Immunology 1978 34 149
The regulation of the immune response of mice to Haemophilus influenzae type b capsular polysaccharide
C. J. LEE, F. G. MALIK & J. B. ROBBINS Bureau of Biologics, National Institutes of Health Building 29, Bethesda, Md. 20014, U.S.A.
Received 25 November 1976; accepted for publication 12 May 1977
Summary. The regulation of age-related antibody response to Haemophilus influenzae type b polysaccharide (HITB-PS) was studied by measuring the splenic plaque forming cells (PFC) following immunization with this capsular polysaccharide. The magnitude of PFC response to HITB-PS was found to be dose-related, enhanced by Freund's complete adjuvant and influenced by the genetic strain of mice. Priming with a low dose of HITB-PS did not induce a state of immunological unresponsiveness. Treatment with antilymphocyte serum significantly increased the PFC response to HITB-PS. Athymic nude mice showed an enhanced ability to induce both IgG and IgA-PFC responses as well as a significant increase in the biosynthesis of protein and mitogenicity in spleen cells. These findings suggest that the immune response to HITB-PS is regulated by the suppressor T cell. The magnitude of the IgM-PFC response induced by HITB-PS in mice increased gradually from two weeks of age and reached a plateau at 8 weeks. Treatment with fetuin resulted in the inhibition of direct IgM and IgG-PFC responses to HITB-PS; the suppressive effect on the immune response was more profound and lasting in young than in adult mice.
INTRODUCTION
Haemophilus influenzae type b capsular polysaccharide (HITB-PS) has been found to be a polymer of ribose and ribitol phosphate containing other chemical components such as amino acids, hexosamines, and sugars. This capsular polysaccharide is mitogenic for both B and T cells from adult human peripheral blood lymphocytes (Lee & Robbins, 1977). Many studies have reported that a lower antibody level, shorter duration of response and smaller percentage of responders were observed in infants immunized with pure HITB-PS (Robbins et al., 1973, Smith et al., 1973, Anderson et al., 1972). The response and the magnitude of antibody formation to the bacterial capsular polysaccharide was shown to be due to the activities of two different types of T lymphocytes; amplifiers and suppressor T cells (Gershon, 1974, Morse, 1976). The effect of antilymphocyte serum (ALS) in increasing the magnitude of the antibody response to type III pneumococcal polysaccharide was found to be due to the inhibition of suppressor T cells (Baker et al., 1974). It has been shown in several species, including mice and man, that suppressor cells comprise the majority of the newborn and infant T cell population (Olding & Oldstone, 1976, Mosier & Johnson, 1975). The lower immune response of human infants and neonates to HITB-PS may be due to a
Correspondence: Dr C. J. Lee, Bureau of Biologics National Institutes of Health Building 29, Bethesda, Md. 20014, U.S.A.
149
C. J. Lee, F. G. Malik & J. B. Robbins
150
suppressor effect as well as to the presence of foetal metabolites produced during early life. The lymphocytes synthesizing IgG and IgA antibodies were observed in response to immunization with bacterial capsular polysaccharide and the numbers of these cells increased greatly after treatment with ALS (Barthold et al., 1974). These findings suggest that the magnitude of not only the IgM, but also the IgG and IgA antibody responses may be regulated by T cell activity and that ALSinduced stimulation is not due to the reversal or change of antibody-mediated feedback inhibition. The present study reports the effects of immunization with HITB-PS on specific plaque forming cells and the regulatory factors concerned in the agerelated variations in the immune response.
MATERIALS AND METHODS HITB capsular polysaccharide and experimental animals The preparation of HITB, strain 'Rab', capsular polysaccharide used in the present study was described in earlier papers (Lee & Robbins, 1977, Rodrigues et al., 1971). Several strains of mice were used: C3H/HeJ, C57BL/6, DBA (Microbiological Associates, Bethesda, Md.), AKR, A/J (Jackson Laboratory, Ma.), BALB/C (Rodent and Rabbit Production Section, National Institute of Health, Bethesda, Md.). Athymic nude and thymus-bearing littermate control mice were obtained from Laboratory Supply Co. (Indianapolis, Ind.). General physiological and immunological characteristics of nude mice have been reported (Wortis, 1974, Pantelouris, 1971).
Plaque forming cell (PFC) assay of antibody response Mice were immunized intraperitoneally with an optimally immunogenic dose (5 pg) of HITB-PS in 0-1 ml Freund's complete adjuvant, and the antibody response determined 4 days later. Splenic plaque forming cells were detected by the technique of localized haemolysis in gel (Jerne, Nordin & Henry, 1963, Dresser & Greaves, 1973). Sheep erythrocytes sensitized with HITB-PS by the chromium chloride coupling procedure (Baker, Stashak & Prescott, 1969) were used as indicator cells. The cell mixtures in the gel plates were incubated overnight at 37°. Complement was added and the plates again incubated for 1 h at 37°. The number of direct IgM plaque forming cells was counted. For the determination of indirect PFC specific for IgG and IgA antibody production, after the determination of direct IgM PFCs as described above, the gel plates were treated with goat anti-mouse IgG or IgA antiserum (1: 100 dilution) with complement, and incubated for 1 h at 37°. Gel plates incubated for the same additional period of time with complement, which did not contain IgG or IgA antiserum, served as controls. PFC counts were made on both types of
300
8
Materials The sources of the reagents used were: Freund's complete adjuvant, fetuin (Grand Island Biological Co., N.Y.); goat anti-mouse IgG and IgA antiserum (Meloy Laboratories, Va.); rabbit anti-mouse lymphocyte serum, sheep erythrocytes (Micro-
biological Associates, Bethesda, Md.); hydrocortisone, poly AU, E. coli lipopolysaccharide, cyclic GMP, and dibutyl cyclic AMP (Sigma, St Louis, Mo.); 14C-amino acid mixture (14C uniformly labelled, 50-125 pCi amino acid/mCi of mixture), 3H-thymidine (6-3H, sp. act. 7-4 Ci/mmole) (Schwarz/Mann, Orangeburg, N.Y.).
I \+~~~
53200
U_aL
5
100
50 5 0-05 0.5 immunizing dose (/.g) Figure 1. Groups of 40 CH/HeJ strain mice (8 in each dose
0-005
subgroup), 3-4-month-old males, were injected intraperitoneally with various doses (0-005-50 pg) of HITB-PS mixed with 0-1 ml Freund's complete adjuvant or dissolved in 0-1 ml 0-9% sodium chloride solution. The splenic plaque forming cell (PFC) response was determined 4 days later. The value of each direct IgM PFC response is the average of duplicate samples from 8 mice. (0) Adjuvant; (0) in saline; means ± s.e.
Immune response to capsular polysaccharide
151
Table 1. The plaque forming cell (PFC) response to H. influenzae type b polysaccharide (HITB-PS) in various strains of mice. Several strains of 8-week-old mice in groups of 8 were injected intraperitoneally with 5 pg HITB-PS mixed with 0 1 ml Freund's complete adjuvant or dissolved in 0 1 ml 09 % sodium chloride solution. The splenic PFC response was determined 4 days later. The value of each PFC response is the average of duplicate samples from 8 mice Class specific PFC/spleen
HITB-PS preparation Mixedwith adjuvant
Dissolved in saline
Mouse strain C3H/HeJ BALB/C A/J C57BL/6 AKR DBA C3H/HeJ BALB/C A/J
C57BL/6 AKR DBA
Direct IgM
IgG
IgA
140±12-7* 116 ±10-6 63-6 ±8-8 24-6 ±4-8 14-1± 3-6 16-6 ±2-8 104 ± 6-6 82-8 ± 7-4 37 5 ±6-0 25-9 ±2-9 14-1 ±2-2 2-2 ±0 9
44-4±6-2 39-2 ± 8-9 26-7 ±6-7 9 1 ±2-7 9-4 ± 2-2 4-4 ±1-8 19-5 ±4-8
300±5 1 57 5 ± 5-6 10 0±3 0 7 5 ± 1-3 10-0 2-3 25±1-3
21-0 3*6
31-9 ± 3-4
450 ±5-4 15-6 3-3 7 5 ±2-3 8-1 ±2-1 00
13-1±5-4 5 6 ±2-4 3-8 ±1-6 1 9 ±0 9
* Average of duplicate samples from 8 mice ± s.e.m.
plates and the gel plates treated with class-specific antiserum were considered to be specific indirect IgG or IgA PFC (Barthold et al., 1974). Treatment with anti-lymphocyte serum and chemical agents Rabbit anti-mouse lymphocyte serum (0 3 ml) was injected intraperitoneally at the same time as HITB-PS. Various chemical agents; hydrocortisone, fetuin, poly AU, E. coli lipopolysaccharide, cyclic GMP and dibutyl cyclic AMP (50 ,ug each in 0-2 ml 0 9 % sodium chloride solution) were injected intraperitoneally on days -1, 0, +1 relative to the injection of HITB-PS. The PFC assay was performed 4 days after the injection of HITB-PS. In further study of fetuin treatment on the PFC response, the mice were injected intraperitoneally with fetuin daily for 1 week.
"C-amino acid and 3H-thymidine incorporation Mice were injected intraperitoneally with 5 ,ug HITB-PS/0-1 ml adjuvant. Three days after the injection, doses of 4 pCi 'IC-amino acid mixture/ 100 g body weight and 25 ,pCi 3H-thymidine/100 g body weight, each in a volume of 0-2ml 0-9% sodium chloride solution, were injected intraperitoneally. The spleens were removed 24 h after the
injection and homogenized separately in Eagle's medium passing the tissue through a stainless steel screen. Portions of the cell suspensions were precipitated with 10 % trichloroacetic acid in an ice bath for 10 min and washed with 95% ethanol saturated with sodium acetate. Radioactivity was measured in a scintillation spectrometer. Incorporation of 14C and 3H was calculated according to the discriminator-ratio method (Okita et al., 1957). RESULTS
Splenic PFC responses to HITB-PS Fig. 1 shows the response of splenic PFC in mice immunized with HITB-PS. Groups of mice were immunized with various amounts of HITB-PS. The magnitude of the direct IgM PFC response to this polysaccharide increased as the dose increased from 0 005 to 5 pg. Higher doses, 50,ug or more, reduced the PFC response. HITB-PS in Freund's complete adjuvant induced a higher PFC response than when dissolved in normal saline. HITB-PS induced a lower PFC response than other T-cell dependent antigens, e.g. 106 sheep red blood cells induced 92,000 PFC/spleen; whereas HITB-PS induced only 200 to 300 PFC/spleen. Three to 4-
C. J. Lee, F. G. Malik & J. B. Robbins
152
209±140
---_ C200
u
150
c. a-
RI II
F
152± 13-1
150±13-4
II II I
14±7-1 1^00
ponders to HITB-PS. The PFC response of A/J strain was intermediate between these two groups. The numbers of plaque forming cells specific to IgG and IgA were less than the numbers of PFCs found using the direct technique.
II I I
114+
I
I
11
HL 0 005 0C05
Priming dose (/ig) Immunizing dose (l~g) 5-0
5.0
ALS (ml)
5-0 0-3
42±5-5
I
1
.
16±4-0
0.005 0-0J5 5-0 03
+ 0-3
Figure 2. Groups of 48 mice (8 in each subgrou p), 8-week-old males, were injected intraperitoneally with a (0 005 pg) of HITB-PS. Three days later, tlhe mice were injected intraperitoneally with 0 3 ml rabb it anti-mouse lymphocyte serum (ALS). At the same time, doses of 5 pg HITB-PS/0-1 ml Freund's complete adjuvant were injected intraperitoneally. The splenic direct IgM resj ponse termined by the procedures described in the Methods. Mean ± s.e. * P < 0.05; t P
The regulation of the immune response of mice to Haemophilus influenzae type b capsular polysaccharide
C. J. LEE, F. G. MALIK & J. B. ROBBINS Bureau of Biologics, National Institutes of Health Building 29, Bethesda, Md. 20014, U.S.A.
Received 25 November 1976; accepted for publication 12 May 1977
Summary. The regulation of age-related antibody response to Haemophilus influenzae type b polysaccharide (HITB-PS) was studied by measuring the splenic plaque forming cells (PFC) following immunization with this capsular polysaccharide. The magnitude of PFC response to HITB-PS was found to be dose-related, enhanced by Freund's complete adjuvant and influenced by the genetic strain of mice. Priming with a low dose of HITB-PS did not induce a state of immunological unresponsiveness. Treatment with antilymphocyte serum significantly increased the PFC response to HITB-PS. Athymic nude mice showed an enhanced ability to induce both IgG and IgA-PFC responses as well as a significant increase in the biosynthesis of protein and mitogenicity in spleen cells. These findings suggest that the immune response to HITB-PS is regulated by the suppressor T cell. The magnitude of the IgM-PFC response induced by HITB-PS in mice increased gradually from two weeks of age and reached a plateau at 8 weeks. Treatment with fetuin resulted in the inhibition of direct IgM and IgG-PFC responses to HITB-PS; the suppressive effect on the immune response was more profound and lasting in young than in adult mice.
INTRODUCTION
Haemophilus influenzae type b capsular polysaccharide (HITB-PS) has been found to be a polymer of ribose and ribitol phosphate containing other chemical components such as amino acids, hexosamines, and sugars. This capsular polysaccharide is mitogenic for both B and T cells from adult human peripheral blood lymphocytes (Lee & Robbins, 1977). Many studies have reported that a lower antibody level, shorter duration of response and smaller percentage of responders were observed in infants immunized with pure HITB-PS (Robbins et al., 1973, Smith et al., 1973, Anderson et al., 1972). The response and the magnitude of antibody formation to the bacterial capsular polysaccharide was shown to be due to the activities of two different types of T lymphocytes; amplifiers and suppressor T cells (Gershon, 1974, Morse, 1976). The effect of antilymphocyte serum (ALS) in increasing the magnitude of the antibody response to type III pneumococcal polysaccharide was found to be due to the inhibition of suppressor T cells (Baker et al., 1974). It has been shown in several species, including mice and man, that suppressor cells comprise the majority of the newborn and infant T cell population (Olding & Oldstone, 1976, Mosier & Johnson, 1975). The lower immune response of human infants and neonates to HITB-PS may be due to a
Correspondence: Dr C. J. Lee, Bureau of Biologics National Institutes of Health Building 29, Bethesda, Md. 20014, U.S.A.
149
C. J. Lee, F. G. Malik & J. B. Robbins
150
suppressor effect as well as to the presence of foetal metabolites produced during early life. The lymphocytes synthesizing IgG and IgA antibodies were observed in response to immunization with bacterial capsular polysaccharide and the numbers of these cells increased greatly after treatment with ALS (Barthold et al., 1974). These findings suggest that the magnitude of not only the IgM, but also the IgG and IgA antibody responses may be regulated by T cell activity and that ALSinduced stimulation is not due to the reversal or change of antibody-mediated feedback inhibition. The present study reports the effects of immunization with HITB-PS on specific plaque forming cells and the regulatory factors concerned in the agerelated variations in the immune response.
MATERIALS AND METHODS HITB capsular polysaccharide and experimental animals The preparation of HITB, strain 'Rab', capsular polysaccharide used in the present study was described in earlier papers (Lee & Robbins, 1977, Rodrigues et al., 1971). Several strains of mice were used: C3H/HeJ, C57BL/6, DBA (Microbiological Associates, Bethesda, Md.), AKR, A/J (Jackson Laboratory, Ma.), BALB/C (Rodent and Rabbit Production Section, National Institute of Health, Bethesda, Md.). Athymic nude and thymus-bearing littermate control mice were obtained from Laboratory Supply Co. (Indianapolis, Ind.). General physiological and immunological characteristics of nude mice have been reported (Wortis, 1974, Pantelouris, 1971).
Plaque forming cell (PFC) assay of antibody response Mice were immunized intraperitoneally with an optimally immunogenic dose (5 pg) of HITB-PS in 0-1 ml Freund's complete adjuvant, and the antibody response determined 4 days later. Splenic plaque forming cells were detected by the technique of localized haemolysis in gel (Jerne, Nordin & Henry, 1963, Dresser & Greaves, 1973). Sheep erythrocytes sensitized with HITB-PS by the chromium chloride coupling procedure (Baker, Stashak & Prescott, 1969) were used as indicator cells. The cell mixtures in the gel plates were incubated overnight at 37°. Complement was added and the plates again incubated for 1 h at 37°. The number of direct IgM plaque forming cells was counted. For the determination of indirect PFC specific for IgG and IgA antibody production, after the determination of direct IgM PFCs as described above, the gel plates were treated with goat anti-mouse IgG or IgA antiserum (1: 100 dilution) with complement, and incubated for 1 h at 37°. Gel plates incubated for the same additional period of time with complement, which did not contain IgG or IgA antiserum, served as controls. PFC counts were made on both types of
300
8
Materials The sources of the reagents used were: Freund's complete adjuvant, fetuin (Grand Island Biological Co., N.Y.); goat anti-mouse IgG and IgA antiserum (Meloy Laboratories, Va.); rabbit anti-mouse lymphocyte serum, sheep erythrocytes (Micro-
biological Associates, Bethesda, Md.); hydrocortisone, poly AU, E. coli lipopolysaccharide, cyclic GMP, and dibutyl cyclic AMP (Sigma, St Louis, Mo.); 14C-amino acid mixture (14C uniformly labelled, 50-125 pCi amino acid/mCi of mixture), 3H-thymidine (6-3H, sp. act. 7-4 Ci/mmole) (Schwarz/Mann, Orangeburg, N.Y.).
I \+~~~
53200
U_aL
5
100
50 5 0-05 0.5 immunizing dose (/.g) Figure 1. Groups of 40 CH/HeJ strain mice (8 in each dose
0-005
subgroup), 3-4-month-old males, were injected intraperitoneally with various doses (0-005-50 pg) of HITB-PS mixed with 0-1 ml Freund's complete adjuvant or dissolved in 0-1 ml 0-9% sodium chloride solution. The splenic plaque forming cell (PFC) response was determined 4 days later. The value of each direct IgM PFC response is the average of duplicate samples from 8 mice. (0) Adjuvant; (0) in saline; means ± s.e.
Immune response to capsular polysaccharide
151
Table 1. The plaque forming cell (PFC) response to H. influenzae type b polysaccharide (HITB-PS) in various strains of mice. Several strains of 8-week-old mice in groups of 8 were injected intraperitoneally with 5 pg HITB-PS mixed with 0 1 ml Freund's complete adjuvant or dissolved in 0 1 ml 09 % sodium chloride solution. The splenic PFC response was determined 4 days later. The value of each PFC response is the average of duplicate samples from 8 mice Class specific PFC/spleen
HITB-PS preparation Mixedwith adjuvant
Dissolved in saline
Mouse strain C3H/HeJ BALB/C A/J C57BL/6 AKR DBA C3H/HeJ BALB/C A/J
C57BL/6 AKR DBA
Direct IgM
IgG
IgA
140±12-7* 116 ±10-6 63-6 ±8-8 24-6 ±4-8 14-1± 3-6 16-6 ±2-8 104 ± 6-6 82-8 ± 7-4 37 5 ±6-0 25-9 ±2-9 14-1 ±2-2 2-2 ±0 9
44-4±6-2 39-2 ± 8-9 26-7 ±6-7 9 1 ±2-7 9-4 ± 2-2 4-4 ±1-8 19-5 ±4-8
300±5 1 57 5 ± 5-6 10 0±3 0 7 5 ± 1-3 10-0 2-3 25±1-3
21-0 3*6
31-9 ± 3-4
450 ±5-4 15-6 3-3 7 5 ±2-3 8-1 ±2-1 00
13-1±5-4 5 6 ±2-4 3-8 ±1-6 1 9 ±0 9
* Average of duplicate samples from 8 mice ± s.e.m.
plates and the gel plates treated with class-specific antiserum were considered to be specific indirect IgG or IgA PFC (Barthold et al., 1974). Treatment with anti-lymphocyte serum and chemical agents Rabbit anti-mouse lymphocyte serum (0 3 ml) was injected intraperitoneally at the same time as HITB-PS. Various chemical agents; hydrocortisone, fetuin, poly AU, E. coli lipopolysaccharide, cyclic GMP and dibutyl cyclic AMP (50 ,ug each in 0-2 ml 0 9 % sodium chloride solution) were injected intraperitoneally on days -1, 0, +1 relative to the injection of HITB-PS. The PFC assay was performed 4 days after the injection of HITB-PS. In further study of fetuin treatment on the PFC response, the mice were injected intraperitoneally with fetuin daily for 1 week.
"C-amino acid and 3H-thymidine incorporation Mice were injected intraperitoneally with 5 ,ug HITB-PS/0-1 ml adjuvant. Three days after the injection, doses of 4 pCi 'IC-amino acid mixture/ 100 g body weight and 25 ,pCi 3H-thymidine/100 g body weight, each in a volume of 0-2ml 0-9% sodium chloride solution, were injected intraperitoneally. The spleens were removed 24 h after the
injection and homogenized separately in Eagle's medium passing the tissue through a stainless steel screen. Portions of the cell suspensions were precipitated with 10 % trichloroacetic acid in an ice bath for 10 min and washed with 95% ethanol saturated with sodium acetate. Radioactivity was measured in a scintillation spectrometer. Incorporation of 14C and 3H was calculated according to the discriminator-ratio method (Okita et al., 1957). RESULTS
Splenic PFC responses to HITB-PS Fig. 1 shows the response of splenic PFC in mice immunized with HITB-PS. Groups of mice were immunized with various amounts of HITB-PS. The magnitude of the direct IgM PFC response to this polysaccharide increased as the dose increased from 0 005 to 5 pg. Higher doses, 50,ug or more, reduced the PFC response. HITB-PS in Freund's complete adjuvant induced a higher PFC response than when dissolved in normal saline. HITB-PS induced a lower PFC response than other T-cell dependent antigens, e.g. 106 sheep red blood cells induced 92,000 PFC/spleen; whereas HITB-PS induced only 200 to 300 PFC/spleen. Three to 4-
C. J. Lee, F. G. Malik & J. B. Robbins
152
209±140
---_ C200
u
150
c. a-
RI II
F
152± 13-1
150±13-4
II II I
14±7-1 1^00
ponders to HITB-PS. The PFC response of A/J strain was intermediate between these two groups. The numbers of plaque forming cells specific to IgG and IgA were less than the numbers of PFCs found using the direct technique.
II I I
114+
I
I
11
HL 0 005 0C05
Priming dose (/ig) Immunizing dose (l~g) 5-0
5.0
ALS (ml)
5-0 0-3
42±5-5
I
1
.
16±4-0
0.005 0-0J5 5-0 03
+ 0-3
Figure 2. Groups of 48 mice (8 in each subgrou p), 8-week-old males, were injected intraperitoneally with a (0 005 pg) of HITB-PS. Three days later, tlhe mice were injected intraperitoneally with 0 3 ml rabb it anti-mouse lymphocyte serum (ALS). At the same time, doses of 5 pg HITB-PS/0-1 ml Freund's complete adjuvant were injected intraperitoneally. The splenic direct IgM resj ponse termined by the procedures described in the Methods. Mean ± s.e. * P < 0.05; t P
