Vol. 59, No. 10

INFECTION AND IMMUNITY, OCt. 1991, p. 3589-3595 0019-9567/91/103589-07$02.00/0 Copyright © 1991, American Society for Microbiology

Dissociated Development of T Cells Mediating Delayed-Type Hypersensitivity and Protective T Cells against Listeria monocytogenes and Their Functional Difference in Lymphokine Production HIROKI TSUKADA,l12* IKUO KAWAMURA,' MASAAKI ARAKAWA,2 KIKUO NOMOTO,3 AND MASAO MITSUYAMA' Department of Bacteriology' and Department of Medicine (II),2 Niigata University School of Medicine, Niigata 951, and Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812,3 Japan Received 13 May 1991/Accepted 19 July 1991

CD4+ T cells mediating both delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR) were generated in mice after immunization with viable Listeria monocytogenes. In contrast, CD4+ T cells from mice immunized with killed L. monocytogenes in complete Freund's adjuvant were capable of mediating only DTH but not ACR. To determine the functional difference between T cells mediating DTH and T cells mediating ACR, we examined two different populations of T cells for profiles of lymphokine production after stimulation with a specific antigen in vitro. The production of interleukin-2 (IL-2) and IL-3 but not IL-4 was observed in both T cells mediating only DTH and those mediating DTH and ACR. In this respect, both types of T cells could be categorized into the TH1 population, and they produced macrophage chemotactic factor equally well. However, the production of gamma interferon (IFN-'y) was observed only in T cells capable of mediating both DTH and ACR. This result was confirmed not only by an enzyme immunoassay specific for murine IFN--y but also by Northern (RNA) analysis for the detection of IFN--y mRNA. These results suggested that the TH1 population may be subdivided further into two distinct subsets and that the ineffectiveness of the killed bacterial vaccine may be partly explained by the dissociated development of T cell function. as the TH1 type or not, but the dissociated development of TDTH and TACR may raise the possibility that these two T cell populations differ in their profiles of lymphokine production. In the present study, we examined the possible difference in the ability to produce various lymphokines between T cells mediating only DTH and those mediating both DTH and ACR and found that the TACR population is characterized by the ability to produce IFN--y.

Both delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR) are the manifestation of cellmediated immunity to facultative intracellular bacteria. During the course of an active infection with Listeria monocytogenes, DTH and ACR are generated, usually with similar kinetics (23, 27). In contrast, when mice were immunized with killed cells of L. monocytogenes (22) or killed cells or lysates of Mycobacterium tuberculosis (33, 34) along with an adjuvant, only DTH was expressed. Dissociated development of T cells mediating DTH (TDTH) and T cells mediating ACR (TACR) was also observed in an in vitro primary culture system inducing L. monocytogenes-specific T cells (25). Several reports showed that the effect of administration of anti-L3T4 monoclonal antibody to mice was different for DTH and ACR (5, 24). Thus, there is no doubt that DTH and ACR to facultative intracellular bacteria are dissociable phenomena. Although there are findings suggesting that double-negative y/b T cells (15, 32) or CD8+ T cells (18) contribute to cell-mediated immunity to Listeria or Mycobacterium cells, CD4+ T cells appear to play the central role in the expression of DTH and ACR (28, 33). CD4+ T cells have been considered to be responsible for both helper function in antibody production and lymphokine production in DTH (14). On the basis of the lymphokine profile produced by CD4+ T cell clones, two major groups, TH1 and rH2, have been defined (30). THl-type cells are charactenrzed by the ability to synthesize interleukin-2 (IL-2) and gamma interferon (IFN--y) but not IL-4 and are believed to be responsible for DTH (3). It is not yet clear whether TACR are categorized *

MATERIALS AND METHODS Experimental animals. Male mice of the C3H/He strain, raised and maintained under specific-pathogen-free conditions, were used for experiments at 7 to 10 weeks of age. Microorganism. L. monocytogenes EGD, originally obtained from G. B. Mackaness (Trudeau Institute, Saranac Lake, N.Y.), was used throughout the study. The bacteria were grown in tryptic soy broth (Difco Laboratories, Detroit, Mich.) at 37°C for 16 h, washed repeatedly, suspended in phosphate-buffered saline (PBS), and stored at -70°C. Killed cells of L. monocytogenes were prepared by heating a viable bacterial suspension of a known concentration for 90 min at 74°C (25). Culture medium. The medium (complete RPMI medium) consisted of RPMI 1640 (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal calf serum (Flow Laboratories Inc., McLean, Va.), 100 U of penicillin per ml, 100 ,ug of streptomycin per ml, and 5 x 10-2 mM 2-mercaptoethanol. Immunization of mice. One group of mice was intravenously immunized with 2 x 103 viable L. monocytogenes cells. Another group of mice was immunized by subcutane-

Corresponding author. 3589

3590

TSUKADA ET AL.

ous injection of 1 x 108 killed L. monocytogenes cells in complete Freund's adjuvant (CFA; Difco). Mice were used as immune animals 7 days after immunization. Local transfer of DTH and ACR. Local cell transfer was carried out by the method of Mitsuyama et al. (27) with slight modifications. In brief, 10 days after the immunization, single-cell suspensions were prepared from the spleen or draining lymph nodes. After passage over a column packed with nylon wool fiber (Wako Pure Chemicals, Osaka, Japan), 5 x 106 nylon wool-nonadherent T cells were resuspended with 5 x 107 killed L. monocytogenes cells in 0.05 ml of Hanks balanced salt solution. The mixture was injected into the left hind footpads of naive, age- and sex-matched recipient mice, and the degree of DTH was measured as the delayed footpad reaction 24 h later. Immediately after the measurement of the delayed footpad reaction, 105 viable L. monocytogenes cells were inoculated into the same footpads. The number of bacteria in the footpads was determined 24 h after the challenge by plating a serially diluted homogenate on agar plates. Thus, ACR was measured by the capacity to eliminate challenge bacteria at the site of cell transfer. Treatment of nylon wool-nonadherent T cells with antibody and complement. Nylon wool-nonadherent cells were treated with antibody and complement in some experiments. Monoclonal anti-Thy 1.2 antibody and monoclonal anti-Lyt 2.1 (anti-CD8) antibody were obtained from Meiji Nyugyo Inc. (Tokyo, Japan). Monoclonal anti-L3T4 (anti-CD4) antibody (RL 172.4) was purchased from Cedarlane Laboratories Ltd. (Hornby, Ontario, Canada). The cells were incubated with appropriate dilutions of monoclonal antibodies for 45 min at 4°C, washed, and exposed to a 1:10 dilution of Low-Tox-M rabbit complement (Cedarlane) for 45 min at 37°C. The cells were washed and adjusted to the desired concentration. Assay of L. monocytogenes-specific antibody titers in immune mouse serum. Serum was collected from each group of immune mice 7 days after immunization. The serum antibody titer was determined by an enzyme immunoassay (EIA) by a previously described method (1) with some modifications. In brief, a viable L. monocytogenes cell suspension was sonicated for three cycles at 180 W for 5 min with an ultrasonicator (Insonater model 200 M; Kubota, Tokyo, Japan). The supernatant containing solubilized antigens was adjusted to a protein concentration of 25 ,ug/ml in coating buffer. The sonicated antigen (50 pl) was placed in polystyrene microtiter wells, adsorbed to the bottom at 4°C overnight, and used as a coating antigen. After blocking was done with 0.5% bovine serum albumin, test serum diluted 1:100 with PBS-Tween 20 (0.05% [vol/vol]) was placed in individual wells in twofold dilution steps. PBS-Tween 20 was placed in control wells. The binding of a specific antibody to the sonicated bacterial cells was detected with alkaline phosphatase-conjugated goat antibody to mouse immunoglobulin (Sigma Chemical Co., St. Louis, Mo.) and then with a phosphatase substrate. Wells were washed four times for 10 min each time with PBS-Tween 20 before the addition of each principal reagent. The optical density was read at 492 nm on a Microelisa Auto Reader (ETY-III; Toyo Inc., Tokyo, Japan). Preparation of an immune culture supernatant. Spleen cells or draining lymph node cells were passed over a nylon wool column, and nonadherent T cells (5 x 106/ml) were cultured for 48 h in the presence of killed cells of L. monocytogenes (5 x 108/ml) and proteose peptone-induced peritoneal exudate cells (2.5 x 105/ml) as antigen-presenting cells. After incubation, the culture fluid was centrifuged at 1,600 x g for

INFECT. IMMUN.

15 min and at 12,000 x g for 20 min, passed through a 0.22-,um-pore-size membrane filter, and kept at -20°C until use. Assessment of lymphokine activity. IL-2 activity in the culture supernatant was determined by the proliferation of IL-2-dependent HT-2 cells. HT-2 cells (104 cells per well) were cultured with the culture supernatant in a 96-well microplate for 24 h at 37°C. During the last 6 h of culturing, [3H]thymidine was added. The cultures were harvested, and the incorporated radioactivity was counted in a liquid scintillation counter. IL-3 activity was determined in the same way by the proliferation of IL-3-dependent FDC-P2 cells (7). WEHI-3, a cell line known to be a constitutive producer of IL-3 (13), was used as the source of the control supernatant. IL-4 activity in the culture supernatant was determined by a bioassay with CTLL cells. CTLL cells (104 cells per well) were cultured with the sample in the presence of anti-IL-2 antibody S4B6 or anti-IL-4 antibody llBli. Monoclonal antibody S4B6 was a generous gift from Hideo Nariuchi, The

Institute of Medical Science, The University of Tokyo (Tokyo, Japan), and monoclonal antibody llBl was purchased from Texstar Monoclonals (Dallas, Tex.). Proliferation in the presence of S4B6 antibody was regarded as IL-4 activity. The cell proliferation assay was carried out in triplicate wells for each sample. Macrophage-chemotactic factor (MCF) activity in the culture supernatant was assessed by the method of Snyderman et al. (35) with some modifications. A sample diluted 1:1 with medium was placed in the lower wells of blind-well chemotaxis chambers. A polycarbonate membrane filter with 5-pum pores (Nuclepore Corp., Pleasanton, Calif.) was placed between the upper and lower wells. As indicator cells, peritoneal exudate cells were collected from mice injected intraperitoneally with 1.5 ml of proteose peptone 3 days before the assay. The cells (6 x 105/ml) in 500 pul of medium were added to the upper wells. After 90 min of incubation, the filters were removed, washed, air dried, and stained with Giemsa solution. The number of migrating macrophages was counted in five randomly selected high-power fields. The IFN-y titer was determined by an EIA as described by Firestein et al. (8) with some modifications. EIA plates were coated with 1.5 ,ug of rat anti-murine IFN-y monoclonal antibody (LEE Biomolecular Research Inc., San Diego, Calif.). After the plates were washed and blocked, serial twofold dilutions of samples or standard murine recombinant IFN--y were applied to the wells. After 90 min of incubation, the wells were washed and incubated with 100-fold-diluted rabbit anti-IFN-,y polyclonal antibody for 90 min and then with peroxidase-conjugated goat anti-rabbit immunoglobulin G (Cappel, Organon Teknika Corp., West Chester, Pa.). Finally, o-phenylenediamine in phosphate-citrate buffer with H202 was added as a substrate solution, and the reaction was terminated with H2SO4. The A492 was measured, and IFN--y activity was expressed as international units per milliliter determined against a known reference standard recombinant IFN-y. Murine recombinant IFN--y and rabbit anti-IFN--y polyclonal antibody were generous gifts from the Central Research Institute, Daiichi Seiyaku Co. Ltd., Tokyo, Japan. Lymphocyte RNA extraction and detection of IFN--y transcripts. Twelve hours after stimulation of nylon wool-passed immune T cells with killed L. monocytogenes, cells were collected and RNA was extracted by the guanidium thiocyanate-CsCl gradient centrifugation procedure (4). Ten micrograms of total RNA was glyoxylated, subjected to electrophoresis through 1% agarose in 10 mM phosphate buffer (pH 7.0), and transferred to a nylon membrane (Hybond-N;

LYMPHOKINE PROFILE OF T CELLS AGAINST L. MONOCYTOGENES

VOL. 59, 1991

TABLE 1. Local transfer' of DTH and protection by T cells from mice immunized with viable or killed L. monocytogenes Immunization of donor mice

Treatment of spleen cells'

None VLm i.V.e

KLm-CFA s.c.Y

3591

TABLE 2. Antibody titers to solubilized antigens of L. monocytogenes in sera from mice immunized with viable or killed L. monocytogenes, as determined by EIA

DTHc (0.1 mm)

Log10 CFU 24 h after

Immunization of mice

A492"

Ratio

4.6 ± 0.6

5.99 ± 0.13

None KLm-CFA s.c.b VLm i.v.d

0.194 ± 0.027 0.386 ± 0.025C

1.00 1.99

0.268 ± 0.008C

1.38

None C only anti-Thy 1.2 + C anti-CD4 + C anti-CD8 + C

13.5 12.6 6.2 4.4 1Q.8

None C only anti-Thy 1.2 + C anti-CD4 + C anti-CD8 + C

9.2 8.6 4.7 3.8 7.4

± ± ± ± ±

1.2 0.8

1.4f l.lf 1.3

± 0.6 ± 1.0 ± 0.8f

O.5f ± 0.7

4.51 4.48 5.76 5.80 4.66

± ± ± ± ±

0.11 0.14

0.08f O.lO 0.11

5.96 ± 0.21 NDh ND ND ND

a Nonadherent spleen cells (5 x 106) were injected into footpads along with killed L. monocytogenes cells (5 x 107). b Cells were treated either with complement (C) only or with monoclonal antibodies plus C before local transfer. c DTH was measured 24 h after elicitation. Data are expressed as means for five or six mice ± standard deviations. d Viable L. monocytogenes cells (10W) were locally injected into footpads, and the number of CFU in footpads was determined 24 h later. Data are expressed as means for five or six mice ± standard deviations. eVLm, viable L. monocytogenes; i.v., intravenously. f P < 0.01 when compared with the results for nontreated spleen cells from VLm-immune mice. g KLm, killed L. monocytogenes; s.c., subcutaneously. h ND, not done.

Amersham Ltd., Amersham, United Kingdom). The filter membrane was hybridized with a 32P-labeled DNA probe specific for murine IFN--y for 16 h at 65°C. The probe was an 800-bp cDNA insert obtained from Yasunobu Yoshikai (Nagoya University, Nagoya, Japan). Ethidium bromide staining of rRNA served as a molecular marker. The filter membrane was exposed to Kodak X-Omat film at -85°C, and the autoradiogram was developed. Statistical analysis. Data were expressed as means + standard deviations, and Student's t test was used to determine the significant differences between control and experimental values. RESULTS

Dissociated development of TDTH and TACR. Adoptive local transfer of spleen cells from mice immunized with viable L. monocytogenes into footpads of naive recipient mice resulted in the expression of both DTH and local protection upon antigenic elicitation or challenge infection, as shown in Table 1. Negative selection revealed that the effector cells for both DTH and ACR were the CD4+ helper-type T cells. When cells from mice immunized with killed bacteria in CFA were locally transferred, DTH could be expressed at a level comparable to that expressed by cells from mice immunized with viable bacteria; however, a significant level of local protection was never observed (Table 1). Differences in L. monocytogenes-specific antibody titers in immune mouse serum. Titers of serum antibody directed against sonicated antigen of L. monocytogenes were significantly elevated in mice 7 days after immunization with both viable and killed bacteria compared with those in nonimmune control mice. Sera from mice immunized with killed bacteria in CFA showed a higher level of antibody response

a A linear regression was obtained from 1:320 to 1:1,280, and the A492 for 1:640-diluted sera was expressed as the mean of triplicate wells + the standard deviation. I See Table 1, footnote g. c P < 0.01 when compared with the control. d See Table 1, footnote e.

than did those from mice immunized with viable bacteria (Table 2). Differences in the ability to produce IL-2, IL-3, and IL-4. Lymphocytes were obtained from the spleen or draining lymph nodes of mice immunized with either viable bacteria or killed bacteria in CFA. Culture supernatants obtained 2 days after stimulation with killed bacterial antigen were examined for the IL-2, IL-3, and IL-4 activities. The ability to produce a significant level of IL-2 activity was found in cells from both viable bacterium-immune mice and killed bacterium-immune mice when assessed by HT-2 cell proliferation (Fig. 1). Higher activity was detected in the culture supernatant of draining lymph node cells from mice immunized with killed bacteria in CFA. The results of the IL-3 assay, measured as FDC-P2 cell proliferation, were almost the same as those of the IL-2 assay (Fig. 2). It was clear that immunization with killed bacteria generated T cells capable of producing IL-2 and IL-3, just as immunization with viable bacteria did. The activity of IL-4, a lymphokine characteristic of the TH2 type of helper T cells, was determined with CTLL cells, which are capable of responding to both IL-2 and IL-4. In the absence of a neutralizing monoclonal antibody, the proliferation of CTLL cells was stimulated by culture supernatants from both groups. The activity was almost completely abolished by monoclonal S4B6, which neutralizes IL-2, but not at all by monoclonal antibody llBli, which neutralizes IL-4 (Fig. 3). The activity in culture supernatants of IL-4-producing D1OG4 cells was completely eliminated by the same amount of antibody llBli. It was clear that IL-4 was not produced in the cultures. These results showed that there were no differences in the profiles of IL-2, IL-3, and IL-4 production between T cells capable of mediating only DTH and those capable of mediating both DTH and ACR. Differences in the ability to produce MCF and IFN-,y. MCF and IFN--y are believed to be the important lymphokines directly relevant to the expression of DTH and ACR. MCF activity was observed in the culture supernatants of cells from mice immunized with killed bacteria in CFA as well as in those of cells from mice immunized with viable bacteria, as previously described (23). However, IFN--y activity was detected only in cultures of cells from viable L. monocytogenes-immune mnice (Table 3). The difference in the ability to produce IFN-y, a THl-type lymphokine, upon stimulation with a specific antigen was further confirmed by Northern blot analysis. mRNA specific for IFN--y was highly detectable only after antigenic stimulation of spleen cells from mice immunized with viable L. monocytogenes (Fig. 4). Analysis of the T cell population responsible for the production of IFN--y. To determine whether antigen-stimulated

3592

.

INFECT. IMMUN.

TSUKADA ET AL.

Culture sup of

Tcells from mice immunized with

3H-TdR UPTAKE of HT-2 cell iv jp

(103cpm) 14

1

none

HK Lm/CFA scl ...........................................................................................

O 0.1

murine

rlL-2

-I \H

\

10.2

u1

\-

0.4 \\\

FIG. 1. Production of IL-2 as determined by a bioassay with HT-2 cells. Spleen cells or draining lymph node cells from mice immunized with viable or killed L. monocytogenes were passed over a nylon wool column, and nylon wool-nonadherent T cells (5 x 106/ml) were cultured for 48 h in the presence of killed cells of L. monocytogenes (5 x 108/ml) and peritoneal exudate cells (2.5 x 105/ml) as antigen-presenting cells. IL-2 activity was expressed as the incorporation of [3H]thymidine (3H-TdR) into HT-2 cells (104) cultured in the presence of each culture supernatant (sup) (final dilution, 1:4). Murine recombinant IL-2 (rIL-2) was used as the standard. Data were expressed as the mean counts per minute for three wells + the standard deviation. VLm, viable L. monocytogenes; i.v., intravenously; HKLm, heat-killed L. monocytogenes; s.c., subcutaneously.

production of IFN--y is limited only to CD4+ T cells, we performed a negative selection experiment. T cells from mice immunized with viable L. monocytogenes were treated with anti-Thy 1.2 antibody plus complement, anti-CD4 antibody plus complement, anti-CD8 antibody plus complement, and complement only. After stimulation with killed listerial antigen for 48 h, the IFN--y titers in the supernatants of the cultures were assayed. High IFN--y titers were detected only in cultures of T cells treated with anti-CD8 antibody plus

complement (Fig. 5), indicating that CD4+ T cells are the main population responsible for IFN--y production. DISCUSSION The mutual relationship between DTH and ACR in the cell-mediated immune response to facultative intracellular bacteria has been of interest for a long time (27, 31, 38). On the basis of the results obtained in various experimental

3H-TdR UPTAKE of FDC-P2 cell (104cpm)

Culture sup of

Tcells from mice

10

_v_

immunized with

none

VLm iv

HKLm/CFA

medium

_

.-=W

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\Is,\IN,\\\\\\§ i

sc

I...............................

.................................................

...

0:I

dilution of

WEHI-3

culture sup

X.,

FIG. 2. Production of IL-3 as determined by a bioassay with FDC-P2 cells. Spleen cells or draining lymph node cells from mice immunized with viable or killed L. monocytogenes were passed over a nylon wool column, and nylon wool-nonadherent T cells (5 x 106/ml) were cultured for 48 h in the presence of killed cells of L. monocytogenes (5 x 108/ml) and peritoneal exudate cells (2.5 x 105/ml) as antigen-presenting cells. IL-3 activity was expressed as the incorporation of [3H]thymidine (3H-TdR) into FDC-P2 cells (104) cultured in the presence of each culture supernatant (sup) (final dilution, 1:4). A culture supematant of WEHI-3 cells was used as the IL-3 control. Data were expressed as the mean counts per minute for three wells + the standard deviation. Abbreviations are as defined in the legend to Fig. 1.

VOL. 59, 1991

LYMPHOKINE PROFILE OF T CELLS AGAINST L. MONOCYTOGENES

3593

E T

8~ ~ ~ ~

~

~

~

~

~

~

~

~~~

6-

CL~ ~ ~

nodne

~

Va Lm

~~~~~~~Cm

H(KLranvtFA

mediumrS L

1A

Q'.6ukn

r

nOG

sup

3. Production of IL-2 and IL-4 as determined by a bioassay with CTLL cells and neutralizing monoclonal antibodies. Spleen cells draining lymph node cells from mice immunized with viable or killed L. monocytogenes were passed over a nylon wool column, and nylon wool-nonadherent T cells (5 x 10"/ml) were cultured for 48 h in the presence of killed cells of L. monocytogenes (5 x 10'/ml) and peritoneal exudate cells (2.5 x 105/mI) as antigen-presenting cells. IL-2 and IL-4 activities in the above-mentioned culture supernatants (sup) were (H-TdR) into CTLL cells (10) in the presence of each culture~ expressed as the incorporation of culture superrpatant [3H]thymiin (U) or anti-IL-2 antibody S4B6 ( M). A culture supernatant of D1OG4 cells supernatant (final dilution, 1:4) (0) and anti-IL-4

FIG.

or

was used as the IL-4 control. Data were are as defined in the legend to Fig. 1.

antioyllBll

expressed

as

the

mean

counts per

systems, it is now clear that the phenomena are not mediated by the same cells (16, 22, 27, 39). However, the critical difference between T cells mediating only DTH and those mediating ACR along with DTH is still unclear. Various lymphokines are released from antigen-stimulated T cells of mice immunized with listerial antigen (19, 20). The present study was mainly focused on the differences in the profiles of lymphokines produced by CD4+ T cells after stimulation with a specific antigen. On the basis of previous findings (22), TDTH could be generated by immunization with killed bacteria in CFA and TACR could be generated by immunization with viable bacteria. After antigenic stimulation in vitro, both TDTH and TACR produced a significant level of IL-2 and IL-3 but not IL-4. The IL-2 and IL-3 activities were higher in the TDTH culture. In this respect, both T cell populations seemed to be categorized as THltype T cells, since TH1 and TH2 are regarded as nonoverlapping subsets that selectively utilize IL-2 and IL-4, respectively, as their autocrine growth factors (30). There was a possibility that some of the immune T cells were of the TH2 type and may have released TH2-type lymphokines, including IL-4, since small amounts of L. monocytogenes-specific antibody were detected in the sera from both viable bacteTABLE 3. MCF and IFN-y activities in culture supernatants of T cells from mice immunized with viable or killed L. monocytogenes Immunization of mice

None KLm-CFA s.c.c VLm i.v.e

MCF activity (no. of migrating macrophages)a 36 11 113 + 12d 114 ± 23d

mpinute for three wells

+

the standard deviation. Abbreviations

rium- and killed bacterium-immune mice and TH2-type T cells were considered to be functioning as the helper for antibody formation (2, 21, 37). However, no detectable level of IL-4 was produced by T cells from either group of mice, at least in our bioassay; therefore, most of the T cells specific for L. monocytogenes antigens seemed to be differentiated into THl-type cells. Among several lymphokines examined, it was found that the ability to produce IFN-y, which acts on mononuclear phagocytes, causing macrophage activation (36), was characteristic of TACR. There was no difference in the ability to

-E

2 S

--c lBS

IFN-y

-

IFN-y titer in culture(U/ml)b supernatant 21 ± 6 46 ± 11 480 + 22d

a Number of peritoneal macrophages that migrated to the surface of the filter facing the lower chamber containing the supematant of each culture. b Determined by a murine IFN-y-specific EIA. c See Table 1, footnote g. d p < 0.001 when compared with the control. ' See Table 1, footnote e.

FIG. 4. Northern blot analysis of the expression of mRNA specific for IFN--y after antigenic stimulation of T cells from mice immunized with viable or killed L. monocytogenes. RNA was extracted from nylon wool-passed T cells (5 x 10"/ml) after stimulation with killed L. monocytogenes (5 x 108) for 12 h. Each lane was loaded with 10 JLg of RNA. Cont, control; other abbreviations are as defined in the legend to Fig. 1.

3594

TSUKADA ET AL.

INFECT. IMMUN.

treatment of T cells with

IFN-J) titer in the culture sup. (U/i-nl) 1000 2000

no n e

antiThyl

+

C

anti CD4+ C antiCD8+C C= C only

\\

\\

FIG. 5. Titration by an EIA of IFN-y in the culture supernatants (sup) of nylon wool-passed T cells from mice immunized with viable L. monocytogenes after treatment with monoclonal antibodies plus complement (C).

produce MCF, another lymphokine which also acts directly on macrophages (10, 17) and partly contributes to mediating ACR (6, 29). This finding was consistent with our previous observation that the local administration of purified MCF was not sufficient for the expression of local protection against challenge infection and that the synergistic action of MCF and IFN--y was required (10, 11). The general agreement concerning TH1 seems to be that IFN--y is one of the lymphokines that characterize THl-type T cell clones and that DTH is mediated by THl-type cells (3, 30). The present finding that DTH can be expressed by T cells incapable of producing IFN--y may be contradictory to the above-mentioned idea. Fong and Mosmann recently reported that DTH responses, expressed after adoptive transfer of several TH1 clones, were not inhibited by antibody to IFN--y (9). Taking this fact into consideration, it may be possible that there are two types of cells responsible for DTH: one is represented by typical TH1 clones producing lymphokines, including IFN-y, and the other is represented by TH1 clones lacking the ability to produce IFN-y. The developmental relationship between TDTH and TACR examined in the present study is still not clear. TDTH lacking the ability to produce IFN-y may be one subset of THl-type cells differing from the subset of TACR; alternatively, TDTH may be identical to TACR except for differing in the stage of functional development. It is quite difficult to obtain T cells mediating ACR but not DTH; therefore, the latter possibility seems to be plausible. In careful kinetic study of T cells during the course of an active infection, TDTH might be detectable in the early stage, since we previously observed the accumulation of macrophages without functional activation at an early phase of an active infection with L. monocytogenes

(29).

Another question to be answered is why ACR is easily generated in an active infection but not after immunization with killed bacteria (38). In the experiments with L. monocytogenes, the critical difference between viable and killed bacteria seemed to be in the ability to stimulate IL-1 production from macrophages and not in the antigenicity (26). The administration of recombinant IL-1 accelerated the functional development of T cells in mice immunized with killed bacteria without adjuvant but failed to induce TACR (12); therefore, some other factor seems to be critically operating in the final development of TH1 into TACR. Finally, attention should be paid to the fact obtained in the present study that assays of lymphokines other than IFN--y

do not always detect the presence of TACR or protective immunity in the host, since T-cell proliferation and IL-2 assays have been widely used for the assessment of vaccine efficiency and for the detection of "protective antigen." ACKNOWLEDGMENTS We are grateful to the Central Research Institute, Daiichi Seiyaku Co. Ltd. (Tokyo, Japan), for providing us with murine recombinant IFN--y and rabbit anti-IFN--y polyclonal antibody, to Yasunobu Yoshikai (Nagoya University, Nagoya, Japan) for the cDNA probe for IFN--y, and to Hideo Nariuchi (The University of Tokyo, Tokyo, Japan) for the S4B6 monoclonal antibody. This work was supported in part by a Grant-in-Aid for Scientific Research to M.M. (02454176) from the Ministry of Education, Science and Culture of Japan and by a grant provided by Niigata Medical Research Foundation, Niigata, Japan. REFERENCES 1. Black, J. R., W. J. Black, and J. G. Cannon. 1985. Neisserial antigen H.8 is immunogenic in patients with disseminated gonococcal and meningococcal infections. J. Infect. Dis. 151: 650-657. 2. Boom, W. H., D. Liano, and A. K. Abbas. 1988. Heterogeneity of helper/inducer T lymphocytes. II. Effect of interleukin 4- and interleukin 2-producing T cell clones on resting B lymphocytes. J. Exp. Med. 167:1352-1363. 3. Cher, D. J., and T. R. Mosmann. 1987. Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones. J. Immunol. 138:3688-3694. 4. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-

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