INFECTION AND IMMUNITY, OCt. 1991, p. 3439-3445
Vol. 59, No. 10
0019-9567/91/103439-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Endogenous Gamma Interferon-Independent Host Resistance against Listeria monocytogenes Infection in CD4+ T Celland Asialo GM1+ Cell-Depleted Mice AKIO NAKANE,* AKIHIKO NUMATA, YU CHEN, AND TOMONORI MINAGAWA Department of Microbiology, Hokkaido University School of Medicine, Kita 15 Nishi 7, Kita-ku, Sapporo 060, Japan Received 16 April 1991/Accepted 13 July 1991
The effects of in vivo administration of antibodies against T-cell subsets and asialo GM1 (ASGM1)-bearing ceUs on endogenous gamma interferon (IFN-,y) production and host defense in Listeria monocytogenes-infected mice were investigated. Endogenous IFN-y titers in the bloodstreams and spleen extracts of mice on day 2 of infection were partially suppressed by administration of rabbit anti-ASGM1 antibody, but not by anti-CD4 monoclonal antibody (MAb) or anti-CD8 MAb. Of the different combinations of these three antibodies, the most suppressive effect on IFN--y production was observed after administration of anti-CD4 MAb and anti-ASGM1 antibody, although anti-CD8 MAb combined with anti-CD4 MAb partially inhibited IFN-y production. In contrast, antilisterial resistance was suppressed by the administration of anti-CD8 MAb but not by anti-CD4 MAb or anti-ASGM1 antibody. Antilisterial resistance in mice in which both CD4+ cells and ASGM1+ cells had been depleted was performed as efficiently as in normal mice in spite of the fact that endogenous IFN-y production was markedly suppressed. Furthermore, these mice also eliminated L. monocytogenes cells efficiently from the spleens even when they were pretreated with anti-mouse IFN-,y MAb. These results indicate that CD4+ T cells, CD8+ T cells, and ASGM1+ cells are all responsible for endogenous IFN-'y production and that antilisterial resistance and endogenous IFN--y production are not absolutely correlated.
Complete elimination of Listeria monocytogenes, a facultative intracellular pathogen, from the tissues of infected animals is performed by T-cell-dependent mechanisms (18, 27). The contributions of CD4+ and CD8+ T-cell subsets to antilisterial resistance were compared recently by depleting these cells with monoclonal antibodies (MAbs) in vivo (3, 7, 20, 29). The results of these studies suggest that CD8+ T cells are critical for host defense, whereas CD4+ T cells make a minor contribution to antilisterial resistance. Two possible roles of CD8+ T cells in host defense are proposed (12, 13): (i) CD8+ T cells might lyse L. monocytogenesinfected host cells; (ii) CD8+ T cells might produce cytokines involving gamma interferon (IFN-y). The crucial role of endogenously produced IFN--y in antilisterial resistance has been demonstrated by in vivo injection of MAbs against this factor (1, 5, 24). Studies on characterization of cell types which produce IFN--y by stimulation with L. monocytogenes cells in cultures of spleen cells or T-cell clones derived from immune mice demonstrated that both CD4+ T cells and CD8+ cells secrete IFN-y (4, 14, 15, 17, 19). In addition, T-cell-independent production of IFN--y, which might be produced by natural killer (NK) cells, was demonstrated in scid mice (1, 2, 32). However, in vivo analysis of a mechanism of endogenous IFN--y production induced by primary infection with L. monocytogenes has not been reported. We demonstrated previously that endogenous IFN--y is detected in the bloodstreams and spleens of mice on days 1 to 4 of L. monocytogenes infection (26). Therefore, we focused our studies on in vivo analysis of cells participating in endogenous IFN-y production, especially on the possibility of CD8+ T cells as *
IFN--y-producing cells, in L. monocytogenes infection by in vivo injection of antibodies against T-cell subsets and asialo GM1 (ASGM1)-bearing cells, which mainly correspond to NK cells. In this report, we provide evidence that CD4+ T cells, CD8+ T cells, and ASGM1+ cells are all responsible for endogenous IFN-y production. Our results demonstrate further that antilisterial resistance and endogenous IFN--y production are not absolutely correlated. MATERIALS AND METHODS Mice. Female ddY mice (age, 5 to 7 weeks; obtained from SLC, Hamamatsu, Shizuoka, Japan) were used. Bacteria. L. monocytogenes lb 1684 was prepared as described previously (25). The concentration of washed cells was adjusted spectrophotometrically at 550 nm. Mice were infected intravenously with 0.2 ml of a solution containing 2 x 104 CFU of viable L. monocytogenes cells in 0.01 M phosphate-buffered saline (PBS; pH 7.4). Antibodies. Hybridoma cells secreting MAbs directed against CD4 (L3T4) (GK1.5, rat immunoglobulin G2b) (8) and CD8 (Lyt-2) (53-6.72, rat immunoglobulin G2a) (16) antibodies were kindly provided by T. Koike, School of Medicine, Chiba University, Chiba, Japan. A rat anti-mouse IFN--y-secreting hybridoma (R4-6A2, immunoglobulin Gl) (30) was donated by Y. Watanabe, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. These MAbs were prepared from ascites fluid in Pristane-primed CD-1 nulnu mice. Partial purification by 50% (NH4)2SO4 precipitation was followed by exhaustive dialysis against PBS. Rabbit anti-ASGM1 antibody was prepared and partially purified by 50% (NH4)2SO4 precipitation as described previously (22). The antibodies were aliquoted and stored at -700C.
Corresponding author. 3439
3440
NAKANE ET AL.
In vivo depletion of CD4+ and CD8+ cells. We gave each mouse a single 400-pRg intravenous injection of anti-CD4 MAb or anti-CD8 MAb (in 0.2 ml of pyrogen-free saline) on day -1 of L. monocytogenes infection. Normal rat globulin, prepared and purified by (NH4)2SO4 precipitation from the pooled sera of Wistar rats (SLC), was injected as a control. Evaluation of in vivo cell depletion by flow cytometry. Cell suspensions prepared from spleens or mesenteric lymph nodes of infected and noninfected mice treated with MAbs or normal rat globulin in vivo were incubated with biotinylated anti-CD8 antibody (Lyt-2) (53-6.7; Becton Dickinson, Mountain View, Calif.) in 100 ,ul of PBS supplemented with 0.1% bovine serum albumin (fraction V; Sigma Chemical Co., St. Louis, Mo.) and 0.1% NaN3 at 4°C for 30 min. After being washed, cells were treated simultaneously with fluorescein isothiocyanate-labeled streptavidin (Caltag Laboratories, Inc., South San Francisco, Calif.) and phycoerythrinlabeled anti-CD4 (L3T4) (GK1.5; Becton Dickinson) antibody at 4°C for 30 min. The cells were then washed three times and analyzed on FACScan (Becton Dickinson) after gating to exclude macrophages and polymorphonuclear leukocytes (number of events, 10,000). In vivo depletion of ASGM1+ cells. Rabbit anti-ASGM1 antibody has been shown previously to deplete NK cell activity in vivo and in vitro (9, 11). To deplete NK cell activity in vivo, 400 jig of anti-ASGM1 antibody was given intravenously in a volume of 0.2 ml on day -1 of L. monocytogenes infection (22). Normal rabbit globulin, prepared and purified by (NH4)2SO4 precipitation from the serum of a nonimmunized rabbit, was injected as a control. To determine whether NK cell activity was abolished by this procedure, spleens were removed from mice treated with anti-ASGM1 antibody or normal rabbit globulin on days 2 and 7 of infection and used in a standard 4-h 51Cr release assay with labeled YAC-1 targets at a 50:1 effector/target cell ratio (9). In vivo depletion of endogenous IFN--y. To deplete endogenous IFN--y in vivo, we gave each mouse a single intravenous injection of 1 mg of rat anti-mouse IFN--y MAb (in 0.2 ml of pyrogen-free saline) 2 h before L. monocytogenes infection, as described previously (24). Normal rat globulin was injected as a control. Tests for endotoxin contamination. All in vivo effects of antibodies or normal globulins described were verified by using reagents tested by the Limulus amoebocyte lysate assay to contain 90% of CD4+ or CD8+ T cells in the spleens and mesenteric lymph nodes was already observed on day 0 of infection and continued until at least day 10 for anti-CD4 MAb and until at least day 17 for anti-CD8 MAb. Single injections of 400 ,ug of either anti-ASGM1 antibody or normal rabbit globulin were given on day -1 of infection, and the efficacy of the antibody in depleting NK cells was determined by reduction of cytolytic activities of spleen cells against YAC-1 targets. Marked reduction of NK cell activity continued at least until day 7 of infection (normal rabbit globulin-treated mice versus anti-ASGM1 antibody-treated mice [percent lysis]; 23.1 + 1.8 versus 1.6 ± 0.5 [day 2 of infection], 29.3 ± 2.7 versus 2.9 ± 0.7 [day 7 of infection]). Effect of MAbs against T-cell subsets or anti-ASGM1 antibody on antilisterial resistance. Mice were injected with MAbs against T-cell subsets or anti-ASGM1 antibody the day before infection with a sublethal dose of the bacterium (2 x 104 CFU; 0.1 50% lethal dose). The number of L. monocytogenes cells in the spleens of the treated mice was determined early (day 2) and late (day 6) in infection (Fig. 2). The growth of bacterial cells in the organs of the antibodytreated mice was not significantly different from that in the corresponding normal globulin-treated controls in the early stage of infection (P > 0.05). In contrast, elimination of bacteria from the spleens was significantly blocked in antiCD8 MAb-treated mice (P < 0.01), but not in anti-CD4 MAb- or anti-ASGM1-treated animals (P > 0.05), in the late stage of infection. The suppressive effect in anti-CD8 MAbtreated mice was equivalent to that in the groups which received a combination of anti-CD4 MAb and anti-CD8 MAb or a combination of these two MAbs and anti-ASGM1
antibody. Effect of MAbs against T-cell subsets or anti-ASGMl antibody on endogenous IFN-,y production. IFN--y in sera and
VOL. 59, 1991
IFN-y IN L. MONOCYTOGENES INFECTION
is,
:
a-
a
1
0.03% A
12.02%
i~~~~~~~~~~~~~~~
83.23i
3441
a
4.72%
B
0.04%
0
92. 33±
7. 63%
a
0C a 0M
.'1- I'"""
L I'
~100
10
.103
102
a
10ig a 4
-
1la
CD8 _11.65%
a
0.04% C
0.
a1'
.049%
0.00%
D
a
a
a
1a3
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-88.15?.
l.S
99. 34?
0.16% IV
im
11-
02
0.17%
a
.7, ~2~ YVL '103
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CD8
32
103
104
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FIG. 1. Fluorocytometric analysis of spleen cells from L. monocytogenes-infected mice which had been injected with normal rat globulin (A), anti-CD4 MAb (B), anti-CD8 MAb (C), or a combination of both MAbs (D) on day -1 of infection. Three mice per group were sacrificed on day 7 of infection, and spleen cells were analyzed for T-cell marker expression with a fluorescence-activated cell sorter. Mice treated with normal rat globulin and those which were untreated gave essentially identical results. Only the results from mice treated with normal rat globulin are shown.
spleen extracts obtained from the antibody-treated mice or the corresponding normal globulin-treated mice 48 h after infection was determined (Fig. 3). IFN--y production in the bloodstreams and spleens of anti-CD4 MAb-treated mice or anti-CD8 MAb-treated mice was not significantly different from that in normal rat globulin-treated animals (P > 0.05). In contrast, the titers of endogenous IFN--y were significantly reduced in anti-ASGM1-treated mice (serum, P < 0.05), in mice which received a combination of anti-CD4 MAb and anti-CD8 MAb (serum, P < 0.05; spleen, P < 0.001), and in mice which received an injection with all three antibodies simultaneously (serum, P < 0.01; spleen, P < 0.001). To analyze cell types responsible for production of endogenous IFN--y further, IFN--y in sera and spleen extracts prepared from mice which had been injected with different combinations of anti-CD4 MAb, anti-CD8 MAb, and antiASGM1 antibody was assayed (Fig. 4). Although IFN--y production was markedly suppressed in all groups which received different combinations of antibody (serum, P < 0.01 or P < 0.001; spleen, P < 0.001), mice which had been injected with a combination of anti-CD4 MAb and anti-CD8 MAb or with a combination of anti-CD8 MAb and antiASGM1 antibody could significantly produce endogenous IFN--y in comparison to mice which received a combination of anti-CD4 MAb and anti-ASGM1 antibody (serum, P
Vol. 59, No. 10
0019-9567/91/103439-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Endogenous Gamma Interferon-Independent Host Resistance against Listeria monocytogenes Infection in CD4+ T Celland Asialo GM1+ Cell-Depleted Mice AKIO NAKANE,* AKIHIKO NUMATA, YU CHEN, AND TOMONORI MINAGAWA Department of Microbiology, Hokkaido University School of Medicine, Kita 15 Nishi 7, Kita-ku, Sapporo 060, Japan Received 16 April 1991/Accepted 13 July 1991
The effects of in vivo administration of antibodies against T-cell subsets and asialo GM1 (ASGM1)-bearing ceUs on endogenous gamma interferon (IFN-,y) production and host defense in Listeria monocytogenes-infected mice were investigated. Endogenous IFN-y titers in the bloodstreams and spleen extracts of mice on day 2 of infection were partially suppressed by administration of rabbit anti-ASGM1 antibody, but not by anti-CD4 monoclonal antibody (MAb) or anti-CD8 MAb. Of the different combinations of these three antibodies, the most suppressive effect on IFN--y production was observed after administration of anti-CD4 MAb and anti-ASGM1 antibody, although anti-CD8 MAb combined with anti-CD4 MAb partially inhibited IFN-y production. In contrast, antilisterial resistance was suppressed by the administration of anti-CD8 MAb but not by anti-CD4 MAb or anti-ASGM1 antibody. Antilisterial resistance in mice in which both CD4+ cells and ASGM1+ cells had been depleted was performed as efficiently as in normal mice in spite of the fact that endogenous IFN-y production was markedly suppressed. Furthermore, these mice also eliminated L. monocytogenes cells efficiently from the spleens even when they were pretreated with anti-mouse IFN-,y MAb. These results indicate that CD4+ T cells, CD8+ T cells, and ASGM1+ cells are all responsible for endogenous IFN-'y production and that antilisterial resistance and endogenous IFN--y production are not absolutely correlated.
Complete elimination of Listeria monocytogenes, a facultative intracellular pathogen, from the tissues of infected animals is performed by T-cell-dependent mechanisms (18, 27). The contributions of CD4+ and CD8+ T-cell subsets to antilisterial resistance were compared recently by depleting these cells with monoclonal antibodies (MAbs) in vivo (3, 7, 20, 29). The results of these studies suggest that CD8+ T cells are critical for host defense, whereas CD4+ T cells make a minor contribution to antilisterial resistance. Two possible roles of CD8+ T cells in host defense are proposed (12, 13): (i) CD8+ T cells might lyse L. monocytogenesinfected host cells; (ii) CD8+ T cells might produce cytokines involving gamma interferon (IFN-y). The crucial role of endogenously produced IFN--y in antilisterial resistance has been demonstrated by in vivo injection of MAbs against this factor (1, 5, 24). Studies on characterization of cell types which produce IFN--y by stimulation with L. monocytogenes cells in cultures of spleen cells or T-cell clones derived from immune mice demonstrated that both CD4+ T cells and CD8+ cells secrete IFN-y (4, 14, 15, 17, 19). In addition, T-cell-independent production of IFN--y, which might be produced by natural killer (NK) cells, was demonstrated in scid mice (1, 2, 32). However, in vivo analysis of a mechanism of endogenous IFN--y production induced by primary infection with L. monocytogenes has not been reported. We demonstrated previously that endogenous IFN--y is detected in the bloodstreams and spleens of mice on days 1 to 4 of L. monocytogenes infection (26). Therefore, we focused our studies on in vivo analysis of cells participating in endogenous IFN-y production, especially on the possibility of CD8+ T cells as *
IFN--y-producing cells, in L. monocytogenes infection by in vivo injection of antibodies against T-cell subsets and asialo GM1 (ASGM1)-bearing cells, which mainly correspond to NK cells. In this report, we provide evidence that CD4+ T cells, CD8+ T cells, and ASGM1+ cells are all responsible for endogenous IFN-y production. Our results demonstrate further that antilisterial resistance and endogenous IFN--y production are not absolutely correlated. MATERIALS AND METHODS Mice. Female ddY mice (age, 5 to 7 weeks; obtained from SLC, Hamamatsu, Shizuoka, Japan) were used. Bacteria. L. monocytogenes lb 1684 was prepared as described previously (25). The concentration of washed cells was adjusted spectrophotometrically at 550 nm. Mice were infected intravenously with 0.2 ml of a solution containing 2 x 104 CFU of viable L. monocytogenes cells in 0.01 M phosphate-buffered saline (PBS; pH 7.4). Antibodies. Hybridoma cells secreting MAbs directed against CD4 (L3T4) (GK1.5, rat immunoglobulin G2b) (8) and CD8 (Lyt-2) (53-6.72, rat immunoglobulin G2a) (16) antibodies were kindly provided by T. Koike, School of Medicine, Chiba University, Chiba, Japan. A rat anti-mouse IFN--y-secreting hybridoma (R4-6A2, immunoglobulin Gl) (30) was donated by Y. Watanabe, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. These MAbs were prepared from ascites fluid in Pristane-primed CD-1 nulnu mice. Partial purification by 50% (NH4)2SO4 precipitation was followed by exhaustive dialysis against PBS. Rabbit anti-ASGM1 antibody was prepared and partially purified by 50% (NH4)2SO4 precipitation as described previously (22). The antibodies were aliquoted and stored at -700C.
Corresponding author. 3439
3440
NAKANE ET AL.
In vivo depletion of CD4+ and CD8+ cells. We gave each mouse a single 400-pRg intravenous injection of anti-CD4 MAb or anti-CD8 MAb (in 0.2 ml of pyrogen-free saline) on day -1 of L. monocytogenes infection. Normal rat globulin, prepared and purified by (NH4)2SO4 precipitation from the pooled sera of Wistar rats (SLC), was injected as a control. Evaluation of in vivo cell depletion by flow cytometry. Cell suspensions prepared from spleens or mesenteric lymph nodes of infected and noninfected mice treated with MAbs or normal rat globulin in vivo were incubated with biotinylated anti-CD8 antibody (Lyt-2) (53-6.7; Becton Dickinson, Mountain View, Calif.) in 100 ,ul of PBS supplemented with 0.1% bovine serum albumin (fraction V; Sigma Chemical Co., St. Louis, Mo.) and 0.1% NaN3 at 4°C for 30 min. After being washed, cells were treated simultaneously with fluorescein isothiocyanate-labeled streptavidin (Caltag Laboratories, Inc., South San Francisco, Calif.) and phycoerythrinlabeled anti-CD4 (L3T4) (GK1.5; Becton Dickinson) antibody at 4°C for 30 min. The cells were then washed three times and analyzed on FACScan (Becton Dickinson) after gating to exclude macrophages and polymorphonuclear leukocytes (number of events, 10,000). In vivo depletion of ASGM1+ cells. Rabbit anti-ASGM1 antibody has been shown previously to deplete NK cell activity in vivo and in vitro (9, 11). To deplete NK cell activity in vivo, 400 jig of anti-ASGM1 antibody was given intravenously in a volume of 0.2 ml on day -1 of L. monocytogenes infection (22). Normal rabbit globulin, prepared and purified by (NH4)2SO4 precipitation from the serum of a nonimmunized rabbit, was injected as a control. To determine whether NK cell activity was abolished by this procedure, spleens were removed from mice treated with anti-ASGM1 antibody or normal rabbit globulin on days 2 and 7 of infection and used in a standard 4-h 51Cr release assay with labeled YAC-1 targets at a 50:1 effector/target cell ratio (9). In vivo depletion of endogenous IFN--y. To deplete endogenous IFN--y in vivo, we gave each mouse a single intravenous injection of 1 mg of rat anti-mouse IFN--y MAb (in 0.2 ml of pyrogen-free saline) 2 h before L. monocytogenes infection, as described previously (24). Normal rat globulin was injected as a control. Tests for endotoxin contamination. All in vivo effects of antibodies or normal globulins described were verified by using reagents tested by the Limulus amoebocyte lysate assay to contain 90% of CD4+ or CD8+ T cells in the spleens and mesenteric lymph nodes was already observed on day 0 of infection and continued until at least day 10 for anti-CD4 MAb and until at least day 17 for anti-CD8 MAb. Single injections of 400 ,ug of either anti-ASGM1 antibody or normal rabbit globulin were given on day -1 of infection, and the efficacy of the antibody in depleting NK cells was determined by reduction of cytolytic activities of spleen cells against YAC-1 targets. Marked reduction of NK cell activity continued at least until day 7 of infection (normal rabbit globulin-treated mice versus anti-ASGM1 antibody-treated mice [percent lysis]; 23.1 + 1.8 versus 1.6 ± 0.5 [day 2 of infection], 29.3 ± 2.7 versus 2.9 ± 0.7 [day 7 of infection]). Effect of MAbs against T-cell subsets or anti-ASGM1 antibody on antilisterial resistance. Mice were injected with MAbs against T-cell subsets or anti-ASGM1 antibody the day before infection with a sublethal dose of the bacterium (2 x 104 CFU; 0.1 50% lethal dose). The number of L. monocytogenes cells in the spleens of the treated mice was determined early (day 2) and late (day 6) in infection (Fig. 2). The growth of bacterial cells in the organs of the antibodytreated mice was not significantly different from that in the corresponding normal globulin-treated controls in the early stage of infection (P > 0.05). In contrast, elimination of bacteria from the spleens was significantly blocked in antiCD8 MAb-treated mice (P < 0.01), but not in anti-CD4 MAb- or anti-ASGM1-treated animals (P > 0.05), in the late stage of infection. The suppressive effect in anti-CD8 MAbtreated mice was equivalent to that in the groups which received a combination of anti-CD4 MAb and anti-CD8 MAb or a combination of these two MAbs and anti-ASGM1
antibody. Effect of MAbs against T-cell subsets or anti-ASGMl antibody on endogenous IFN-,y production. IFN--y in sera and
VOL. 59, 1991
IFN-y IN L. MONOCYTOGENES INFECTION
is,
:
a-
a
1
0.03% A
12.02%
i~~~~~~~~~~~~~~~
83.23i
3441
a
4.72%
B
0.04%
0
92. 33±
7. 63%
a
0C a 0M
.'1- I'"""
L I'
~100
10
.103
102
a
10ig a 4
-
1la
CD8 _11.65%
a
0.04% C
0.
a1'
.049%
0.00%
D
a
a
a
1a3
la2
CD8
-88.15?.
l.S
99. 34?
0.16% IV
im
11-
02
0.17%
a
.7, ~2~ YVL '103
la
CD8
32
103
104
CD8
FIG. 1. Fluorocytometric analysis of spleen cells from L. monocytogenes-infected mice which had been injected with normal rat globulin (A), anti-CD4 MAb (B), anti-CD8 MAb (C), or a combination of both MAbs (D) on day -1 of infection. Three mice per group were sacrificed on day 7 of infection, and spleen cells were analyzed for T-cell marker expression with a fluorescence-activated cell sorter. Mice treated with normal rat globulin and those which were untreated gave essentially identical results. Only the results from mice treated with normal rat globulin are shown.
spleen extracts obtained from the antibody-treated mice or the corresponding normal globulin-treated mice 48 h after infection was determined (Fig. 3). IFN--y production in the bloodstreams and spleens of anti-CD4 MAb-treated mice or anti-CD8 MAb-treated mice was not significantly different from that in normal rat globulin-treated animals (P > 0.05). In contrast, the titers of endogenous IFN--y were significantly reduced in anti-ASGM1-treated mice (serum, P < 0.05), in mice which received a combination of anti-CD4 MAb and anti-CD8 MAb (serum, P < 0.05; spleen, P < 0.001), and in mice which received an injection with all three antibodies simultaneously (serum, P < 0.01; spleen, P < 0.001). To analyze cell types responsible for production of endogenous IFN--y further, IFN--y in sera and spleen extracts prepared from mice which had been injected with different combinations of anti-CD4 MAb, anti-CD8 MAb, and antiASGM1 antibody was assayed (Fig. 4). Although IFN--y production was markedly suppressed in all groups which received different combinations of antibody (serum, P < 0.01 or P < 0.001; spleen, P < 0.001), mice which had been injected with a combination of anti-CD4 MAb and anti-CD8 MAb or with a combination of anti-CD8 MAb and antiASGM1 antibody could significantly produce endogenous IFN--y in comparison to mice which received a combination of anti-CD4 MAb and anti-ASGM1 antibody (serum, P
