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The Journal of Immunology, 2002, 169: 3293-3300.
Copyright © 2002 by The American Association of Immunologists

Enhanced Type 1 Immunity After Secondary Viral Challenge in Mice Primed as Neonates1

Shaza A. Fadel, Daniel A. Ozaki and Marcella Sarzotti2

Department of Immunology, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The goal of infant immunization against viral infection is to develop protective long term memory responses. Priming neonatal mice with a low dose of Cas-Br-E murine leukemia virus (Cas) results in adult-like, type 1 protective responses. However, other studies suggest that Ag priming of neonates leads to an increase in type 2 secondary responses even when primary responses were type 1. We assessed whether type 1 CD8+ T cell-mediated responses developed in murine neonates are maintained after secondary challenge with Cas in adulthood. Despite the induction of significant anti-viral CD8+-mediated cytotoxic T lymphocyte and IFN-{gamma} responses, initial neonatal priming led to a lower frequency of virus-specific T cells compared with adult priming. Adult frequencies were reached in mice primed as neonates only after secondary challenge in adulthood. A nonspecific and transient CD4+-mediated IL-4 response was present in all groups after secondary challenge with Cas or medium, indicating that this rise in type 2 cytokine production was not unique to mice that had been primed as neonates. Rather, type 1 anti-viral memory CD8+ T cell responses developed in neonatal mice are stable, protective, and enhanced after secondary challenge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neonatal mice are exposed to many Ags after birth while their immune system continues to develop. Classical experiments demonstrating neonatal tolerance to allogeneic grafts (1) and increased susceptibility of neonates to infection led to the perception that the neonatal immune system is immature and incompetent. Certainly, several studies have supported the idea that cellular immune responses are deficient soon after birth as a result of decreased absolute numbers of lymphocytes, compromised APC function, or Th2-biased cytokine regulation (2, 3, 4).

Tolerance is not the only outcome of Ag exposure in neonatal mice. Despite the deficiencies noted above, neonatal mice have been shown to generate type 1 (promoting cytotoxicity and IFN-{gamma} production) or type 2 (IL-4, IL-10) T cell responses to various immunogens (5, 6, 7, 8, 9). CTLs of 5-day-old and adult mice exhibit comparable cytotoxicity on a per T cell basis (10). Whereas at least 106 viral particles are needed to induce antiviral Ab- and complement-mediated lysis, CTLs can be activated by as little as 100 viral particles (11). This CTL sensitivity allows for the establishment of adult-like neonatal responses to low doses of virus (5, 12). Furthermore, transferring adult APCs into newborns enables neonatal mice to develop adult-like anti-HY-specific CTL (8). Therefore, the production of adult-like cytokine, proliferative, and CTL responses requires priming conditions uniquely suited to the neonatal host (5, 6, 13, 14, 15).

Long term memory responses after neonatal priming are essential for effective immunization. Murine neonates have been shown to develop memory T cell responses with adult-like type 1 immunity (5, 6, 9, 12, 13, 16). However, other groups have reported an inherent type 2 bias in neonates that leads to an increase in or a predominance of type 2 secondary responses, even when primary responses were type 1 (4, 17, 18, 19). This would qualitatively compromise the effectiveness of vaccines when type 1 responses are essential in clearing pathogens. These conflicting reports bring into question the stability and longevity of T cell responses after neonatal priming.

To study neonatal CD8+ responses, we used a natural murine pathogen, Cas-Br-E murine leukemia virus (Cas).3 When (>=1000 PFU) Cas infect newborn mice (1–4 days of age) they become carriers and develop a slow onset (6–8 wk postinfection) of a neurodegenerative disease (5, 13, 20). These mice are not immunosuppressed, but they fail to generate protective T cell immunity. Adult mice exposed to the same dose of Cas develop protective CD8+ CTL activity and IFN-{gamma} production. Decreasing the initial exposure dose of Cas (<=1 PFU) enables neonates to develop seemingly adult-like protective, CD8+-mediated, type 1 immune responses (5). We hypothesized that optimal priming of murine neonates, which results in adult-like CD8+ T cell responses, will lead to committed type 1 memory CD8+ T cells. In the present study we challenged adult mice that had been primed as neonates with Cas and found that anti-viral CD8+-mediated type 1 responses were stable and enhanced after secondary exposure to the priming virus.


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

NFS/N mice were obtained from Charles River/NCI Biological Testing Branch (Frederick, MD). Pregnant mice were kept in pathogen-free conditions and monitored daily for delivery at the Duke University Medical Center vivarium. NFS/N mice are Fv-1nn, H-2sq4, NK1.1- and express no ecotropic murine leukemia virus and only low levels of endogenous xenotropic murine leukemia virus. Cas virus was grown on SC-1 or NIH-3T3 fibroblast cells, titrated by XC plaque assay (21), and monitored in vivo by paralysis induction (20). A low dose of virus used to prime neonates is equivalent to <=1 PFU, and a high dose of virus is >=1000 PFU (5). Mice were injected i.p. at 4 days of age with 0.03 ml of a high dose (carriers) or a low dose (neonates) of Cas. Mice were also injected with medium on day 4 or were left untreated (naive). Adults (>=21 days of age) were injected i.p. with 0.05 ml of a high dose of Cas. All mice were rechallenged with either >1000 PFU (infectious dose) or 5 PFU (booster dose) of Cas at least 8 wk after priming. In parallel, naive adult mice received a virus challenge once (adult 1000 PFU or adult 5 PFU). The protocol for viral injections is summarized in Fig. 1Go. Secondary responses were tested 6 days after viral rechallenge. All protocols for animal studies were approved by the institutional animal care and use committee of Duke University.



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  		FIGURE 1. Summary of the injection protocol. Neonatal mice (day 4 after birth) were primed with <=1 PFU Cas (neonate), infected with >=1000 PFU Cas (carrier), or sham-inoculated with medium or left uninjected (naive). Adult mice (>21 days after birth) were primed with >=1000 PFU Cas (adult). Neonates, carriers, and adults received an in vivo secondary challenge with a boosting (5 PFU) or infectious dose (>=1000 PFU) of Cas at least 8 wk after initial exposure to Cas. Naive mice that are exposed to Cas once at the same time as the secondary challenge received either an infectious dose (adult 1000 PFU) or a boosting dose (adult 5 PFU) of Cas.

 
In vitro stimulation of spleen cells

Spleens were obtained from mice and stored in complete MEM (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS, 25 mM HEPES buffer, 5 x 10-5 M 2-ME, 1% nonessential amino acids, and 1% penicillin-streptomycin-glutamine (Life Technologies) on ice until mechanical separation was performed (13). Single-cell suspensions were treated with an ammonium chloride potassium chloride (ACK) buffer to lyse erythrocytes (22). Cell cultures were set up in 24-well tissue culture plates for CTL stimulation as previously described (16). Cells in one set of plates were cocultured with irradiated NS467 cells. NS467, a Cas-infected pre-B cell line, was subjected to 10,000 rad using a cesium irradiator (Mark I; J. L. Shepherd and Associates, San Fernando, CA). A control set of plates received 1 ml complete medium as a control. Cultures were incubated at 37°C in 5% CO2 for 5 days. On day 5 of culture, effector cells were harvested from replicate wells, pooled, washed, and counted for use in a standard 51Cr cytotoxicity assay. Cytotoxicity to Cas infection cannot be detected without in vitro restimulation (5).

Cytotoxicity assays

Effector cells from 5-day cultures were harvested, counted, and aliquoted in triplicate wells with 51Cr-labeled (Perkin-Elmer/NEN Life Sciences, Boston, MA) NS467 target cells at varying E:T cell ratios in 96-well microtiter plates. After a 4-h incubation at 37°C, 100 µl cell-free supernatant was collected from each well, and radioactivity was detected in a 1272 Clinigamma (LKB Wallac, Turku, Finland) gamma counter. Cytotoxicity was determined by comparing 51Cr released in supernatants from wells with both effector and radiolabeled target cells (experimental), and from wells with radiolabeled target cells alone (spontaneous), as well as 51Cr incorporated into labeled target cells (total) using the following formula: (experimental - spontaneous/total - spontaneous) x 100. Data are expressed as the percent cytotoxicity or as lytic units per 1 x 107 spleen cells required for 30% cytotoxicity (LU30/107) as previously described (13). CTL responders are defined as mice whose cells display a percent cytotoxicity that is 2 SD above the mean of sham-inoculated (naive) mice and that exhibit >20 LU30/107 cells (5, 13).

ELISPOT assay

ELISPOT assays were performed using modified standard procedures (23). Briefly, 96-well filtration plates (Cellular Technology, Cleveland, OH) were coated with 0.2 µg/well purified anti-mouse IFN-{gamma} (clone RA-6A2) or anti-mouse IL-4 (clone 11B11) Abs (all Abs used for ELISPOT were obtained from BD PharMingen, San Diego, CA). Coated plates were washed with sterile PBS and blocked for 1.5 h with PBS/1% BSA. Serial dilutions of responder cells (in duplicates) starting at 0.5–1 x 106 cells in HL-1 serum-free medium (BioWhittaker, Walkersville, MD) were added to the wells, followed by stimulation with 5 x 105 irradiated NS467 cells or HL-1 serum-free medium. Cultures were incubated for 24 h at 37°C in 5% CO2. After incubation, cells were extensively washed with PBS, followed by PBS/1% BSA/0.05% (v/v) Tween 20. Each well then received 0.2 µg/ml biotinylated anti-IFN-{gamma} (clone XMG1.2) or anti-IL-4 (clone BVD6-24G2) Ab diluted in PBS/0.05% Tween 20/0.1% BSA. After overnight incubation, unbound secondary Ab was removed by washing with PBS/0.05% Tween 20. HRP-conjugated streptavidin (Southern Biotechnology Associates, Birmingham, AL) was diluted 1/2000 in PBS/0.05% Tween 20/0.1% BSA, and 100 µl of this solution was added to each well. After a 1.5-h incubation, plates were washed with PBS. ELISPOT plates were developed by the addition of 0.03% 3-amino-9-ethyl-carbazole (Pierce, Rockford, IL) diluted in 0.1 M acetate buffer, pH 5, containing 0.05% H2O2. Reactions were allowed to proceed for 10–40 min and were halted by extensive washing in water. Plates were dried, and spots were counted using an ELISPOT Imager (Cellular Technology). Virus-specific cytokine responses were determined by subtracting spots in wells with medium alone from the number of spots in wells stimulated with NS467. Positive IFN-{gamma} and IL-4 responses were defined as 2 SD above mean spot values from naive mice (26 and 9 spots/1 x 106 splenocytes, respectively). Irradiated NS467 cells produced no IFN-{gamma} or IL-4 (data not shown).

Flow cytometry and intracellular staining

Splenocytes were resuspended in PBS/BSA buffer (PBS, 1% BSA, and 0.1% NaN3). Cell suspensions were incubated in the dark for 10 min on ice with Fc Block (BD PharMingen) to prevent Ab binding to Fc receptors and with 7-aminoactin omycin-D (Molecular Probes, Eugene, OR) to enable exclusion of dead cells during analysis (22). The following BD PharMingen mAbs and reagents were used for flow cytometry: anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), and anti-pan-NK marker DX5. Anti-CD62 ligand (CD62L) mAb (LAM1-116) was provided by Drs. Tedder and Steeber, Duke University Medical Center (24). mAbs were either biotinylated or directly conjugated to FITC, PE, Red-613, or allophycocyanin. Cell samples were analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA). Between 20,000 and 50,000 events/sample were collected and were analyzed using FlowJo software (TreeStar, San Carlos, CA).

To enumerate cytokine-producing cells, intracellular cytokine staining was performed by culturing 1 x 106 freshly isolated splenocytes in flat-bottom 96-well plates. Cells were either left untreated or were stimulated with NS467 for 6 h at 37°C in 5% CO2. Monensin (Golgi-Stop; BD PharMingen) was added for the duration of the culture period to facilitate intracellular cytokine accumulation. Cell surface staining for CD8 and CD62L (described above) was then performed, followed by intracellular cytokine staining using the Cytofix/Cytoperm kit (BD PharMingen) according to the manufacturer’s recommendations. For intracellular staining, the Abs used were anti-IFN-{gamma} (clone XMG1.2) and anti-IL-4 (clone BVD4-1011; BD PharMingen).

CD4+ and CD8+ T cell purification

Splenocytes were enriched for either CD4+ or CD8+ subpopulations using T cell subset enrichment columns (R&D Systems, Minneapolis, MN) following the manufacturer’s recommendations. The purity of the subpopulations exceeded 95%.

Adoptive transfer

Donor mice were either 8 wk or 9 mo old. Splenocytes from mice primed as neonates or adults were collected after a 5-day culture with irradiated NS467, whereas splenocytes from naive adult mice were freshly isolated. After removal of dead cells by centrifugation on Ficoll (Nycomed, Oslo, Norway), 5 x 106 donor cells were injected i.p. into 2-day-old recipient mice. Recipients were infected 24 h later with >1000 PFU Cas i.p. on the opposite side of the abdomen. Examinations of the hosts for clinical symptoms of neurologic disease were performed 8–34 wk after adoptive transfer as previously described (20). Clinical symptoms included tremor, hind limb weakness, and paralysis. The presence of Cas in the host was determined by RT-PCR. Spleens and brains were removed from the hosts. RNA was extracted from spleen and brains using TRIzol (Life Technologies) and was converted to cDNA using Moloney murine leukemia virus reverse transcriptase (Life Technologies). The presence of Cas was detected by PCR using Cas envelope-specific primers as previously described (13).

Statistical analysis

Cytotoxicity and ELISPOT values >=2 SD above those exhibited by naive or sham-inoculated mice were considered significant. Data from experimental groups were also compared using Student’s t test. Statistical significance was determined by p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Secondary cytotoxic responses to Cas

Neonatal mice develop significant adult-like CTL responses after priming with a low dose of Cas virus, whereas neonates that receive a high, infectious dose of Cas (>=1000 PFU, carriers) do not exhibit cytotoxicity toward Cas-infected targets (NS467, Fig. 2GoA) (5). CTL responses induced by priming neonates with a low dose of Cas are not significantly different from cytotoxic responses following priming of adults (Fig. 2GoA).



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  		FIGURE 2. Adult-like cytotoxic responses are detected in mice that were primed as neonates, before and after rechallenge. Mice were exposed to Cas as neonates or adults once (see Fig. 1Go). Naive mice were sham-inoculated with medium as neonates or left uninjected. CTL responses were measured in the spleen at >=2 wk after primary challenge (A). At >=8 wk after priming, mice were rechallenged with >=1000 PFU of Cas (B) or with 5 PFU of Cas (C and D). CTL responses were measured in the spleen 6 days (B and C) or 3 wk (D) after secondary challenge. Naive mice were also challenged once with Cas (adult 1000 PFU, adult 5 PFU) when the experimental groups received the secondary challenge. For all experiments cytotoxicity was measured 5 days after in vitro restimulation with NS467 cells by a standard 51Cr release assay. The cytotoxicity of naive lymphocytes from mice that were not rechallenged was not significant (A and D). Values represent the mean cytotoxicity ± SEM.

 
We first tested whether priming murine neonates would compromise their T cell responses to an infectious dose of Cas (>=1000 PFU) in adulthood. Mice that had been primed with Cas as neonates or adults, or naive mice, were challenged with an infectious dose of Cas at least 8 wk after priming. Six days after secondary challenge, mice that were previously primed with Cas developed significant CTL activity compared with mice that were exposed to Cas only 6 days earlier (adult 1000 PFU; Fig. 2GoB). Cas carriers failed to exhibit significant cytotoxicity toward NS467 even when rechallenged as adults (Fig. 2GoB). Mice that had been primed as neonates developed CTL activity comparable to that of adult-primed mice when rechallenged with an infectious dose of Cas.

Since memory T cells have a greater and faster ability to respond to low doses of Ag than their naive counterparts (25), we measured responses to a rechallenge with a low viral dose (5 PFU, boosting dose) (26). Unless otherwise specified, the experiments described hereafter used a boosting dose as a rechallenge. Six days after rechallenge only mice that were primed with Cas as neonates or as adults exhibited significant CTL activity (Fig. 2GoC). Three weeks after challenge, primary virus-specific cytotoxicity was detectable in adult mice challenged only with the boosting dose (adult 5 PFU; Fig. 2GoD). Therefore, focusing on 6 days after rechallenge with Cas distinguished between a developing primary CTL response and a secondary response, especially since priming of naive cells has been described as a component of the memory response (27). Importantly, mice primed as neonates or adults exhibited similar cytotoxicity.

CTL activity was compared by measuring the splenocyte lytic units, which reflects the number of effector cells per 1 x 107 splenocytes required for 30% Cas-specific lysis. Lytic units were calculated from mean cytotoxicity values before (Fig. 2GoA) and after secondary challenge (Fig. 2GoC). This analysis allowed us to compare adult vs neonatal responses. However, since the accuracy of the LU calculation depends on the slope of the cytotoxicity curves, we could not compare primary with secondary responses by this method because their cytotoxicity curves were not parallel. After initial priming, a 3.2-fold greater number of effector cells from mice primed as neonates was required to achieve the same cytotoxicity as that in adult-primed mice (317 LU30/107 after adult priming and 100 LU30/107 after neonatal priming; Fig. 2GoA). A threshold of <20 LU30/107 was measurable in naive mice. However, 6 days following the secondary challenge with a boosting dose of Cas only a 1.4-fold greater number of effector cells was required for neonatally primed mice to achieve the same cytotoxicity as adult-primed mice (75 LU30/107 after adult priming and 55 LU30/107 after neonatal priming; Fig. 2GoC). These results suggest that there are initially fewer Cas-specific CTL in mice primed as neonates than in those primed as adults, and that this difference decreases following a booster injection.

Measuring the virus-specific memory pool

Increased IFN-{gamma} production is consistently found in mice that exhibit Cas-specific CTL responses (28). Thus, we determined the frequency of virus-specific IFN-{gamma}-producing spleen cells before and after rechallenge with the virus. ELISPOT analysis was used, since it provides a sensitive, single-cell quantitation of Cas-specific cells ex vivo (9, 29). Splenocytes from mice primed as neonates or adults were harvested >=2 wk after priming or 6 days after secondary challenge. Splenocytes were stimulated for 24 h with NS467 or medium alone in the ELISPOT plates. IFN-{gamma}-producing cells per 1 x 106 splenocytes were then enumerated. As predicted by the CTL responses, neonatally primed or adult-primed mice had significantly increased frequencies of Cas-specific IFN-{gamma}-producing splenocytes before and after viral rechallenge compared with naive mice (Fig. 3GoA). However, after initial priming the frequency of virus-specific IFN-{gamma} producers in neonatally primed mice was 41% of that in adult-primed mice (1/15,385 and 1/6,369, respectively; p = 0.01; Fig. 3GoA). After the secondary challenge the frequencies of virus-specific IFN-{gamma} producing splenocytes in neonatally primed mice were not significantly different from those in adult-primed mice (1/6,536 and 1/5,155, respectively; p > 0.05). This result was consistent with the analysis of CTL activity.



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  		FIGURE 3. Frequency of Cas-specific IFN-{gamma}- and IL-4-producing splenocytes in mice primed as neonates or adults after a boosting dose of Cas. Mice that had been primed as neonates or adults (primary) were then rechallenged with a boosting dose of Cas (secondary) at least 8 wk postpriming. Naive mice (primary) were challenged once with a boosting dose of Cas (adult 5 PFU; secondary). Spleens were harvested at least 2 wk after priming and 6 days after secondary challenge (A and B) or 3 wk after secondary challenge (C). Splenocytes were then restimulated with NS467 cells or medium for 24 h in an ELISPOT assay. Values represent the mean ± SEM spots of IFN-{gamma} (A and C) or IL-4 (B and C) per 1 x 106 splenocytes. The average frequency of IFN-{gamma}-producing cells is reported in the accompanying table. *, p = 0.01, by Student’s t test. Four to 19 mice were used in each group in B.

 
Neonates are reported to have an inherent bias to type 2 responses that is enhanced with secondary challenge in adulthood (4, 17). Thus, we examined whether virus-specific type 2 responders were present after neonatal priming and preferentially increased after secondary challenge. Since the number of IFN-{gamma}-producing splenocytes increases between neonatal priming and secondary challenge in adulthood, one might have expected a reciprocal change in IL-4 production. However, virus-specific IL-4-producing splenocytes were detected at very low levels >=2 wk after initial priming with Cas (Fig. 3GoB) and were increased in frequency in all groups 6 days after the rechallenge (Fig. 3GoB). There was no significant difference between the frequency of IL-4-producing splenocytes in mice primed with Cas as neonates and in adult mice that were challenged with Cas only 6 days previously. The IL-4 response was transient, as IL-4-producing splenocytes were no longer detectable above medium background 3 wk after rechallenge, whereas IFN-{gamma}-producing splenocytes persisted at increased levels (Fig. 3GoC).

Virus-specific type 1 responses are mediated by CD8+ memory cells

Our group previously reported that Cas-specific CTL and IFN-{gamma} responses are mediated by CD8+ T cells, while IL-4 production is mediated by CD4+ T cells (5, 22). Six days after in vivo rechallenge, splenocytes from adult-primed and neonatally primed mice were fractionated into CD4+ and CD8+ subsets and tested by ELISPOT for IFN-{gamma} and IL-4 production (Fig. 4GoA). Enrichment of CD8+ cells, but not CD4+ cells, resulted in a 4- to 5-fold increase in the number of IFN-{gamma}-producing cells in spleens of neonatally primed/boosted and adult-primed/boosted mice over the frequencies seen in unfractionated splenocytes (Fig. 4GoA). IL-4-producing cells were enriched in CD4+ splenocytes from control mice more than in CD4+ splenocytes from mice that had been primed and rechallenged with Cas (Fig. 4GoB). Furthermore, IL-4 responses were increased at similar frequencies in mice primed with medium as adults or neonates and rechallenged with medium alone (Fig. 4GoC). Therefore, IL-4-producing cells in these rechallenged mice were neither Cas specific nor age dependent. The IL-4 response was not a result of NK T cells, as the CD4-enriched population was negative for DX-5, a pan-NK cell marker (data not shown). The CD8+-enriched population did not produce IL-4 (Fig. 4GoA).



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  		FIGURE 4. CD8+ cells produce virus-specific IFN-{gamma} in adult-primed boosted and neonatally primed boosted mice. The frequencies of IFN-{gamma}- and IL-4-producing splenocytes were enumerated after enriching for either CD8+ cells (A) or CD4+ (B and C) cells from mice that had been primed as neonates or adults with Cas and then rechallenged with a boosting dose of Cas >=8 wk after priming (A and B). Mice that were sham-inoculated with medium as neonates or adults were challenged with Cas or with medium alone (C; n = 2–4/group; mean IL-4 spots ± SD). Six days after rechallenge, splenocytes were restimulated with NS467 cells or medium alone for 24 h in an ELISPOT assay. Values represent the mean ± SEM (A and B) number of spots per 1 x 106 spleen cells.

 
Flow cytometric analysis of cytokine-producing cells has been used to detect virus-specific cells in many systems (30, 31). The negative to low expression of CD62L (CD62Llow/-) has been useful in identifying functional memory T cell populations (25). We investigated whether the CD8+ IFN-{gamma}-producing splenocytes displayed a memory phenotype. Six days after secondary challenge most of the CD8+ IFN-{gamma}-producing cells in adult-primed and neonatally primed mice were CD62Llow/- memory cells (Fig. 5Go). This virus-specific population accounted for 1 and 0.84% of CD8+ lymphocytes in mice primed as adults and neonates, respectively (Fig. 5GoB).



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  		FIGURE 5. IFN-{gamma} is produced by Cas-specific CD8+ memory cells in boosted mice. Six days after mice were rechallenged with a boosting dose of Cas, their splenocytes were cultured with NS467 cells for 6 h in the presence of monensin. The cells were harvested and stained for CD8, IFN-{gamma}, and CD62L. Analysis was performed after gating on the CD8+ population. A, Plots represent IFN-{gamma} production and CD62L expression of CD8+ cells from mice primed as neonates or adults. The plots shown are representative of three experiments with at least three mice per experimental group. The percentages of CD62Llow/- CD8+ cells that produce IFN-{gamma} upon restimulation with NS467 cells are summarized from the three experiments. Restimulating the cells with EL-4, an allogeneic cell line, for 6 h shows background IFN-{gamma} production ranging from 0.18 to 0.22% in neonatally primed mice and from 0.23 to 0.31% in adult-primed mice (data not shown). Value represent the mean percentage of CD8+ cells ± SEM.

 
Longevity of neonatal memory

Data from the in vitro cytotoxicity and cytokine responses suggested that mice that were primed as neonates exhibited type 1 responses qualitatively similar to those of mice primed as adults despite a quantitative difference in the number of effectors generated after priming. Thus, we investigated whether the lower frequency of virus-specific CD8+ memory cells generated after neonatal priming compromised long-term protection in vivo. We previously reported that transferring splenocytes from mice that had been primed as neonates protects carrier host mice from developing Cas-induced neurodegenerative disease (5). We wondered whether protection decayed more rapidly in splenocytes from mice that were primed as neonates compared with adults, since the latter had a higher frequency of memory cells after initial priming. Therefore, we transferred 5 x 106 splenocytes from 8-wk-old or 9-mo-old mice primed as neonates or adults into neonatal hosts, which were then infected with >=1000 PFU Cas the following day. Host mice were examined weekly for clinical symptoms of neurological disease (tremor, hind limbs weakness, and paralysis). Cas-immune cells from mice primed as neonates or adults protected the carrier mice from developing neurological disease even when the donors were 9 mo old (Fig. 6GoA). Furthermore, RT-PCR amplification of the Cas envelope region could not detect virus in the brains and spleens of asymptomatic hosts >=32 wk after transfer (Fig. 6GoB). Transfer of naive splenocytes delayed the onset, but did not protect the recipient mice from disease.



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  		FIGURE 6. Long-lasting protection from neurological disease is provided by splenocytes from mice that were primed as neonates or adults. Splenocytes (5 x 106) from either 8-wk-old or 9-mo-old mice primed as neonates or adults were adoptively transferred into neonates 2 days after birth of the host. Mice were then infected with >=1000 PFU Cas on day 3 after birth. A, Mice were examined weekly for clinical symptoms of neurologic disease (tremor, hind limb weakness, and paralysis). Values represent cumulative percentage of mice that developed neurologic disease. B, RT-PCR was used to detect the presence of virus in the brains (B) and spleens (S) of host mice. Asymptomatic mice did not show the presence of Cas >=32 wk after transfer (adult 9 mo (lanes 1–4), adult 8 wk (lanes 5–8), neonate 9 mo (lanes 9–12), neonate 8 wk (lanes 13–16)). Cas is detected in the brains and spleens of carrier mice that received only naive spleen cells (lanes 17 and 18) or no donor cells (lanes 19 and 20). Hypoxanthine phosphoribosyltransferase is also amplified using the same cDNA in separate reactions as a control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neonatal mice can exhibit diverse, mature immune responses and are not always tolerized by exposure to Ags. Yet, they remain more susceptible to infections than adults. Much of this susceptibility is ascribed to decreased lymphocyte numbers, reduced APC function, or biased cytokine regulation (2, 3, 4). Many groups have shown that optimizing neonatal priming can essentially overcome these deficiencies and lead to qualitatively adult-like T cell responses (5, 8, 9, 12, 15, 32). However, the stability and longevity of this response have been questioned by reports of altered cytokine profiles with secondary antigenic challenge (4, 17, 18, 19).

Here we show that mice primed with optimal doses of Cas as neonates maintain virus-specific CD8+ type 1 responses when challenged with the virus in adulthood. Only mice that previously developed CTL activity to Cas responded with significant specific cytotoxicity 6 days after the secondary challenge (Fig. 2Go, B and C). The fact that mice that were primed as neonates exhibit CTL activity similar to that after priming adults even when challenged with an infectious dose of Cas indicates that neonatal exposure does not compromise the efficiency of CTLs in adulthood. After virus inoculation, it takes 10–15 days to induce Cas-specific CTL responses in both murine adults and neonates (5). The similar kinetics suggest that CTLs are primed during the neonatal period rather than by delayed activation induced by lingering virus in older mice (5). As expected, Cas carrier mice, which did not develop CTL activity after neonatal infection with Cas, failed to exhibit significant cytotoxicity toward NS467 even when rechallenged as adults (Fig. 2Go, A and B). Therefore, activating CTL effectors in the neonatal period is by no means a disadvantage to the murine CD8+ T cell response; the CTLs remain effective and specific in adulthood.

After initial priming, a 3.2-fold greater number of effector cells from mice primed as neonates is required to achieve the same cytotoxicity as that in adult-primed mice. However, only a 1.4-fold greater number of effector cells is required for boosted neonatally primed mice to achieve the same cytotoxicity as mice primed as adults. We could not directly compare LU values of primary with secondary responses because the slopes of the killing curves were not parallel. Greater cell death in the 5-day restimulation cultures of splenocytes from rechallenged mice may have caused a lower cytotoxicity at the lower E:T cell ratios, which resulted in steeper killing curve slopes and lower LU values. However, this effect was not evident in the ELISPOT experiments, where splenocytes were cultured for 24 h ex vivo. Yet, LU comparisons between neonatal and adult priming were similar to the ELISPOT frequencies, which indicate 59% fewer virus-specific cells after initial priming of neonates vs adults. Following secondary viral challenge of mice primed with Cas, the frequencies of Cas-specific IFN-{gamma}-producing splenocytes in mice that were primed as neonates (1/6,536) were not statistically different from those that had been primed in adulthood (1/5,155), which is in agreement with the LU analysis. Therefore, type 1 responses were actually enhanced after secondary challenge of mice that had been primed as neonates.

The lower frequency of virus-specific memory T cells in mice primed as neonates vs adults suggests a smaller Cas-specific memory T cell pool that reflects the magnitude of the initial clonal burst during priming (30, 33). With the smaller number of lymphocytes present in peripheral lymphoid tissues in murine neonates (1% of adult lymph node and 10% of adult spleen (2)), one would expect a smaller activated T cell pool in neonates than in adults. After priming with Cas, the frequency of virus-specific IFN-{gamma}-producing cells in neonates is 41% of that measured after priming adults. Similarly, 50 days after neonatal infection with polyoma virus, the number of Ag-specific CD8+ cells was 13% of the adult CD8 memory cell population. However, these cells exhibited adult-like cytotoxic function (31). In both cases the smaller size of the resulting memory pool in mice that had been primed as neonates compared with that in adults reflects the small magnitude of the initial Ag-specific clonal burst of neonatal T cells (33). Thus, despite a quantitatively smaller memory pool, type 1 responses remain efficient and adult-like in qualitative terms.

To confirm that IFN-{gamma} is produced by memory cells, we assessed the phenotype of cytokine producers in the spleens 6 days after boosting mice primed with Cas. Most of the virus-specific IFN-{gamma} after viral rechallenge is produced by CD8+ cells, which is concordant with our previous reports on the response of mice primed with Cas as neonates (Fig. 4Go) (5). Furthermore, this IFN-{gamma}-producing population is mostly CD62Llow/-, indicating cytokine production by committed virus-specific type 1 memory CD8+ cells (Fig. 5Go).

In contrast to IFN-{gamma} responses, IL-4 production after secondary challenge with Cas was transient and was not virus specific (Fig. 3Go, B and C, and Fig. 4GoC). The maintenance of type 1 responses in mice primed with Cas did not result from a deficiency in IL-4 production in this mouse strain. Nonspecific IL-4 was increased within 6 days after challenge with virus or medium, and it subsided within 3 wk after challenge. Even though we cannot rule out that the increase in nonspecific IL-4 production is caused by the FCS in the medium used for injections, it is interesting to note that a sharp increase in the frequency of IL-4-producing splenocytes is detected in the first weeks after initial priming of adults (unpublished results). This is reminiscent of bystander recall IL-4 produced during a type 1 response to OVA (23). The physiological role of such nonspecific IL-4 during the induction of secondary responses to Cas remains to be determined. It is unlikely that committed memory type 1 CD8+ cells would be affected by IL-4, since type 1 committed CD8+ cells resist switching to type 2 cytokine production upon stimulation in vivo and in vitro (34, 35). However, IL-4 may compromise the ability of naive T cells to become type 1 responders at the time of secondary challenge (27). This is supported by evidence of increased susceptibility to disease in mice that received exogenous IL-4 during the initial priming with Cas (2) and other studies that documented the inhibitory effect of IL-4 on developing type 1 CD8+ cells (36, 37). Therefore, IL-4 may regulate the expansion of primary virus-specific effectors and the virus-specific memory pool as seen in another viral system (38). The data do not suggest a virus-specific switch in the secondary cytokine profile that is inherent to neonatal priming.

Our data differ from those of investigators who have documented immune deviation toward type 2 responses in mice primed as neonates but not as adults. However, in those systems primary type 1 responses either were not highly polarized (type 2 responses were present or dominant) (4, 17, 18, 39, 40) or were defined as weak generation of Th1/CTL responses by the authors (19). This difference is unlikely to be strain specific, because NFS/N mice produce IL-4 (5, 22). More likely, priming conditions and types of Ag influence the magnitude and strength of the neonatal T cell response. Recently, adult-like CD4+ and CD8+ T cell responses were generated in neonates by priming with a HSV-1 that could undergo only one round of replication (32). The strategy of priming neonatal CD4+ and CD8+ T cells using a viral vector with limited ability to replicate was also successful in another system (6). Limited viral replication may control the amount of virus presented to T cells in the neonate, much like decreasing the initial priming dose of Cas. Neonatal DNA immunization, which produces low sustained Ag synthesis, has been another successful strategy that leads to long-lasting CD8+ T cell responses (15, 16, 26). This further emphasizes the need for determining ways to optimize neonatal priming, which will largely influence the stability of a secondary response.

Our data suggest that a boosting strategy may allow mice that were primed as neonates to develop memory cell populations quantitatively similar to those seen in adults. However, our in vitro cytotoxicity data argue that a quantitative difference, if present, may not affect the quality of type 1 effector function. This conclusion is supported by our in vivo comparison of function, since adoptive transfer of splenocytes from adult mice that had been primed with Cas as neonates conferred protection from disease in mice that received a full dose of Cas as neonates (carriers; Fig. 6GoA) (5). This protection is present when donor cells are obtained 8 wk or 9 mo after initial neonatal or adult priming (Fig. 6Go, A and B) despite the lower number of Cas-specific memory CD8+ cells in the mice primed as neonates. The quality of their long-lasting memory response was efficient in controlling viral replication in the host’s target organs and in preventing the onset of neurological disease (20). Since an optimal number of splenocytes were transferred in these experiments, we are determining whether transfer of limiting numbers of donor Cas-specific memory cells would reveal a difference in the quality of protection between cells of mice primed in adulthood compared with those primed during the neonatal period. We note that donor cells may control viral replication at least in part by assisting the development of a host-derived immune response (41, 42). Nevertheless, our data suggest that the maturity of the neonatal immune response may be underestimated if only based on quantitative differences.

In conclusion, our results indicate that the Cas-specific type 1 responses in mice primed as neonates are CD8+ mediated, specific, and enhanced after secondary viral challenge. The data suggest that initiating CD8+ T cell responses early in life does not compromise the host’s immunity. Rather, it efficiently protects the murine neonatal host from infection until the host develops adult levels of virus-specific responders.


   Acknowledgments
 
We thank Annie M. Garcia and Drs. Doug Steeber, Mike Krangel, You-Wen He, and Louise M. Markert for their thoughtful input in these studies and critical reading of the manuscript. We are also appreciate the technical expertise of Dr. Shui Cao as well as Dr. Mike Cook and Lynn Martinek (Duke Comprehensive Flow Cytometry Facility). We thank Natalie Dugger (Baltimore Veterans Affairs Medical Center) for performing the XC plaque assay.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant CA65388 (to M.S.) Back

2 Address correspondence and reprint requests to Dr. Marcella Sarzotti, Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 27710. E-mail address: msarzott{at}duke.edu Back

3 Abbreviations used in this paper: Cas, Cas-Br-E or Cas-Br-M murine leukemia virus; CD62L, CD62 ligand; LU, lytic unit. Back

Received for publication April 24, 2002. Accepted for publication July 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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