for 18 hr at 4°C. After incubation, the excess, unbound anti-mouse immunoglobulin was removed by washing three times with 15 ml of sterile PBS. Spleen cells were diluted to a concentration of 5 X 1 06/ml in RPM1 1640 medium containing 10% FBS and 2 mM Hepes but lacking sodium bicarbonate (Hepes medium). A IO-ml portion of the spleen cell suspension was added to each anti-mouse immunoglobulin-coated dish. The dishes were incubated for 1 hr at 4°C with gentle agitation of the cell suspension every 30 min. The nonadherent cells (sIg-) were removed with a pipette, washed twice, and suspended in complete medium.

Separation of T-cell subsets by panning. T lymphocytes, bearing various cell surface antigens, were selectively obtained as follows. Ig--enriched spleen cells were incubated for 1 hr at 4°C on plastic dishes coated with anti-L3T4 monoclonal antibodies (mAb) (the anti-L3T4 mAb was from an ascites produced in NFR nude mice injected with hybridoma clone GK 1.5 purchased from the American Type Culture Collec- tion). Nonadherent cells were removed from the dishes by gentle rinsing and discarded. The adherent cells were eluted by vigorous pipetting with a Pasteur pipet. The eluted cells were Ig-, L3T4+, Lyt l+ as measured by the flow Cytofluorograf. Alternatively, the Lyt 2+-enriched population was prepared by panning of L3T4-- enriched cells on anti-Lyt 2 mAb-coated dishes. Nonadherent cells were removed from the dishes by gentle rinsing and discarded. The adherent cells were eluted by vigorous pipetting with a Pasteur pipet. The eluted cells were Ig-, L3T4-, Lyt 2+ as examined by the flow Cytofluorograf. These cells were washed twice and suspended in complete medium. Both the L3T4+, Lyt l+ and the L3T4-, Lyt 2+ cells were irradiated (so that they themselves did not proliferate in response to allogeneic stimulator cells) before addition to the CTL-generating system.

Flow cytofluorometry. Flow cytofluorometric analysis of cell surface markers sIg, Thy 1, Lyt 1, and Lyt 2 was conducted as detailed before (26). In addition, a phycoerythrin-conjugated anti-L3T4 mAb (Becton-Dickinson) was used in labeling for L3T4 markers.

Statistics. Significance was determined by Students’ t test. P values less than 0.05 were considered significant. All experiments were conducted a minimum of four times.


5’Cr BALE/c

PT Ipg)

E:T RATIO

FIG. 1. Suppression of in vitro primary and secondary CTL responses by PT. Spleen cells from the NFR/ N mice (A) unprimed or (B) primed 4 weeks earlier with BALB/c allogeneic spleen cells were cultured against allogeneic BALB/c stimulator spleen cells. PT was added to the cultures at different concentrations. Cytotoxic effector cell activity was tested on BALB/c spleen cells (48-hr Con A blasts labeled with “Cr). Spontaneous release was 20%. Each value represents the mean + SEM of five animals. SEM was less than 5%. These data are representative of five replicate experiments.

RESULTS

Suppression of the in Vitro CTL Response by PT

In order to investigate the effect of Pertussis toxin on the CTL response, NFR/N (H-2q) mouse spleen cells were cultured against two different alloantigens, (BALB/ cN, H-2d) or (AKR/N, H-2k), in the presence of various concentrations of PT ranging from 0.05 to 4 rg/5 X lo6 cells/2 ml. The CTL responses against the allogeneic spleen cell targets were measured after 5 days of incubation using a 4-hr 5’Cr-release assay. Figure 1 A, representative of five separate experiments, indicates that PT at concentrations ranging from 0.05 to 1 pg/culture markedly suppressed the CTL response against BALB/c alloantigen. PT also suppressed the CTL response against AKR allogeneic stimulators (data not shown). PT heated to 100°C for 30 min did not suppress the CTL response. The viable cell number in the presence of PT did not differ appre- ciably from that of control cells (data not shown).

The effect of PT on the secondary CTL response was studied by priming NFR/N mice with 5 X lo7 BALB/c allogeneic spleen cells injected intraperitoneally. After 4 weeks, spleen cells were collected from these alloantigen-primed mice and cultured


-PT

PT ADDED AT TIME [hrl

100

1

B

PT REMOVED FROM CULTURES [hr]

FIG. 2. Kinetics of PT suppression on the CTL response. Responder spleen cells were cultured against allogeneic BALB/c stimulator spleen cells. (A) PT was added to cultures at various intervals during the 5- day incubation period. (B) Alternatively, PT (1 &ml) was added to cultures at time 0, and then free PT was removed (by washing cells 3X) at various time periods (O-l 20 hr). Cytotoxic effector cell activity was tested on BALB/c spleen cells (4%hr Con A blasts labeled with “Cr) at an ET ratio of 10: 1. Spontaneous release was 24%. Dam are expressed as the means + SEM of triplicate samples. These data are representative of five replicate experiments.

against BALB/c allogeneic stimulator spleen cells and in the presence or absence of different concentrations of PT. When alloantigen-primed mouse spleen cells were exposed to the sensitizing alloantigen (BALB/c), there was an elevated CTL response (Fig. 1B). Addition of PT (1 &culture) decreased this secondary CTL response by about 50%. Suppression of this secondary CTL response was even more profound if the concentration of PT/culture was raised to either 2 or 4 &culture (Fig. 1 B). Thus, PT markedly suppressed the secondary CTL response in a dose-dependent manner.

Kinetics of the Suppressive Effect of PT on Responder Spleen Cells following Exposure to Alloantigen

The suppressive effect of PT was investigated during the early and late stages of the CTL response. PT, 1 &culture, was added to the CTL-generating system at various intervals ranging from 0 to 120 hr. Results shown in Fig. 2A indicate a marked sup-


Pl

FIG. 3. PT effect on cloned CTL lines. Cloned CT L lines were cultured for 5- 10 days in the presence of PT (1 &culture), and then tested for CTL activity on “Cr-labeled targets syngeneic to their respective specificities. Data are expressed as the means + SEM of triplicate samples. These data are representative of five replicate experiments.

pression of the CTL response when PT was added any time within 24 hr of initiation of cultures. However, suppression of the CTL response was not observed when PT was added later than 48 hr. Alternatively, PT was added to the cultures at time 0 and then removed at various time periods during the 5-day culture. Results shown in Fig. 2B indicate that suppression of the CTL response by PT was abrogated or reduced only if PT was removed from cultures prior to the first 2 hr of incubation, suggesting that suppression by PT is reversible only if PT is removed during the early stage of culture.

PT was also tested on cloned CTL lines which have been established in our laboratory. These cloned CTL lines are restricted to different specificities (anti-Db, anti-H- 2d, and anti-H-2qMTNP) (P. K. Arora et al., submitted for publication). PT did not suppress the cytotoxic activity of any of these cloned CTL lines (Fig. 3). These observations suggest that PT acts at the early stage(s) of CTL generation, perhaps at the precursor T-cell level.

To determine whether soluble factor(s) released from PT-exposed cells were re- sponsible for suppression of the CTL response observed, spleen cells were incubated with PT for 24 hr, washed to remove free PT, and then cultured for different time periods ranging from 2 to 120 hr. Supernatant fluids collected up to 120 hr from these cultures had no suppressive effect on the CTL response of freshly initiated CTL (data not shown).

To determine whether suppression of the CTL response by PT were due to an indirect effect on the antigen, allogeneic stimulator spleen cells were exposed to PT for 4 hr, either before or after irradiation. Data shown in Fig. 4 indicate that preexposure of alloantigen to PT (either before or after irradiation) prior to addition into the CTL culture system did not result in suppression of the CTL response. Addition of allogeneic stimulator cells (a number higher than the optimal dose) to cultures did not abrogate suppression by PT (data not shown), further indicating that immunosuppression by PT was not due to depletion of the antigen in the cultures. On the


m

+ 1 2 4 1 2 4 PT

TIME OF PRE-TREATMENT WITH PT Ihr)

FIG. 4. PT effect on responder and/or stimulator spleen cells. Responder or stimulator spleen cells were incubated with PT for 4 hr and washed three times with HBSS. The following combinations were cultured for 5 days: PT-treated responder cells + untreated stimulator cells, untreated responder cells + PT-treated stimulator cells. Cytotoxic effector cell activity was tested as described in Fig. 2. Data are expressed as the means + SEM of triplicate samples. SEM was less than 5%. These data are representative of five replicate experiments.

other hand, preexposure of responder spleen cells to PT resulted in marked suppression of the CTL response (Fig. 4), indicating that immunosuppression by PT observed in the system was due to an interaction of PT with the responder spleen cells.

Eflect of PT on Adherent vs Nonadherent Responder Spleen Cells

Responder spleen cells were incubated with and without PT. After 4 hr of incubation, the nonadherent cells were separated from the adherent macrophages by re- peated washing of the monolayers. Combinations of PT-treated and -untreated adherent and nonadherent cell populations were tested as shown in Fig. 5. Adherent cells, exposed to PT for 4 hr, exerted no suppression of the CTL response when compared with the control groups. When only the nonadherent cells in the responder cell population were exposed to PT for 4 hr, the CTL response was markedly suppressed when compared with the control group. Suppression of the CTL response was like- wise observed when both cell populations were exposed to PT and combined. We also observed that treatment of adherent and nonadherent responder cells sepa- rately with PT showed suppression only when nonadherent cells were treated (data not shown).

Eflect of PT on Spleen Cell Subpopulations Fractionated by Anti-Immunoglobulin Panning

Anti-immunoglobulin panning was used to deplete surface immunoglobulin-bearing (sIg+) lymphocytes from surface immunoglobulin negative (sIg-) lymphocytes prior to further enrichment of lymphocytes bearing L3T4+, Lyt 1 + or L3T4-, Lyt 2+ surface antigens. These enriched spleen cell populations were analyzed by flow cytofluorometry following staining with the respective fluorescein-labeled mono-


2.5 5 10 20 EFFECTOR: TARGET CELL RATIO

FIG. 5. PT effect on nonadherent and/or adherent responder spleen cells. Responder spleen cells were incubated with PT. After 4 hr, nonadherent cells were separated from adherent macrophage monolayers as described under Materials and Methods. The following responder cell combinations were cultured against allogeneic BALB/c stimulator spleen cells: whole spleen cells, untreated nonadherent and adherent cells, untreated nonadherent + PT-treated adherent cells, PT-treated nonadherent cells + untreated adherent cells, PT-treated nonadherent and adherent cells. Cytotoxic effector cell activity was determined as described in Fig. 2. Data are expressed as the means + SEM of triplicate samples. SEM was less than 5%. These data are representative of five replicate experiments.

clonal or the second step affinity-purified antibodies (Fig. 6). The fractionated spleen cells were then tested for their response to PT. Preliminary experiments to test for possible mitogenic effects of PT on sIg- and sIg+ populations showed the greatest mitogenic responses in T-cell-enriched populations (Fig. 7). The proliferative response to PT was also examined in sIg- populations that were further depleted of either L3T4+, Lyt l+ or L3T4-, Lyt 2+ cells. When, the L3T4+, Lyt l+ and L3T4-, Lyt 2+ populations were tested for the proliferative response to PT it was observed that the L3T4-, Lyt 2+ cells exhibited the greatest proliferation (Fig. 7).

Finally the effect of the L3T4+, Lyt I+ and L3T4-, Lyt 2+ subsets on PT-induced suppression of the CTL response of spleen cells was investigated. The data in Fig. 8 show that when L3T4-, Lyt 2+ cells were exposed to PT, irradiated (so that they themselves did not proliferate in response to allogeneic stimulator spleen cells), and then added to the CTL-generating system, there was marked suppression of the CTL response, and this suppression was dependent on the number of PT-treated L3T4-, Lyt 2+ cells added, whereas addition of L3T4+, Lyt I+ cells (preexposed to PT) to the CTGgenerating system did not result in suppression of the CTL response (Fig. 8).

DISCUSSION

PT, an oligomeric protein exotoxin produced by phase I B. pertussis, has been reported to enhance immune responsiveness to a variety of antigens (27, 28). The best adjuvant effect was obtained when B. pertussis vaccine was injected simulta- neously with the antigen (27, 28). If administered a few days before or after antigen B. pertussis vaccine may suppress the immune response. Similar to the effect of B. pertussis, PT suppressed immune responsiveness if the timing of injection of PT with

MODULATION OF CTL RESPONSES BY PERTUSSIS TOXIN

T 0 lllmbh

1 IPIWJNO FL. lee@ 100

E

::

E

I: T 0

1 ItlIlUNO FL. 1000

iHiltiNb iL: 100

liii0

1 INWJNO FL. 1000

FIG. 6. Immunofluorescence by flow cytofluorometry of cells fractionated by anti-immunoglobulin panning (Materials and Methods). Fractionated (nonadherent) cells were stained with FITC-conjugated goat anti-mouse IgG (A) or anti-Thy 1.2 (B) antibodies. These enriched T cells were further fractionated by anti-L3T4 panning into adherent, eluted (C, E, and G), and nonadherent (D, F, and H) cell populations (Materials and Methods). Fractionated cells were stained with phycoerythrin-conjugated anti-L3T4 (C and D), FITC-conjugated anti-Lyt 1 (E and F), and FIT&conjugated anti-Lyt 2 (G and H) monoclonal antibodies. Each cytofluorogram shows the fluorescence of stained (shaded area) vs unstained (solid line) cells.

respect to the antigen was varied ( 18, 19). PT, therefore, may suppress the immune response during the course of infection.

We investigated one aspect of the immunomodulatory potential of purified pertussis toxin. PT was found to suppress both the primary and the secondary in vitro CTL responses against alloantigens (Fig. 1). This suppressive effect was eliminated by heat- ing PT at 80-100°C for 30 min. Dose-response studies showed that suppression by PT occurred at concentrations greater than 0.05 &culture. Maximal suppression of the 5-day in vitro CTL response to alloantigens was achieved by the addition of PT to a final concentration of 1 &culture. A direct toxic effect of PT on lymphocytes was ruled out, since viable cell counts were unaltered after 5 days.

Several other findings are noteworthy toward understanding the mechanism of PT- induced suppression of the CTL response. A sharp decline of the CTL response occurred when PT was added at an early stage (2-24 hr) of CTL generation. When PT was added to cultures later than 48 hr after antigenic stimulation, no effect on the


FIG. 7. Proliferative response ([3H]thymidine incorporation) to PT (1 &ml) ofwhole spleen cells (WSC) (O), sIg- spleen cells ( ), and sIg-, L3T4-, Lyt2* (H) and sIg-, L3T4+, Lyt 1’ (0) spleen cells. Data are expressed as mean CPM + SEM of quadruplicate samples.

CTL response occurred (Fig. 2A). Results of an experiment (Fig. 2B) in which PT was added to cultures at time 0, and then removed from cultures (by washing the cells 3X) at different time periods, indicated that suppression by PT was abrogated only if PT was removed from cultures within the first 2 hr. Thus, suppression is revers-

CELLS ADDED TO THE *CTL GENERATION SYSTEMS:

TYPE

% SPECIFIC LYSIS

wsc

WT4- + WT4+ 3x106 I

KiT4-,Ly12+1 3.0 x ld

wr4- ,bt*+lPT 0.3 x ld

[WT4- ,~~,+IpT 1.0 x 108

UT4- ,bt2+lPT 3.0 x 16

lWT4+.W,+l 3.0 x ld

fL3T4+.Lvt,+l~ 3.0 x lb

FIG. 8. Effect of PT on responder spleen cells fractionated by panning. Responder spleen cells were incubated with PT. After 4 hr, nonadherent cells were separated from adherent macrophage monolayers. Nonadherent cells were separated into Igf and Ig- cells by panning as described under Materials and Meth- ods. Nonadherent T cells (Ig-) were further separated into L3T4+, Lyt l+ and L3T4-, Lyt 2+ cells by panning as described under Materials and Methods. The ‘staining pattern of both L3T4+, Lyt l+, and L3T4-, Lyt 2+ cell populations is shown in Fig. 7 (C, E, and G) and (D, F, and H), respectively. PT-treated or -untreated L3T4+, Lyt l+ and PT-treated or -untreated L3T4-, Lyt 2+ cells were irradiated (so that they themselves do not proliferate in response to allogeneic stimulator cells) and added at varying cell numbers into the CTL-generating system (3 X lo6 responder spleen cells + 1 X lo6 allogeneic stimulator spleen cells). Data are expressed as means + SEM of triplicate samples. These data are representative of five replicate experiments.


ible if PT is removed from cultures within the first few hours. Addition of PT to the 4-hr “G-release assay system did not result in suppression of the CTL response (data not shown), suggesting that the cytolytic capacity of CTL effecters was not affected. PT, when added to fully mature CTL clones (against alloantigens or against self +x) (23), did not suppress their cytotoxic response (Fig. 3). Thus an early stage(s) of the process of CTL generation such as antigen presentation, or T cell-T cell interaction, and proliferation of antigen-sensitive precursor CTL may be the susceptible site(s) for the PT-induced suppression. Our results obtained with PT are similar to those reported for the lymphocytosis-promoting factor (LPF) (20). Moreover, experiments from other laboratories have demonstrated that LPF suppresses the in vitro anti- SRBC antibody response, and is most effective if it is added during the inductive phase of the PFC response. We observed that the suppression of the CTL response by PT was greatest also when it was added on Day 0 or 1 of culture (Fig. 2A). Johnson et al. (29, 30) demonstrated that staphylococcal enterotoxin-A (SEA) also signifi- cantly suppressed the PFC response when it was added on Day 0 or 1 of culture but not when it was added later than Day 2 of culture.

Supematant fluids obtained from spleen cells (preexposed to PT) or from the CTL- generation system (preexposed to PT), when added to fresh CTL cultures, did not suppress the CTL response (data not shown).

Addition of an increasing number of allogeneic stimulator spleen cells (more than the optimal dose) in the CTL-generating system did not abrogate suppression by PT (data not shown), suggesting that immunosuppression by PT was not due to depletion of the antigen. Preexposure of allogeneic spleen cells (antigen) to PT did not result in suppression of the CTL response, whereas preexposure of responder spleen cells to PT resulted in marked suppression of the CTL response (Fig. 4). These results suggest that immunosuppression by PT observed in this system was due to a direct interaction of PT with the responder spleen cells (Fig. 4). It has been well established that PT can modulate cyclic nucleotides and thus interfere with T-cell activation (3 1). Whether suppression of the CTL response observed is due to PT acting on the signal- transducing mechanisms of the responder T cells (acting directly on CTL precursors or helper T lymphocytes (HTL)) needs further examination.

It is generally understood that most immunosuppressive substances either kill or inhibit the proliferation or the reactivity of immunocompetent cells. It has been demonstrated in several systems that the immune responses may be suppressed by the generation of suppressor T lymphocytes (STL) (32-37). In order to determine the cell type that was responsive to PT, spleen cells were fractionated into sIg+ (B lymphocytes) and sIg- (T lymphocytes) subpopulations by anti-immunoglobulin panning. Our results indicate that the cells undergoing maximal proliferation in response to PT are of the sIg- type (Fig. 7). These results are in agreement with the reports of Vogel et al. (38) and Kong and Morse (39) who identified T lymphocytes as the prolif- erating cells in response to PT. Further fractionation of the sIg- population into L3T4+, Lyt If cells (T helper/inducer) or L3T4-, Lyt 2+ cells (T suppressor/cytotoxic) indicates that the L3T4-, Lyt 2+-enriched population undergoes the greatest proliferative response in the presence of PT. This result is consistent with a conclusion that PT induces immunosuppression by stimulating an increase of STL. Further, cells which proliferated in response to PT and the PT-induced STL failed to show any CTL activity when tested directly on the targets (data not shown).


Suppressor cell activity has been associated with the Lyt 2+ subpopulation of T lymphocytes (39, 40). Our results demonstrate that the greatest suppression of the CTL response occurs when the L3T4-, Lyt 2+ subset is added into the CTL-generating system. In view of our observations in total, we hypothesize that the L3T4-, Lyt 2+ subset is the major cell type which generates the PT-induced suppression of the cytotoxic T-lymphocyte response.

It was not possible to conclude from these experiments whether STL had any effect directly on CTL or whether the observed inhibition of CTL responses resulted from loss of help. Preliminary experiments involving the addition of crude IL-2 to spleen cells in cultures were inconclusive in that the CTL responses were all elevated, but the responses of the PT-treated groups remained lower than controls (unpublished observations).

The mechanism of action of the STL described here is not known. Preliminary experiments suggest that STL are of he L3T4-, Lyt 2+ phenotype. It is unlikely that they are cytotoxic for either the CTL effecters or the allogeneic stimulator cells as based on the negative results from direct examination of their cytotoxic potential (data not shown).

It is possible that STL act through interaction with the antigen-presenting cell (APC), thereby preventing the activation of HTL, as opposed to actively suppressing HTL. Alternatively, they might suppress HTL directly, either through the action of a soluble factor or through cellular interaction at the site of antigen recognition. The ability both to use a monoclonal HTL population for the induction of CTL responses and to suppress or prevent the activation of those HTL should enable a better understanding of the mechanism(s) of suppression of the CTL responses by pertussis toxin and perhaps other microbial agents.

ACKNOWLEDGMENTS

We thank Dr. John B. Robbins for critically reviewing the manuscript. We thank Dr. Carl Hansen, geneticist, Small Animal Section, NIH, for his continuing cooperation. We acknowledge the expertise of Mr. Michael Walker of this laboratory in the operation of the Cytofluorograf System 50HH/2 150.

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Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin

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