The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Muthukkumar, S.
Right arrow Articles by Stein, K. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Muthukkumar, S.
Right arrow Articles by Stein, K. E.
The Journal of Immunology, 2000, 165: 4803-4813.
Copyright © 2000 by The American Association of Immunologists

The Ability of B Cells and Dendritic Cells to Present Antigen Increases During Ontogeny

Subramanian Muthukkumar1, Julia Goldstein and Kathryn E. Stein2

Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The immune response to polysaccharide (PS) Ags in mice is delayed during ontogeny even when administered in a thymus-dependent (TD) form. In this study, Neisseria meningitidis group C PS-tetanus toxoid conjugate (MCPS-TT) vaccine was used to examine whether the delay in the development of Ab responses to TD PS conjugate vaccines in neonatal mice is due to defective Ag presentation. The results show that B cells and dendritic cells (DC) from 3- and 7-day-old mice were severely defective in presenting TT and MCPS-TT to Ag-specific T cell clones. The ability of these cells to present Ag reaches adult levels by 4 wk. The development of anti-MCPS and anti-TT Abs in neonatal mice parallels the functional ability of their APC to present Ag. DC from neonatal mice expressed very low levels of MHC class II, costimulatory molecules B7.1, B7.2, and CD11c but high levels of monocyte-specific markers F4/80 and CD11b and granulocyte marker, Ly6G. Significant changes in the expression of these markers were observed as the age of the mice increased. MHC class II, B7.1 and B7.2, and CD11c all increased with age, reaching adult levels between 3 and 4 wk, concurrent with the function of APC. These results demonstrate that one reason neonates fail to produce high titers of anti-PS Abs even when immunized in a TD form is that their B cells and DC are not fully functional.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infants <2 years of age are at greatest risk for infection with encapsulated bacteria such as Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. (1, 2). Anti-polysaccharide (PS)3 Abs have been found to play crucial roles in protecting hosts from invasive diseases caused by these organisms (3, 4, 5, 6), and the high level of susceptibility to these encapsulated organisms is mainly due to the inability of neonates to respond to PS Ags (2, 7, 8, 9, 10, 11). Meningococcal infections, often to epidemic proportions, caused by several serogroups have been reported in many parts of the world (12, 13). Meningococcal vaccines containing purified group C and other capsular PS are poorly immunogenic in young children and also have been found to cause tolerance (8, 14, 15). A marked improvement in the immunogenicity of PS has been achieved by conjugation to protein carriers (16, 17). Vaccination of infants routinely with H. influenzae type B (Hib) PS-protein conjugates has markedly reduced the incidence of invasive hemophilus disease wherever it has been used in the world (18). Hib PS conjugate vaccines induce T cell-dependent responses with increased Ab titers, increased Ab avidity, and prime for immunologic memory (19). Similar improvement in the immunogenicity of meningococcal (20, 21, 22) and pneumococcal PS (23) has been reported when they were coupled to protein carriers, although McCool et al. (24) have reported that serotypes of the pneumococcal capsule can influence the response to both the PS and the protein carrier. Thus, unresponsiveness to thymus-independent (TI) PS can be overcome by the use of thymus-dependent (TD) protein conjugate vaccines (25).

In mice, the ability to respond to TI-2 PS Ags has been shown to correlate with the presence of a late-developing subset of B lymphocytes (26, 27). This late-developing subset of murine B lymphocytes, characterized by the expression of Lyb-5 and Lyb-3 surface Ags, has been shown to be required for the immune response to the PS Ag, dextran, whether it was administered in a TI or a TD form (28). In that study, 3- to 4-wk-old mice produced adult levels of Ab in response to the TD form of dextran compared with a peak response at 12 wk of age when dextran alone was used as the immunogen. Mice younger than 3 wk old failed to produce a significant anti-dextran response, even when dextran was given in a TD form, strongly suggesting that there is an age-related response to TD as well as TI forms of PS. Although the equivalent of the murine late-developing subset of B lymphocytes has not been described, a similar developmentally associated increase in Ab responses to two Hib PS-protein conjugate vaccines has been reported in human infants (29, 30). Thus, the murine model was valuable in predicting the developmental delay of human immune responses to PS and suggested that it could be used to further explore the basis for the development of responses to TD forms of PS.

Although the mechanism of the unresponsiveness of human and murine neonates to PS Ags is not thoroughly understood, there are two likely possibilities. The first is that the unresponsiveness to PS in neonates is due to the delay in the maturation of B cells. The second is that the unresponsiveness is due to a delay in the maturation of other cell types such as APCs and T cells, which, in turn, may be required for the induction of anti-PS responses and for the maturation of B cells as well. Several intrinsic defects in neonatal T and B cells have been reported. For example, in contrast to adult B cells, which are B220high sIgMlow, and sIgDhigh, neonatal B cells are B220low, sIgMhigh, and sIgD- (31). In addition, immature B cells are refractory to anti-IgM-induced proliferation (32), the Ag-triggered signaling pathway is not coupled to the inositol-phospholipid cascade in immature B cells (33), and reduced levels of src-kinases have been reported in these cells (34). Similar to normal neonatal cells, B cells from adult CBA/N mice, which have an X-linked immunodeficiency (xid) (4) due to a mutation in the protein kinase, Btk, also do not respond to TI-2 PS Ag (35, 36, 37). T cells from neonatal animals produce low levels of cytokines such as IL-2, proliferate poorly in response to anti-CD3 stimulation (38), and are biased toward Th2 responses (39). However, administration of CpG oligodeoxynucleotides in neonatal mice has been shown to circumvent the Th2 polarization in response to vaccines but failed to fully redirect the Th2 responses established by neonatal priming (40). Both murine (41) and human (42) newborn T cells expressed low levels of CD40 ligand upon activation, suggesting the inability of these cells to interact with CD40 on B cells to induce Ig class switch.

The limited ability of neonates to mount immune responses to PS-protein conjugates might be primarily attributable to defective APC. Among APC, dendritic cells (DC) have been shown to play a key role in determining the type of immune response (43, 44). Through the production of IL-12, DC preferentially direct the development of Th1 responses (45, 46) and, indeed, such responses are lacking in neonates (39), suggesting neonatal DC might be defective in stimulating T cells. Recently, human cord blood DC have been shown to be less effective in supporting the proliferation of T cells in response to mitogens (47). It has been demonstrated that addition of exogenous cytokines such as IL-1, -6, and -12 restored anti-PS responses in neonates and enhanced those in adults (48, 49), suggesting that APC that activate T cells or TI-2 PS-specific B cells are deficient in neonates. Furthermore, IL-2 production by neonatal T cells following anti-CD3 mAb stimulation was greatly enhanced by costimulation with anti-CD28 mAb (38), suggesting that newborn T cells have a greater requirement for accessory cell signals. However, little information is available regarding the ability of neonatal APC to present Ag, to prime Ag-specific T cells, or to participate in cellular interactions that are critical for the induction of immune responses. To further understand the development of Ab responses to PS, we have examined the Ag-presenting capacity of B cells and DC isolated from neonatal mice to present tetanus toxoid (TT) and Neisseria meningitidis group C PS-TT conjugate (MCPS-TT) to TT-specific T cell clones. Our studies demonstrate that both B cells and DC undergo maturation in their ability to present Ags during ontogeny.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Eight-week-old and pregnant female BALB/cAnN (BALB/c) and C57BL/6N mice were purchased from Charles River Breeding Laboratories (Wilmington, MA) through the National Institutes of Health Small Animal Section. All animal protocols were approved by the Center for Biologics Evaluation and Research Animal Care and Use Committee.

Reagents

MCPS prepared from N. meningitidis strain C11 was obtained from Merck Sharp and Dohme (lot 1815T; West Point, PA). MCPS-TT vaccine was kindly provided by Dr. H. Jennings, National Research Council (Ottawa, Ontario, Canada) (50). TT (lot TLC-16-1) manufactured by Aventis Pasteur (Willowdale, Toronto, Canada) was provided by Laboratories of Bacterial Toxins, Center for Biologics Evaluation and Research, Food and Drug Administration. Pigeon cytochrome c (PCC) was purchased from Sigma (St. Louis, MO).

Immunization protocol

Adult (8–12 wk old) and neonatal BALB/c mice of various ages were immunized i.p. with 10 µg of MCPS as free PS or in the form of MCPS-TT (containing 40 µg of TT) or 40 µg of TT. All three Ags were given in 5% (1:20 dilution from stock) Maalox (William H. Rorer, Fort Washington, PA) as an adjuvant. Maalox stock solution contains magnesium hydroxide (4%) and aluminum hydroxide (4.5%). In some control groups, mice were immunized with 50 µg PCC. Entire litters were immunized if the mice were younger than 3 wk old at the time of immunization. For Ab titration, all groups of mice were bled 4 wk after immunization and were prebled 1 wk before immunization in adult and 4-wk-old mice. In mice younger than 4 wk old, unimmunized, age-matched litters were used as the source of preimmune sera. In some experiments, Ag-primed APC were prepared from spleens 7 days after immunization.

Production of TT-specific T cell clones

T cell clones were produced as described (51) with minor modifications. Mice were immunized i.p with 10 µg of MCPS-TT in 5% Maalox as an adjuvant. Thirty days after the primary immunization, mice were boosted with the same dose of MCPS-TT. Seven days later, draining inguinal and popliteal lymph nodes were removed and single-cell suspensions were prepared. These lymph node cells (2 x 106) were cultured with irradiated (2500 rad) syngenic spleen cells (6 x 106) in 24-well plates (Costar, Cambridge, MA) in DMEM supplemented with 2 mM glutamine, 100 U/ml penicillin-streptomycin, 5 x 10-5 M mercaptoethanol, 12 mM HEPES, and 10% FCS. MCPS-TT (2.5 µg/ml) and TT (10 µg/ml) were added in separate cultures. After 7 days, responding T cell blasts were harvested from the wells and purified from the dead cells by Ficoll density gradient centrifugation. T cells were then put in resting cultures at 2 x 105 cells/well with 2 x 106 irradiated syngenic spleen cells for another 7 days in 48-well plates. An initial reduction in the number of T cells was noticed in these cultures, whereas the fraction of Ag specific cells was apparently rising. The cells were then cultured with MCPS-TT or TT in the presence of 2 x 106 irradiated syngenic spleen cells for an additional 7 days. A large number of T cell blasts were noticed in these cultures, and 50-µl aliquots from each well were taken and cultured in 96-well plates (Costar) with 1 x 106 irradiated syngenic spleen cells with MCPS-TT or TT for 72 h. Cell proliferation in response to these Ags was assessed by [3H]thymidine incorporation before cloning.

The rested T cells were counted and diluted to 5 cells/ml. One hundred microliters of this dilution of cells was added to each well (96-well plate; Costar) along with 1 x 106 irradiated syngenic spleen cells in the presence of MCPS-TT or TT. IL-2 (80 U/ml; PharMingen, San Diego, CA) was added in the cloning wells. After 9–14 days, the wells showing growth were expanded into large wells (24-well plate) with additional Ag, irradiated syngenic spleen cells, and IL-2. After 1 wk of further expansion, the cells were harvested and washed twice, then rested 1 wk with 4 x 106 irradiated syngenic spleen cells. These T cell clones were expanded by rounds of stimulation and rest without IL-2 and tested for Ag specificity in the standard proliferation assay using various related and unrelated protein and PS Ag (data not shown). Among an array of TT-specific T cell clones obtained, two clones, P1/7 and P4/19, were used in this study as indicator cells for the ability of B cells and DC to present Ag.

Purification of APC

Total splenic APC were prepared using a standard protocol. Briefly, cells were dispersed by pressing the spleens against the bottom of a tissue culture dish containing incomplete DMEM using the flat surface of a syringe plunger. Cells were left undisturbed for 1 min to let the debris settle, then the cells were collected and washed by centrifugation. The viability of the cells was determined by trypan blue dye exclusion. B cells were purified as described elsewhere (52). Briefly, plastic-adherent cells were removed by incubating 1.5 x 108 spleen cells in 15 ml of DMEM supplemented with 5% FCS on a 150 x 25 mm tissue culture dish for 1.5 h at 37°C. The nonadherent cells were collected and depleted of T cells by treating them with a mixture of anti-T cell Abs (anti-Thy1, anti-CD4, and anti-CD8) followed by complement-mediated lysis. The purity of the B cells was verified by flow cytometry using anti-B220 Abs (RA3-6B2; PharMingen). B220+ cells were >90% in various experiments. Splenic DC were enriched following the protocol of Steinman and coworkers (53). Briefly, after mechanical dissociation, splenic fragments were subjected to mild collagenase D (type IV; Boehringer Mannheim, Indianapolis, IN) digestion at 37°C for 30 min to release DC. Low-density cells were selected by centrifugation on a 35% BSA gradient (Sigma) and cultured in plastic dishes for 1–2 h, then the nonadherent cells were removed. After another round of removal of nonadherent cells, adherent cells were incubated at 37°C for 16–18 h. DC that detached during this incubation were harvested and washed, and the purity was assessed by flow cytometry using anti-CD11c Ab (HL3; PharMingen). In most experiments, >95% of the cells were CD11c+ when the source of the spleen was an adult BALB/c mouse.

Purification of CD4+ and CD8+ T cells

CD4+ and CD8+ T cells from C57/B6N mice were purified by negative selection using commercially available columns. Cell suspensions were prepared according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Briefly, RBC-depleted spleen cells were incubated with a mixture of appropriate mAb for 10 min at room temperature. After removing the unbound Abs by washing, the cells were then loaded onto the anti-Ig-coated glass bead column, incubated for 15 min, and eluted. The eluted cells were washed, and the purity of CD4+ and CD8+ cells was assessed by flow cytometry.

Ag presentation assay

Ag presentation was measured by culturing TT-specific T cells (2 x 104/well) with irradiated (2500 rad) syngenic total splenic APC or purified B cells (2 x 105/well) or DC (1 x 105/well) in 96-well plates (Costar) in the presence or absence of TT (10 µg/ml) or MCPS-TT (2.5 µg PS and 10 µg TT/ml) in DMEM supplemented with 10% FCS. Cultures were incubated at 37°C with 5% CO2 for 120 h and pulsed with 1 µCi of [3H]thymidine (sp. act. 2 Ci/mmol; NEN Life Science Products, Boston, MA) during the last 4 h of culture. The cultures were harvested on glass-fiber filters, and the incorporated radioactivity was measured in a Betaplate scintillation counter (LKB-Pharmacia, Piscataway, NJ).

Flow cytometry

Cells were washed twice in DMEM supplemented with 5% FCS and resuspended in 100 µl of FACS buffer (PBS, 5% FCS, and 0.1% NaN3). To block the nonspecific binding with FC receptors, cells were treated with 1 µg of anti-FcR Ab (2.4G2) and incubated for 10 min at 4°C. FITC or biotin-conjugated Abs against CD11c (HL3), CD45R (RA3/6B2), CD80/B7.1 (16-10A1), CD86/B7.2 (GL1), class I (34-2-12), and class II (39-10-8) molecules (PharMingen) were added at 1 µg/106 cells and incubated for 30 min on ice. For two-color analysis of DC FITC-conjugated anti-CD11c and PE-conjugated anti-F4/80 (F4/80) (Serotec, Oxford, U.K.), CD11b (M1/70), Ly6G (RB6-8C5), or CD8{alpha} (53-6.7) (PharMingen) were used. As appropriate, after washing, the cells were incubated with FITC-avidin (1 µg) for an additional 30 min. After the incubation, cells were washed and resuspended in the above indicated medium. Live cells were gated using forward and size scatter in a FACScan (Becton Dickinson, Mansfield, MA) flow cytometer, and 106 events per sample were acquired. Level of receptor expression was determined on either FL1 or FL2. Percentage of cells expressing a particular surface marker was determined using Cell Quest software. Fluorochrome-conjugated isotype-matched rat mAbs were used as negative controls.

Fluorescence ELISA

Anti-MCPS and anti-TT Ab levels were estimated by fluorescence ELISA as described elsewhere (54).

Mixed lymphocyte reactions

CD4+ or CD8+ T cells (1 x 105) isolated from C57/B6N mice were cultured with irradiated spleen cells (2 x 105) obtained from neonatal or adult BALB/c mice for a total of 72 h in DMEM supplemented with 10% FCS in 96-well flat-bottom plates (Costar). These cultures were pulsed with [3H]thymidine and harvested as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-MCPS and anti-TT Ab responses in neonatal mice

The development of the immune response to MCPS and TT determinants was examined by measuring serum Ab levels in neonatal mice that were immunized with either MCPS, TT, or MCPS-TT. As shown in Fig. 1Go, anti-MCPS IgM Abs were detected in the control sera of 3-day-old unimmunized mice, and the level raised to ~2-fold in 7-day-old mice. However, no further increase in the levels of background anti-MCPS IgM was observed as the age of the mice reached 4 wk. Immunization with either MCPS or MCPS-TT did not cause any significant increase in anti-MCPS IgM levels in 3- and 7-day-old mice, but a marginal response began to appear in sera from 4-wk-old mice. In 3-day-old mice the levels of IgG1 anti-MCPS response increased to 3-fold over preimmune sera after immunization with MCPS-TT, and the titers increased 10- to 100-fold in mice 7–28 days old. Thus a clear age-associated development of IgG1 anti-MCPS Abs was evident from the mice immunized with MCPS-TT. An age-associated increase in the levels of IgG3 anti-MCPS was observed in MCPS as well as MCPS-TT immunized mice (Fig. 1Go, IgG3). Neither preimmune nor TT immune sera showed significant IgG1 or IgG3 anti-MCPS Abs. These results indicated a developmental delay in the induction of Ab response to PSs whether they were administered in TI or TD form.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. Ontogeny of anti-MCPS and -TT responses in sera from mice immunized at different ages. Mice were immunized with MCPS ({square}), TT ({diamond}), or MCPS-TT ({circ}) at different ages, as indicated. Anti-MCPS and -TT Ab levels of indicated isotypes were analyzed in preimmune sera ({triangleup}) and postimmune sera obtained 4 wk after immunization. IgM anti-MCPS and -TT responses in adult mice were not significantly higher than those in 28-day-old mice. IgG3 anti-MCPS titers in MCPS- and MCPS-TT-immunized adult mice were 1900 ± 155 and 3200 ± 230, respectively. Adult and 28-day-old mice that received MCPS showed similar IgG1 anti-MCPS response but the adult mice that were immunized with MCPS-TT mounted a higher IgG1 response (16,410 ± 1,305). IgG1 anti-TT titers in TT- and MCPS-TT-immunized adult mice were 83,450 ± 4,220 and 26,940 ± 1,700, respectively. IgM and IgG3 anti-TT titers were low and similar in adult and 28-day-old mice. The titers represent mean ± SD of 10–20 mice in each age group. Deviations smaller than the symbols are not visible.

 
Because both TI and TD forms of MCPS failed to induce a strong anti-MCPS Ab response in 3-day-old neonates, it was of interest to examine the response to the carrier protein, TT, in these mice. The same sera described above from all age groups of mice also were tested for anti-TT Ab levels. As shown in Fig. 1Go, an age-associated increase in the titers of IgM, IgG1, and IgG3 anti-TT were evident from the groups of mice that were immunized with TT or MCPS-TT. Although a clear developmentally-associated delay was noticed, the IgM anti-TT response showed a greater increase with age than the IgM anti-MCPS response. A 10- to 15-fold increase in anti-TT IgG1 response was observed in 3-day-old mice when compared with the levels in preimmune sera (probably background in the assay), but the difference was not as high as in 28-day-old mice, suggesting a developmental increase in the immune response to TT as well as MCPS. TT-immunized animals showed slightly higher anti-TT IgM and IgG1 titers than the MCPS-TT group, as might be expected where some epitopes of the proteins are lost in the conjugation to PS. In contrast to IgM and IgG1 anti-TT responses, the development of the IgG3 response to TT, like the IgG3 anti-MCPS response, was much slower as seen by very low titers of this isotype in 3- and 7-day-old mice. These data demonstrate a developmental delay in the immune response to TI and TD Ags, with differences in the kinetics of the response in different IgG subclasses.

Ag presentation by neonatal spleen cells

As neonatal mice failed to induce adult-like anti-MCPS and anti-TT Ab responses, we examined the ability of neonatal APC to present Ag. To study this, the capacity of neonatal spleen cells to stimulate TT-specific proliferation of a T cell clone, P1/7, was examined. The P1/7 proliferative response is indicative of mutual interactions between these T cells and APC and it includes Ag processing and presentation by the APC. Total spleen cells isolated from neonatal mice of various age groups ranging from 3 to 28 days were examined for their ability to present TT and are presented in Fig. 2Go as the percentage of the adult response for each experiment. A marked defect in the ability of neonatal spleen cells to present TT to P1/7 T cells was evident when the APC were derived from 3- and 7-day-old mice, and the responses were only 15 and 23% of adult responses, respectively. However, the ability of neonatal APC to present Ag improved with age, and >90% of the adult response was observed when APC were taken from 4-wk-old mice.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2. Ag presentation by neonatal spleen cells. P1/7 T cells (2 x 104) were cultured for 5 days with total spleen cells (2 x 105) isolated from neonatal mice of various age groups as indicated. TT (10 µg/ml) was used as the Ag. [3H]Thymidine incorporation was measured during the last 4-h culture period. Each data point denotes an independent experiment. An adult control was run in triplicate for each experiment and used to calculate the percentage of the adult response for each age group. The mean of adult values for all 15 experiments shown in Fig. 2Go was 18,259 cpm and the range was 10,119–27,332 cpm.

 
Ag presentation by neonatal B cells

To identify whether the inability to stimulate T cell proliferation is a total spleen cell phenomenon or whether a particular type of APC is functionally impaired in neonates, we chose to examine the ability of B cells to present TT. As described above, B cells purified from adult and neonatal mice of various age groups ranging from 3 to 28 days were cultured with P1/7 T cells in the presence of TT. The proliferation of P1/7 cells was assessed as a measure of the ability of B cells to present TT. As shown in Fig. 3GoA, neonatal B cells, particularly from 3- and 7-day-old mice were only weakly able to stimulate TT-specific proliferation of P1/7 T cells. For instance, the level of P1/7 T cell proliferation stimulated by B cells from 3-day-old mice was <20% of the adult response, indicating a severe functional impairment in their ability to present Ag. As with whole spleen cells, adult-like Ag presentation was not seen in B cells until the mice were 28 days old. Cells isolated from mice younger than 28 days old always stimulated T cells less well than cells from adult mice.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. A, Ag presentation by neonatal B cells. P1/7 T cells (2 x 104) were cultured for 5 days with B cells (2 x 105) isolated from neonatal mice of various age groups as indicated. TT (10 µg/ml) was used as the Ag. [3H]thymidine incorporation was measured during the last 4-h culture period. Each data point denotes an independent experiment. An adult control was run in triplicate for each experiment and used to calculate the percentage of the adult response for each age group. The mean of adult values for all 18 experiments shown in (A) was 29,911 cpm and the range was 12,757–55,936 cpm. B, Presentation of MCPS-TT by neonatal B cells. P4/19 T cells (2 x 104) were cultured for 5 days with B cells (2 x 105) isolated from neonatal mice of various age groups, as indicated. MCPS-TT (2.5 µg/ml) was used as the Ag. [3H]Thymidine incorporation was measured during the last 4-h culture period. Each data point denotes an independent experiment. An adult control was run in triplicate for each experiment and was used to calculate the percentage of the adult response for each age group. The mean of adult values for all 15 experiments shown in (B) was 14,462 cpm and the range was 8,222–22,316 cpm.

 
To ensure that the impairment seen in APC was not a function of the response of a particular T cell clone, the ability of neonatal B cells to present Ag was examined using another T cell clone, P4/19, which reacts with TT and MCPS-TT. In these experiments, MCPS-TT was used as the Ag (Fig. 3GoB). A similar impairment in the ability of neonatal B cells to present Ag was found with the P4/19 T cell clone, as was seen with P1/7. The ability of 3- and 7-day-old neonatal B cells to present Ag was only 10 and 20% of adult cells, respectively, and B cells isolated from 28-day-old mice behaved almost like adult mice (8–12 wk old) in their ability to present Ag.

Ag presentation by neonatal DC

DC have been shown to play a key role in priming naive Th cells (55, 56, 57), therefore, the ability of neonatal and adult DC to stimulate TT-specific proliferation of the P1/7 T cell clone was examined. The range of numbers of DC chosen for the proliferation experiments was based on published data from various studies describing the potency of different APC to stimulate Th cells (58, 59, 60) and by a titration experiment using various numbers of DC in our system. Fig. 4Go shows the P1/7 proliferative response induced by splenic DC derived from neonatal mice of various ages ranging from 3 to 28 days as a percentage of the adult DC response. DC from 3-day-old mice showed a major impairment in their ability to stimulate T cells. Their ability to present Ag appears to improve relatively faster than B cells as seen by DC isolated from 7-, 14-, and 21-day-old mice (Fig. 4Go). However, DC isolated from mice that were <28 days old were less effective in stimulating the proliferation of P1/7 T cells when compared with their adult counterparts. The results were essentially similar when another TT-reactive T cell clone, P4/19, was used (data not shown). These results clearly indicate that DC from neonatal mice are not fully competent to present Ag before 4 wk of age.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 4. Ag presentation by neonatal DC. P1/7 T cells (2 x 104) were cultured for 5 days with DC (1 x 105) isolated from neonatal mice of various age groups as indicated. TT (10 µg/ml) was used as the Ag. [3H]Thymidine incorporation was measured during the last 4-h culture period. Each data point denotes an independent experiment. An adult control was run in triplicate for each experiment and was used to calculate the percentage of the adult response for each age group. The mean of adult values for all 12 experiments shown in Fig. 4Go was 45,526 cpm and the range was 18,178–63,747 cpm.

 
Expression of MHC and costimulatory molecules on B cells and DC

The activation of Ag-specific Th cells requires two important signaling events. The first signal involves a well-known cognate interaction between the TCR and Ag/MHC class II complexes generated by APC. The second signal is provided by the interaction between costimulatory molecules such as B7.1 and B7.2 expressed on APC and CD28 on T cells (61). Thus the expression of adequate levels of B7.1 and B7.2 molecules on the surface of APC are critical for effective Ag presentation. Constitutive expression of high levels of these costimulatory molecules and abundant expression of MHC class II on the surface of DC contribute to their potency as highly effective APC (56, 57).

The failure of effective Ag presentation by neonatal B cell and DC preparations led us to compare the expression levels of MHC molecules B7.1 and B7.2 on these cells (Table IGo). CD11c and CD45 expression also were examined as DC and B cell lineage markers, respectively. The expressions of B7.1 and B7.2 in adult B cell preparations were 34 and 43% positive cells, respectively. In contrast, in neonatal B cell preparations, particularly from 3- and 7-day-old mice, B7.1- and B7.2-positive cells were extremely infrequent (Table IGo). Nonetheless, most of the cells in B cell preparations from neonatal mice of different age groups (7–21 days) expressed class I molecules similar to that of adult B cells except for cells from 3-day-old mice in which 64% cells were class I positive. In contrast, B cell preparations from 3-day-old mice almost completely failed to express class II molecules. Analysis of cells from 3- to 21-day-old mice showed a clear age-associated increase in the number of B cells expressing class II molecules. In contrast, most of the B cell preparations from neonatal and adult mice expressed CD45. B cells from neither neonatal nor adult mice expressed CD11c (<0.5%), a marker for DC, indicating a lack of DC contamination in the B cell preparations (Table IGo). The total number of cells recovered in B cell preparations from 3-, 7-, 14-, 21-day-old and adult mice were 3.2 x 106, 8.9 x 106, 19.6 x 106, 33.0 x 106, and 39.3 x 106 per spleen, respectively.


View this table:
[in this window]
[in a new window]
  		Table I. Expression of surface Ags on B cells and DCs1

 
Adult DC preparations were 88% CD11c+. DC from 3-day-old mice prepared in the same manner as that of adults almost completely lacked the expression of CD11c (7%), B7.1 (5%), B7.2 (15%), and class II (0.3%), however, expression of these markers gradually increased in DC obtained from 7-, 14-, and 21-day-old mice. Similar to B cells, class I expression appeared on DC during the early period of neonatal life, as 72% of the DC isolated from 3-day-old mice were positive for these molecules. The total number of cells recovered in DC preparations from 3-, 7-, 14-, 21-day-old and adult mice were 0.9 x 105, 1.7 x 105, 2.4 x 105, 5.3 x 105, and 9.4 x 105 per spleen, respectively. The development of class II expression in DC was more or less similar to B cells, as observed by fewer class II-positive DC in 3- and 7-day-old mice, were 0.3 and 12%, respectively. However, DC appear to attain the adult level expression of class II somewhat faster than B cells. No significant difference in the level of expression (mean fluorescence index) was observed for any of these surface Ags between neonatal and adult mice (data not shown). Altogether, the lack of expression of class II as well as costimulatory molecules B7.1 and B7.2 on neonatal B cells and DC strongly points to their inability to present Ag as effectively as adult cells.

MLR by neonatal APC

It was of interest to examine whether the neonatal APC that are defective in presenting TT to specific T cells also fail to stimulate class I- and II-restricted MLR. This was examined by culturing either CD4+ or CD8+ T cells from C57/BL6 (H2b) mice as responder cells with irradiated neonatal total splenic APC from 3- and 7-day-old or adult BALB/c (H2d) mice as stimulator cells in a 72-h culture. As shown in Fig. 5Go, APC from 3-day-old (cpm 398) and 7-day-old (cpm 1370) mice failed to stimulate the proliferation of allogenic CD4+ T cells, whereas adult APC from the same background induced a strong MLR (cpm 6199). Interestingly, the same APC from 3- and 7-day-old BALB/c mice induced a robust MLR in allogenic CD8+ T cells, comparable to the response seen with adult stimulator cells. The results of these experiments confirmed that the class II-restricted Ag presentation is impaired in neonatal APC. The differential effect of neonatal APC to stimulate class I- and II-restricted allogenic MLR is likely due to the difference in the expression of class I and II molecules on neonatal APC.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 5. MLR by neonatal APC. CD4+ T cells and CD8+ T cells (1 x 105) isolated from C57/B6 mice were cultured with irradiated (2500 rad) spleen cells (2 x 105) isolated from adult or neonatal BALB/c mice of 3 or 7 days of age for 72 h. [3H]Thymidine incorporation was measured during the last 4-h culture period. cpm ± SE of triplicates from a single experiment are shown.

 
Neonatal DC are morphologically similar to adult DC

The fact that neonatal DC isolated in the same manner as adult cells failed to express the DC-specific marker CD11c (Table IGo) led us to question whether the cells isolated from 3-day-old neonates were, in fact, DC. To address this question, cells isolated from 3-day-old mice were cultured for an additional 48 h in complete DMEM without the addition of any cytokines. The morphology of these neonatal DC and similarly cultured adult DC were then examined under phase contrast microscopy. DC preparations from both neonates and adults displayed similar morphological features with several elongated dendrites, which are characteristic of DC (Fig. 6Go), suggesting that cells isolated from neonates are dendritic type cells.



View larger version (87K):
[in this window]
[in a new window]
  		FIGURE 6. Morphology of DC. DC were isolated from 3-day-old neonatal and adult mice as described in Materials and Methods and cultured for an additional 48 h in six-well plates. The elongated dendrites characteristic of DC were seen in neonatal and adult DC preparations. Picture shows x100 magnification under phase contrast microscopy.

 
Phenotypic characteristics of DC at various stages of development

Although DC preparations from neonatal and adult mice were morphologically similar, most of DC from 3- or 7-day-old mice lacked the expression of the DC-specific marker, CD11c. As spleen is believed to contain a large number of monocytoid DC precursors, monocyte-specific markers such as F4/80 and CD11b, granulocyte marker, Ly6G, and CD8{alpha} were used to further characterize the DC through different stages of ontogeny. Two-color analysis of DC obtained from 3-, 7-, 14-, 21-, and 28-day-old mice with CD11c and F4/80, CD11b, Ly6G, or CD8{alpha} revealed three patterns of marker development on DC (Fig. 7Go). A large number of DC from 3-day-old mice expressed F4/80 and CD11b. As the age of the mice increased, the number of DC expressing these molecules gradually diminished whereas the number of CD11c+ cells increased. However, 28-day-old and adult mice showed significant numbers of CD11c+/F4/80+ and CD11c+/CD11b+ cells, but CD11c-/F4/80+ or CD11c-/CD11b+ cells were almost absent in DC preparations from adult mice. The percentage of cells expressing individual markers is shown in Table IIGo. It should be noted that DC preparations from 3-, 7-, and 14-day-old mice contain a significant number of F4/80- cells. As evident from 21-, 28-day-old, or adult mice, these F4/80- cells also matured into CD11c+ DC (Fig. 7Go). The second pattern of marker development in DC is with reference to the expression of CD11c vs Ly6G. Most of the neonatal DC expressed Ly6G and its expression gradually diminished as the age of the mice increased. The expression of Ly6G in DC is indirectly proportional to CD11c expression during development. The third pattern of cell surface marker development in DC showed that CD8{alpha}+ (single positive) cells were almost completely absent in DC populations throughout the course of development, including adult DC. However, a significant proportion of DC expressing both CD11c and CD8{alpha} were found in adult mice, and the gradual appearance of this double-positive population was clearly evident from the DC preparations of 3-, 7-, 14-, 21-, and 28-day-old mice (Fig. 7Go and Table IIGo). These two-color FACS data indicate a clear pattern of development of DC populations in neonatal mice with maturation DC evident at 21–28 days old.



View larger version (65K):
[in this window]
[in a new window]
  		FIGURE 7. Phenotypic characteristics of DC at various stages of development. Two-color FACS analysis was performed to characterize the expression of CD11c vs F4/80, CD11b, Ly6G, and CD8{alpha} on DC during the course of development. Splenic DC preparations from mice of the indicated age groups were examined. FITC-conjugated anti-CD11c and PE-conjugated anti-F4/80, CD11b, Ly6G, and CD8{alpha} were used.

 

View this table:
[in this window]
[in a new window]
  		Table II. Expression of CD11c vs other myeloid lineage-associated markers on DCs during ontogeny1

 
Effect of Ag priming on ability of B cells to present Ag

To determine whether Ag priming would improve the ability of neonatal B cells to present Ag, 7-day-old neonates and adult mice were immunized with TT. B cells purified from these Ag-primed mice 7 days after immunization were used as APC to stimulate TT-specific proliferation of the P1/7 T cell clone. As shown in Fig. 8Go, and as expected, naive adult B cells induced TT-specific proliferation in P1/7 cells (cpm 13,432). Interestingly, the TT-primed adult B cells presented Ag more effectively and caused a significant increase in the proliferation of T cells (cpm 23,614). However, B cells obtained from MCPS-primed animals failed to cause such an augmented proliferation in P1/7 cells. To further examine the specificity of the Ag priming, B cells from PCC-primed mice were used. PCC-primed B cells presented TT effectively and comparable to B cells from TT-primed mice, suggesting that any TD response (TT or PCC) but not a TI-2 response (MCPS) can augment the ability of B cells to present Ag. Because the TT-primed B cells from 7-day-old mice were obtained 1 wk after immunization, at 14 days of age, B cells from 14-day-old unprimed mice were used as an age-matched control. Naive B cells from 14-day-old mice caused lower TT-specific proliferation in P1/7 T cells (cpm 7692) than adult B cells, however, as seen with adult cells, both TT- and PCC-primed B cells were better at presenting TT than unprimed cells. Again, as with adult cells, MCPS failed to improve the Ag-presenting capacity of neonatal B cells. These results suggest that the effect of Ag priming on the ability of B cells to present Ag is a bystander effect and that both adult and neonatal B cells are susceptible to such a bystander influence.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 8. Presentation by Ag-primed B cells. Adult and 7-day-old neonatal mice were immunized i.p. with MCPS, TT, or MCPS-TT. Control mice received PCC. Seven days after immunization, B cells were isolated from Ag-primed and unprimed control mice of all groups. These B cells (2 x 105) were used to stimulate the proliferation of P1/7 T cells (2 x 104) in a 5-day culture in the presence or absence of TT (10 µg/ml). [3H]Thymidine incorporation was measured during the last 4-h culture period. cpm ± SE of triplicates were shown from a representative experiment of four.

 
Effect of irradiation on neonatal B cell ability to present Ag

One reason that neonatal B cells or DC could be less effective in presenting Ag than adult cells is that they are more sensitive to irradiation than adult cells. To examine whether irradiating the neonatal B cells at 2500 rad inhibited their ability to present Ag, lower doses of irradiation such as 1000 and 1500 rad were used. As shown in Table IIIGo, no difference in the capacity of neonatal B cells to stimulate TT-specific proliferation was observed at any dose of irradiation ranging from 1000 to 2500 rad. Their Ag-presenting ability remained low when compared with adult B cells at all doses of irradiation, suggesting that irradiation dose is not responsible for the defective Ag presentation by neonatal B cells.


View this table:
[in this window]
[in a new window]
  		Table III. Effect of irradiation on Ag presentation by neonatal B cells1

 
To rule out whether any factors derived from irradiated neonatal B cells could inhibit T cell proliferation by adult cells, 3-day-old neonatal B cells were added to cultures containing adult B cells and P1/7 T cells in the presence of TT. The Ag presentation by this cell mixture was compared with those of unmixed B cell populations of the appropriate cell numbers. The presence of neonatal B cells in the cultures did not inhibit the Ag presentation by adult B cells (Table IIIGo). On the contrary, neonatal B cells were found to cause an additive effect in stimulating the proliferation of T cells by adult B cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well known that Abs against the capsular PS play an important role in protecting hosts from invasive diseases caused by encapsulated bacteria (3, 4, 5, 6), and that the immune response to PS Ags is delayed during ontogeny (1, 2, 8, 35). We have shown previously that immunization of neonatal mice with MCPS failed to induce an anti-MCPS Ab response (54). Current studies show that MCPS in a TD form, as MCPS-TT, evoked an anti-MCPS response in neonatal mice, but the levels were significantly lower than the adult response until 3–4 wk of age. Earlier studies also have shown a developmental delay in inducing Ab responses to isomaltohexaosyl PS-keyhole limpet hemocyanin conjugate, a TD form of dextran, in mice (28). A similar developmentally associated increase in Ab responses to Hib PS-protein conjugate vaccines has been reported in human infants (29, 30). To understand the basis of delay in the development of anti-PS responses, whether to TI-2 or TD forms of PS Ags, we have examined the ability of cells from neonatal mice to present Ag to T cells.

Although a large number of studies reported defective T and B cell function in neonates (reviewed in Ref. 62), not much is known regarding the functional ability of neonatal accessory cells. Our results showed that APC isolated from 4-wk-old mice were able to present TT comparable to adult cells, but the APC obtained from mice younger than 4 wk old always showed a reduced ability to stimulate T cells, indicating a developmental delay in the ability of APC to present Ag. These results confirm and extend the finding of Levin and Gershon (63) that neonatal spleen cells were defective in stimulating conalbumin-specific T cell proliferation. Moreover, our results show that both B cells and DC are defective in Ag-presenting capacity, as discussed below, and define the period during development when the function matures.

To determine the function of individual APC populations, purified B cells from neonatal mice were studied. These cells were found to be defective in their ability to present TT to specific T cell clones. Furthermore, the developmental kinetics of the ability of B cells to present Ag was similar to that seen with total spleen cells. B lymphocytes from newborns have been shown to display a decreased ability to stimulate IL-2 secretion from Ag-specific T cell hybridomas, suggesting their inability to interact with T cells compared with their adult counterparts (64). Recently, recruitment of syk tyrosine kinase by the Ig-{alpha} subunit of the B cell receptor has been shown to be critical for the MHC class II-restricted Ag presentation by B cells (65). At present, it is unclear whether immature neonatal B cells fail to recruit syk kinase.

We also have found that neonatal DC are impaired in the function of Ag presentation as seen by a reduction in their ability to stimulate TT-specific proliferation in the P1/7 cell line. Similar results were obtained when another TT-reactive cell line, P4/19, was used (data not shown). Our results are consistent with the recent findings that DC isolated from human cord blood were less effective in supporting T cell proliferation in response to mitogen (47). Furthermore, defective regional immunity in the respiratory tract of neonatal rats has been shown to be due to hyporesponsiveness of local DC to GM-CSF (66). This study also demonstrated that inhalation of microbial stimuli or administration of IFN-{gamma} triggers rapid recruitment of DC into the airway epithelium and lung parenchyma in adult rats but this response was markedly attenuated in newborns. All of these data, including our own, indicate that neonatal DC are not functionally mature. In addition to DC and B cells, macrophages isolated from 3- and 7-day-old neonatal mice were also defective in their ability to stimulate TT-specific proliferation in P1/7 T cells (S. Muthukkumar and K. E. Stein, unpublished observation).

Surface characteristics revealed that in contrast to adults, early neonatal DC almost completely lack the expression of costimulatory molecules B7.1 and B7.2 (Table IGo). Similarly, the expression of B7.1 and B7.2 was almost completely absent in neonatal B cells. Expression of class II molecules was also severely reduced in neonatal DC as well as in B cells. These findings strongly correlated with the inability of neonatal DC and B cells to present Ag. The role of B7.1- and B7.2-mediated interaction between accessory or B and T cells in providing activation signals to the latter is well known (61). Expression of CD11c, a known marker for DC, was also found to be developmentally regulated because the cells isolated from 3-day-old mice expressed very low levels of this molecule but expression gradually increased as the mice reached adulthood. Most of the neonatal DC expressed monocyte-specific markers F4/80 and CD11b and granulocyte marker Ly6G. Significant changes in the expression of these markers were observed as the DC became CD11c positive during the course of development. Neonatal spleen also contains nonmonocytoid DC precursors as evident from a significant number of F4/80- cells in the neonatal DC preparations that matured into CD11c+ cells during development. Neonatal DC appear to attain the functional ability to present Ag faster than the B cells as examined by B cells and DC isolated from neonatal mice of various age groups. This might be predicted from the findings that a higher proportion of DC express MHC class II as well as costimulatory molecules when compared with B cells at a given age of neonatal development.

The functional expression of MHC class I was not altered in either neonatal B cells or DC and the levels were comparable to adults. Interestingly, neonatal APC effectively stimulated class I-restricted MLR by allogenic CD8+ T cells but class II-restricted MLR by CD4+ cells was severely affected, and this can be explained by the very low level expression of class II molecules on neonatal APC. Ag-primed B cells have been implicated as important APC in priming naive CD4+ T cells (67). We found that TT priming caused an augmentation on the ability of B cells to stimulate TT-specific T cells in both adult and 7-day-old mice. This could very well be due to the up-regulation of B7.2-like costimulatory molecules as suggested by others (67). However, this enhancement does not appear to be Ag specific as similar enhancement was observed with B cells from mice immunized with PCC, a nonspecific Ag. In contrast, the TI-2 MCPS-primed B cells failed to stimulate T cells effectively. These results suggest a role for T cells in augmenting the potency of APC to present Ag and that any ongoing TD response can increase the function of APC.

In summary, we have shown that immature neonatal B cells and DC are functionally defective in their ability to present Ag to T cells, a fundamental event in the generation of an immune response to a TD Ag. The functional impairment of these cells correlated with the lack of expression of class II and other costimulatory molecules on B cells and DC and CD11c on DC. The finding that the developmental kinetics of the ability of APC to present Ag parallels the development of Ab response to MCPS as well as to TT in neonatal mice suggest the basis of reduced responsiveness to PS-protein conjugate vaccines during the early period of neonatal life. Therefore, identifying strategies to enhance the potency of neonatal APC would be a valuable approach to improve the immunogenicity of the existing vaccines against encapsulated bacterial pathogens as well as to develop vaccines that can be used at birth.


   Acknowledgments
 
We thank Drs. Amy Rosenberg and Barbara Rellahan for critical reading of this manuscript. Our thanks are also due to Dr. Harold Jennings for providing meningococcal polysaccharide-tetanus toxoid conjugates and Dr. Willie Vann for tetanus toxoid.


   Footnotes
 
1 S.M. is a recipient of a National Research Council Fellowship. Back

2 Address correspondence and reprint requests to Dr. Kathryn E. Stein, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Building 29B Room 3NN16, 29 Lincoln Drive, Bethesda, MD 20892-4555. Back

3 Abbreviations used in this paper: PS, polysaccharide; MCPS, Neisseria meningitidis group C polysaccharide; TT, tetanus toxoid; MCPS-TT, Neisseria meningitidis group C polysaccharide-tetanus toxoid conjugate; DC, dendritic cells, PCC, pigeon cytochrome c; TD, thymus dependent; TI, thymus independent; Hib, H. influenzae type B. Back

Received for publication December 2, 1999. Accepted for publication July 19, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Goldschnieder, I., E. C. Gotschlich, M. S. Artenstein. 1969. Human immunity to the meningococcus. I. The role of humoral Abs. J. Exp. Med. 129:1307.[Abstract]
  2. Peltola, H., H. Kayhty, A. Sivonen, P. H. Makela. 1977. Haemophilus influenzae type b capsular polysaccharide vaccine in children: a double-blind field of study of 100,000 vaccinees 3 mo to five years of age in Finland. Pediatrics 60:730.[Abstract/Free Full Text]
  3. Kabat, E. A., C. P. Miller, H. Kaiser, A. Z. Foster. 1945. Chemical studies on bacterial agglutination. VII. A quantitative study of the type specific and group specific Abs in antimeningococcal sera of various species and their relation to mouse protection. J. Exp. Med. 81:1.[Abstract]
  4. Schneerson, R., L. P. Rodrigues, Jr J. C. Parke, J. B. Robbins. 1971. Immunity to disease caused by Hemophilus influenzae, type b. II. Specificity and some biological characteristics of ‘natural’ infection-acquired and immunization-induced Abs to the capsular polysaccharide of Hemophilus influenzae, type b. J. Immunol. 107:1081.[Abstract/Free Full Text]
  5. Kaijser, B., S. Ahlstedt. 1977. Protective capacity of Abs against Escherichia coli O and K Ags. Infect. Immun. 17:286.[Abstract/Free Full Text]
  6. Egan, M. L., D. G. Pritchard, Jr H. C. Dillon, B. M. Gray. 1983. Protection of mice from experimental infection with type III group B streptococcus using mAbs. J. Exp. Med. 158:1006.[Abstract/Free Full Text]
  7. Smith, D. H., G. Peter, D. L. Ingram, A. L. Harding, P. Anderson. 1973. Responses of children immunized with the capsular polysaccharide of Hemophilus influenzae, type b. Pediatrics 52:637.[Abstract/Free Full Text]
  8. Gold, R., M. L. Lepow, I. Goldschneider, E. C. Gotschlich. 1977. Immune response of human infants to polysaccharide vaccines of group A and C Neisseria meningitidis. J. Infect. Dis. 136:S31.
  9. Parke, J. C., R. Schneerson, J. B. Robbins, J. J. Schlesselman. 1977. Interim report of controlled field trial of immunization with Hemophilus influenzae, type b and group C Neisseria meningitidis in Mecklenburg County, North Carolina (March 1974–March 1976). J. Infect. Dis. 136:S51.
  10. Pincus, D. J., D. Morrison, C. Andrews, E. Lawrence, S. H. Sell, P. F. Wright. 1982. Age-related response to two Hemophilus influenzae, type b vaccines. J. Pediatr. 100:197.[Medline]
  11. Kayhty, H., V. Karanko, H. Peltola, P. H. Makela. 1984. Serum Abs after vaccination with Haemophilus influenzae type b capsular polysaccharide and responses to reimmunization: no evidence of immunologic tolerance or memory. Pediatrics 74:857.[Abstract/Free Full Text]
  12. deMorais, J. S., R. S. Monford, J. B. Risi, E. Antezana, R. A. Feldman. 1974. Epidemic disease due to serogroup C Neisseria meningitidis in SaoPaulo, Brazil. J. Infect. Dis. 129:568.[Medline]
  13. Finley, R. A.. 1976. Prophylaxis against meningococcal disease. JAMA 236:459.[Medline]
  14. Gold, R., M. L. Lepow, I. Goldschneider, T. L. Draper, E. C. Gotschlich. 1975. Clinical evaluation of group A and C meningococcal polysaccharide vaccines in infants. J. Clin. Invest. 56:1536.
  15. Granoff, D. M., R. K. Gupta, R. B. Belshe. 1998. Induction of immunologic refractoriness in adults by meningococcal C polysaccharide vaccination. J. Infect. Dis. 178:870.[Medline]
  16. Avery, O. T., W. F. Goebel. 1931. Chemo-immunological studies on conjugated carbohydrate-proteins. V. The immunological specificity of an Ag prepared by combining the capsular polysaccharide of type III pneumococcus with foreign protein. J. Exp. Med. 54:437.[Abstract]
  17. Stein, K. E.. 1994. Glycoconjugate vaccines: what next?. Int. J. Technol. Assess. Health Care 10:167.[Medline]
  18. Steinhoff, M. C.. 1997. Haemophilus influenzae type b infections are preventable everywhere. Lancet 349:1186.[Medline]
  19. Eskola, J., H. Kayhty. 1996. Ten years’ experience with Haemophilus influenzae type b (Hib) conjugate vaccines in Finland. Rev. Med. Microbiol. 7:231.
  20. Jr Twumasi, P. A., S. Kumah, A. Leach, T. J. D. O’Dempsey, S. J. Ceesay, J. Todd, C. V. Broome, L. B. G. M. Carlone, P. K. Pais, P. K. Holder, et al 1995. A trial of a group A plus group C meningococcal polysaccharide-protein conjugate vaccine in African infants. J. Infect. Dis. 171:632.[Medline]
  21. Richmond, P., R. Borrow, E. Miller, S. Clark, F. Sadler, A. J. Fox, N. Begg, R. Morris, K. Cartwright. 1999. Meningococcal serogroup C conjugate vaccine is immunogenic in infancy and primes for memory. J. Infect. Dis. 179:1569.[Medline]
  22. Rubinstein, L. J., P. A. Garcia-Ojeda, F. Michon, H. J. Jennings, K. E. Stein. 1998. Murine immune responses to Neisseria meningitidis group C capsular polysaccharide and a thymus-dependent toxoid conjugate vaccine. Infect. Immun. 66:5450.[Abstract/Free Full Text]
  23. O’Brien, K. L., M. C. Steinhoff, K. Edwards, H. Keyserling, M. L. Thoms, D. Madore. 1996. Immunologic priming of young children by pneumococcal glycoprotein conjugate, but not polysaccharide, vaccines. Pediatr. Infect. Dis. J. 15:425.[Medline]
  24. McCool, T. L., C. V. Harding, N. E. Greenspan, J. R. Schreiber. 1999. B and T cell immune responses to pneumococcal conjugate vaccines: divergence between carrier and polysaccharide-specific immunogenicity. Infect. Immun. 6:4862.
  25. Stein, K. E.. 1992. Thymus-independent and thymus-dependent responses to polysaccharide Ags. J. Infect. Dis. 165:(Suppl.1):S49.
  26. Ahmed, A., S. Irwin, S. O. Sharrow. 1977. B lymphocyte heterogeneity: development and characterization of alloantiserum which distinguishes B-lymphocyte differentiation alloantigens. J. Exp. Med. 145:101.[Abstract/Free Full Text]
  27. Mosier, D. E., I. M. Zitron, J. J. Mond, A. Ahmed, I. Sher, W. E. Paul. 1977. Surface Ig D as a functional receptor for a subclass of B lymphocytes. Immunol. Rev. 37:89.[Medline]
  28. Stein, S. E., D. A. Zopf, C. B. Miller, B. M. Johnson, P. K. A. Mongini, A. Ahmed, W. E. Paul. 1983. The immune response to a thymus-dependent form of B512 dextran requires the presence of Lyb5+ lymphocytes. J. Exp. Med. 157:657.[Abstract/Free Full Text]
  29. Madore, D. V., C. L. Johnson, D. C. Phipps, L. A. Popjoy, R. Eby, D. H. Smith. 1990. Safety and immunologic response to Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine in 1 to 6-month infants. Pediatrics 85:331.[Abstract/Free Full Text]
  30. Ahonkhai, V. I., L. J. Lukacs, L. C. Jonas, H. Matthews, P. P. Vella, R. W. Ellis, J. M. Staub, K. T. Dolan, C. M. Rusk, G. B. Calandra, R. J. Gerety. 1990. Haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate) (PedvaxHIBTM): clinical evaluation. Pediatrics 85:676.[Abstract/Free Full Text]
  31. Osmond, D. G.. 1982. Bone marrow and B cell differentiation. Adv. Exp. Med. Biol. 149:3.[Medline]
  32. Chang, T. L., G. Capraro, R. E. Kleinman, A. K. Abbas. 1991. Anergy in immature B lymphocytes: differential responses to receptor-mediated stimulation and T helper cells. J. Immunol. 147:750.[Abstract]
  33. Yellen, A. J., W. Glenn, V. P. Sukhatme, X. Cao, J. G. Monroe. 1991. Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes: developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice. J. Immunol. 146:1446.[Abstract]
  34. Weksler, R. J, J. G. Monroe. 1995. Immature B lymphocytes are deficient in expression of the src-family kinases p59fyn and p55fgr. J. Immunol. 154:1919.[Abstract]
  35. Mosier, D. E., J. J. Mond, E. A. Goldings. 1977. The ontogeny of thymic independent Ab responses in vitro in normal mice and mice with X-linked B cell defect. J. Immunol. 119:1874.[Abstract/Free Full Text]
  36. Paul, W. E., B. Subbarao, J. J. Mond, D. G. Sieckmann, I. M. Zitron, A. Ahmed, D. E. Mosier, I. Scher. 1979. B lymphocyte development and activation: analysis with the mutant strain. B. Pernis, and H. J. Vogel, eds. Cells of Ig Synthesis 383. Academic Press, New York.
  37. Bondada, S., M. Garg. 1994. Thymus independent Ags. E. C. Snow, ed. Handbook of B and T Lymphocytes 343. Academic Press, New York.
  38. Adkins, B., A. Ghanei, K. Hamilton. 1994. Up-regulation of murine neonatal T helper cell responses by accessory cell factors. J. Immunol. 153:3378.[Abstract]
  39. Adkins, B., R. Q. Du. 1998. Newborn mice develop balanced Th1/Th2 primary effector responses in vivo but are biased to Th2 secondary responses. J. Immunol. 160:4217.[Abstract/Free Full Text]
  40. Kovarik, J., P. Bozzotti, L. Love-Homan, M. Pihlgren, H. L. Davis, P. H. Lambert, A. M. Kreig, C. A. Siegrist. 1999. CpG oligodeoxynucleotides can circumvent the Th2 polarization of neonatal responses to vaccines but may fail to fully redirect the Th2 responses established by neonatal priming. J. Immunol. 160:1611.
  41. Flamand, V., V. Donckier, F. X. Demoor, A. Le Moine, P. Matthys, M. L. Vanderhaeghen, Y. Tagawa, Y. Iwakura, A. Billiau, D. Abramowicz, M. Goldman. 1998. CD40 ligation prevents neonatal induction of transplantation tolerance. J. Immunol. 160:4666.[Abstract/Free Full Text]
  42. Durandy, A., G. De Saint Basile, B. Lisowska-Grospierre, J. F. Gauchat, M. Forveille, R. A. Kroczek, J. E. Bonnefoy, A. Fischer. 1995. Undetectable CD40 ligand expression on T cells and low B cell responses to CD40 binding agonists in human newborns. J. Immunol. 154:1560.[Abstract]
  43. Steinman, R. M., M. Pack, K. Inaba. 1997. Dendritic cells in the T cell areas of lymphoid organs. Immunol. Rev. 156:25.[Medline]
  44. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  45. Sornasse, T., V. Flamand, G. De Becker, H. Bazin, F. Tielmans, K. Thielemans, J. Urbain, O. Leo, M. Moser. 1992. Ag-pulsed dendritic cells can efficiently induce an Ab response in vivo. J. Exp. Med. 175:15.[Abstract/Free Full Text]
  46. Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C.-Y. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, A. O’Garra. 1995. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J. Immunol. 154:5071.[Abstract]
  47. Petty, R. E., D. W. C. Hunt. 1998. Neonatal dendritic cells. Vaccine 16:1378.[Medline]
  48. Chelvarajan, R. L., N. L. Gilbert, S. Bondada. 1998. Neonatal murine B lymphocytes respond to polysaccharide Ags in the presence of IL-1 and IL-6. J. Immunol. 161:3315.[Abstract/Free Full Text]
  49. Buchanan, R. M., B. P. Arulanandam, D. W. Metzger. 1998. IL-12 enhances Ab responses to T-independent polysaccharide vaccines in the absence of T and NK cells. J. Immunol. 161:5525.[Abstract/Free Full Text]
  50. Jennings, H. J., C. Lugowski. 1981. Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J. Immunol. 127:1011.[Abstract]
  51. Berkower, I., H. Kawamura, L. A. Matis, J. A. Berzofsky. 1985. T cell clones to two major T cell epitopes of myoglobin: effect of I-A/I-E restriction on epitope dominance. J. Immunol. 135:2628.[Abstract]
  52. Brorson, K., M. Brunswick, S. Ezhevsky, D. G. Wei, R. Berg, D. Scott, K. E. Stein. 1997. xid affects events leading to B cell cycle entry. J. Immunol. 159:135.[Abstract]
  53. Swiggard, W. J., R. M. Nonacs, M. D. Witmer-Pack, R. M. Steinman, and K. Inaba. 1991. Enrichment of dendritic cells by plastic adherence and EA rosetting. In Current Protocols in Immunology. J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, and W. Strober, eds. Wiley, New York, p. 3.7.1.
  54. Rubinstein, L. J., K. E. Stein. 1988. Murine immune response to the Neisseria meningitidis group C capsular polysaccharide. I. Ontogeny. J. Immunol. 141:4352.[Abstract]
  55. Inaba, K., R. M. Steinman. 1985. Protein-specific helper T-lymphocyte formation initiated by dendritic cells. Science 229:475.[Abstract/Free Full Text]
  56. Steinman, R. M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271.[Medline]
  57. Cassell, D. J., R. H. Schwartz. 1994. A quantitative analysis of APC function: activated B cells stimulate naive CD4 T cells but are inferior to dendritic cells in providing costimulation. J. Exp. Med. 180:1829.[Abstract/Free Full Text]
  58. Metlay, J. P., E. Pure, R. M. Steinman. 1989. Distinct features of dendritic cells and Ig activated B cells as stimulators of the primary mixed leukocyte reaction. J. Exp. Med. 169:239.[Abstract/Free Full Text]
  59. Croft, M., D. D. Duncan, S. L. Swain. 1992. Response of naive Ag-specific CD4+ T cells in vitro: characteristics and APC requirements. J. Exp. Med. 176:1431.[Abstract/Free Full Text]
  60. Levin, D., S. Constant, T. Pasqualini, R. Flavell, K. Bottomly. 1993. Role of dendritic cells in the priming of CD4+ T lymphocytes to peptide Ag in vivo. J. Immunol. 151:6742.[Abstract]
  61. Linsley, P. S., J. A. Ledbetter. 1993. The role of CD28 receptor during T cells responses to Ag. Annu. Rev. Immunol. 11:191.[Medline]
  62. Bona, C., A. Bot. 1997. Neonatal immuno-responsiveness. Immunologist 5:5.
  63. Levin, D., H. Gershon. 1989. Ag presentation by neonatal murine spleen cells. Cell. Immunol. 120:132.[Medline]
  64. Morris, J. F., J. T. Hoyer, S. K. Pierce. 1992. Ag presentation for T cell IL-2 secretion is a late acquisition of neonatal B cells. Eur. J. Immunol. 22:2923.[Medline]
  65. Lanker, D., V. Briken, K. Adler, P. Weiser, S. Cassard, U. Blank, M. Viguier, C. Bonnnerot. 1998. Syk tyrosine kinase and B cell Ag receptor (BCR) Ig-