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Neonatal Immunity Develops in a Transgenic TCR Transfer Model and Reveals a Requirement for Elevated Cell Input to Achieve Organ-Specific Responses¹

Department of Microbiology, University of Tennessee, Knoxville, TN 37996

	Abstract

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In recent years, it has become clear that neonatal exposure to Ag induces rather than ablates T cell immunity. Moreover, rechallenge with the Ag at adult age can trigger secondary responses that are distinct in the lymph node vs the spleen. The question addressed in this report is whether organ-specific secondary responses occur as a result of the diversity of the T cell repertoire or could they arise with homogeneous TCR-transgenic T cells. To test this premise, we used the OVA-specific DO11.10 TCR-transgenic T cells and established a neonatal T cell transfer system suitable for these investigations. In this system, neonatal T cells transferred from 1-day-old DO11.10/SCID mice into newborn (1-day-old) BALB/c mice migrate to the host’s spleen and maintain stable frequency. The newborn BALB/c hosts were then given Ig-OVA, an Ig molecule carrying the OVA peptide, and challenged with the OVA peptide in CFA at the age of 7 wk; then their secondary responses were analyzed. The findings show that the lymph node T cells were deviated and produced IL-4 instead of IFN- and the splenic T cells, although unable to proliferate or produce IFN-, secreted a significant level of IL-2. Supply of exogenous IL-12 during Ag stimulation restores both proliferation and IFN- production by the splenic T cells. This restorable form of splenic unresponsiveness referred to as IFN--dependent anergy required a transfer of a high number of neonatal DO11.10/SCID T cells to develop. Thus, the frequency of neonatal T cell precursors rather than repertoire diversity exerts control on the development of organ-specific neonatal immunity.

	Introduction

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A half-century ago, it was shown that neonatal exposure to an alloantigen induced a form of tolerance permissive for grafting tissues that otherwise would be rejected (1). Since then, significant progress has been made to show that injection of Ag during the neonatal stage induces rather than suppresses immunity (2, 3, 4, 5, 6), but such responses are biased and lead to tolerance of Ag during a subsequent encounter (7). In most cases, neonatal exposure to Ag changes the phenotype of the T cells from Th1 to Th2 (3, 4, 6) and diverts the response to develop in the spleen instead of the lymph node (3, 4). Indeed, mice given soluble proteins or peptides in IFA on the day of birth and challenged with the same Ag in CFA later at an adult age develop Th2 instead of Th1 T cell responses, and such responses arise in the spleen instead of the lymph node (3, 4). Although the exact mechanism for the apparent selective maintenance of the Th2 cells is unknown, it has been suggested that the primary Th1 cells that arise upon neonatal exposure to Ag are more vulnerable to apoptosis (8). Also, recent investigations have envisioned the involvement of regulatory (9) and CD4⁺Thy1^- (10) T cells to maintain such biased neonatal immunity. Furthermore, although neonatal tolerance has proved effective in conferring resistance against autoimmunity (11, 12, 13, 14), the mechanism initiating and perpetuating the biased neonatal immunity is still undefined. Recent investigations reported that factors such as the type of APCs (5, 15); the adjuvant into which the Ag is emulsified (4, 14); and the dose (6, 14), form (16, 17), in vivo availability (18), and continuous supply (19) of Ag control the induction of neonatal tolerance. Our own investigations using Igs as a vehicle for peptide delivery revealed yet another bias in neonatal immunity, namely, development of organ-specific responses in the lymph node vs the spleen (7, 13, 14) as opposed to spleen alone in free peptide systems (3, 4, 12). Indeed, Ig-PLP1 (20, 21) a chimera harboring the proteolipid protein (PLP)⁵ 139–151 sequence, or PLP1, given to SJL mice in saline on the day of birth, induced an organ-specific regulation of T cells characterized by a deviation in the lymph node and a novel form of IFN--dependent anergy in the spleen (7, 13, 14). Specifically, mice given Ig-PLP1 on the day of birth and challenged with PLP1 peptide at 7 wk of age developed PLP1-specific T cells in the lymph node that produced IL-4 instead of IL-2, and in the spleen, the cells, although nonproliferative and unable to produce IFN-, secreted significant amounts of IL-2. Furthermore, when supplied with IFN- or the IFN- inducer IL-12, these splenic cells regained proliferative and IFN- responsiveness (13, 22). This form of neonatal immunity, which circumvents the use of adjuvant, confers protection against autoimmunity and allows resistance to the induction of experimental allergic encephalomyelitis (EAE) (7, 13). However, free PLP1 peptide given to mice on the day of birth in saline instead of Ig-PLP1 had no effect on the adult response to a challenge with PLP1 in CFA, and such animals were not protected against EAE (7, 14). Also, PLP1 in IFA given on the day of birth, although protective against EAE, generated a response to immunization with peptide in CFA characterized by a deviated T cell response in the spleen, and unresponsiveness in the lymph node (14). Consequently, delivery of peptide on Ig circumvents the use of adjuvant and confers to the peptide the ability to protect against autoimmunity by a unique mechanism involving lymph node deviation and IFN--dependent splenic anergy (13, 22). In this report, we wished to address the question of whether such organ-specific regulation of neonatal immunity is related to the diversity of the T cell repertoire or whether it occurs when the neonatal immune system comprises a homogeneous population of TCR-transgenic T cells. To this aim, an Ag-specific TCR-transgenic neonatal T cell transfer system was developed which allowed for T cell homing and homeostasis within the host’s spleen. In this system, 1-day-old BALB/c newborns were given neonatal DO11.10-transgenic T cells (23) carrying a TCR specific for aa 323–339 of OVA, and homing analysis indicated that efficient transfer of the donor cells to the host’s spleen did occur. In addition, the frequency of DO11.10 cells reached 3% among host’s splenic CD4 lymphocytes by day 6 after transfer. Furthermore, when the newborn mice that received the neonatal DO11.10 T cells were given Ig-OVA, an Ig carrying the OVA peptide, and challenged with the OVA peptide at the age of 7 wk, they developed lymph node deviation and IFN--dependent splenic anergy similar to the Ig-PLP1/SJL system (13, 14). However, to achieve such organ-specific neonatal immunity and induce the IFN--dependent anergy, a high T cell transfer input was required. Moreover, we found that OVA peptide presentation on Igs interferes with the up-regulation of CD40 ligand (CD40L) on T cells, and such interference may play a role in the development of organ-specific neonatal immunity.

	Materials and Methods

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Mice

BALB/c mice (H-2^d) were purchased from Harlan Sprague Dawley (Indianapolis, IN). DO11.10-transgenic mice expressing a TCR specific for chicken OVA_323–339 in the context of the MHC class II molecule, I-A^d were previously described (23). DO11.10 mice were bred onto the SCID background to avoid rearrangement of the endogenous TCR -chain. The mice were maintained and bred in our pathogen-free animal facility. To coordinate delivery between 1-day-old donor DO11.10/SCID and newborn (1-day-old) host BALB/c mice, we maintain a significant number of breeding sets. All experimental procedures were conducted according to the guidelines of the institutional animal care committee.

Peptides

HPLC-purified (90% purity) OVA_323–339 (SQAVHAAHAEINEAGR) and the control hemagglutinin (HA)_110–120 (SFERFEIFPKI) peptides were purchased from Research Genetics (Huntsville, AL). The OVA peptide is presented to T cells in association with I-A^d class molecules, whereas HA peptide is recognized by T cells in the context of I-E^d molecules.

Ig chimeras

OVA peptide was expressed on an IgG2b Ab, and the resulting chimera was designated Ig-OVA. The genes used to construct this chimera are those coding for the L and H chains of the antiarsonate Ab, 91A₃, which was used to generate Ig-PLP1 (20). The procedures used to engineer and express Ig-OVA are similar to those described for Ig-PLP1 (20). In brief, the 91A₃V_H gene (24) was subcloned into the EcoRI site of the pUC19 plasmid and used as template DNA in PCR mutagenesis reactions to generate 91A₃V_H fragments carrying OVA_323–339 nucleotide sequence in place of the D segment within complementarity determining region 3 region. The resulting 91A₃V_H-OVA fragment was then analyzed by nucleotide sequencing to ensure that the D segment was deleted and a nucleotide sequence that will encode full OVA peptide was inserted instead. Subsequently, the 91A₃V_H-OVA was subcloned into a PSV2-gpt-2b expression vector in front of the exons coding for the constant region of a BALB/c 2b. This PSV2-gpt-91A₃V_H-OVA-2b plasmid was then cotransfected into non-Ig-producing SP2/0 B myeloma cells with an expression vector carrying the parental 91A₃ L chain (25), pSV2-neo-91A₃L. Transfectants producing Ig-OVA were selected in the presence of geneticin and mycophenolic acid. All the cloning and sequencing procedures were similar to those used to generate Ig-PLP1 (20). Ig-W, the parental 91A₃ IgG2b, which does not carry any peptide, was also used in this study. Large scale cultures of transfectants were conducted in DMEM containing 10% iron-enriched calf serum (BioWhittaker, Walkersville, MD). The Ig chimeras were purified from culture supernatant on columns made of rat anti-mouse -chain coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ). To avoid cross-contamination, separate columns were used to purify the chimeras.

Antibodies

The anti-B7.1 (1G10, rat Ig-G2a), anti-B7.2 (2D10, rat Ig-G2b), 33D1 (rat IgG2b), and rat anti-mouse L chain mAbs were obtained from American Type Culture Collection (ATCC; Manassas, VA). Rabbit anti-mouse 2b Abs were purchased from Zymed Laboratories (San Francisco, CA). FITC-labeled anti-mouse IgG2a, FITC-labeled anti-mouse B220 (RA3-6B2), FITC-labeled anti-mouse CD11b (M1/70), PE-labeled anti-CD4 (RM4–5), PerCP-labeled anti-CD4 (RM4–5), biotin-labeled anti-CD40L (MR1), biotin-labeled anti-CD4 (GK1.5), and biotin-labeled hamster IgG were purchased from BD PharMingen (San Diego, CA).

Radioimmunoassay

Capture RIA was used to assess secretion of complete Ig-OVA constructs from SP2/0 transfectants. Microtiter 96-well plates were coated with 2 µg/ml polyclonal rabbit anti-mouse 2b Ab (Zymed Laboratories) overnight at 4°C and then blocked with 2% BSA in PBS for 1 h at room temperature. The plates were then washed with PBS and incubated with 100 µl/well supernatant from SP2/0 cells growing under selective pressure for 2 h at room temperature. After three washes with PBS, captured Ig chimeras were revealed by incubation with 1 x 10⁵ cpm/well ¹²⁵I-labeled rat anti-mouse mAb (ATCC) for 2 h at 37°C. The plates were then washed five times with PBS and counted using an LKB gamma counter (LKB Instruments, Gaithersburg, MD).

Purification of CD4⁺-KJ1-26⁺ T cells

Because the spleen of DO11.10/SCID mice is smaller than that of non-DO11.10/SCID mice, we usually used 60 newborns and 9 adult mice to purify a sufficient number of neonatal and adult T cells. CD4⁺KJ1-26⁺ T cells were purified by negative selection using the mouse CD4 Subset Mini Column Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The purity of T cells was 93% as assessed by double staining with KJ1-26 and anti-CD4 Abs using flow cytometry.

Transfer of neonatal CD4⁺-KJ1-26⁺ T cells into 1-day-old BALB/c mice

Neonatal DO11.10/SCID spleen cells were depleted of RBC by incubation in hypotonic lysis buffer. Subsequently, the percent of CD4⁺KJ1-26⁺ T cells was determined by flow cytometric analysis as described (26), and the splenic cells were resuspended in 50 µl saline so as to contain the equivalent of 2, 10, or 30 x 10³ CD4⁺KJ1-26⁺ T cells. Subsequently, the cell preparations were injected into 1-day-old BALB/c mice i.v. through the anterior facial vein using a 30-gauge needle as described (27).

Neonatal injection of tolerogen and adult immunization with Ag

One-day-old BALB/c recipients of neonatal DO11. 10/SCID T cells were given the tolerogen (Ig chimera) i.p. in 100 µl saline. When the mice reached 7 wk of age, they were immunized s.c. with 125 µg OVA peptide emulsified in 200 µl PBS/CFA (v/v). The reasoning for using 125 instead of the usual 300 µg OVA peptide for immunization is that lower Ag concentration would likely trigger high affinity T cells much more efficiently than low affinity T cells. Consequently, this dose favors recalling of neonatally induced memory cells over priming of naive BALB/c cells in mice tolerized with Ig-OVA. Ten days later, the mice were sacrificed, and their spleens and lymph nodes (axillary, lateral axillary, inguinal, and popliteal) were removed for analysis of proliferative and cytokine responses.

Lymph node and spleen T cell proliferation

Lymph node and spleen cells were incubated in 96-well flat-bottom plates at 4 and 10 x 10⁵ cells/100 µl/well, respectively, with 100 µl stimulator for 3 days. Subsequently, 1 µCi [³H]thymidine was added per well, and the culture was continued for an additional 14.5 h. The cells were then harvested on a Trilux 1450 Microbeta Wallac Harvester, and incorporated [³H]thymidine was counted using the Microbeta 270.004 software (EG&G Wallac, Gaithersburg, MD). The stimulators, OVA and HA_110–120 peptides, were used at 10 µM. A control medium with no stimulator was included for each mouse and used as background.

Measurement of cytokines by ELISA

Spleen cells were incubated in 96-well round-bottom plates at 10 x 10⁵ cells/100 µl/well with 100 µl stimulator for 24 h. Cytokine production was then measured by ELISA according to PharMingen’s instructions using 100 µl culture supernatant. Capture Abs were: rat anti-mouse IL-2, JES6-1A12; rat anti-mouse IL-4, 11B11; and rat anti-mouse IFN-, R4-6A2. Biotinylated anti-cytokine Abs were rat anti-mouse IL-2, JES6-5H4; rat anti-mouse IL-4, BVD6-24G2; and rat anti-mouse IFN-, XMG1.2. The OD₄₀₅ was measured on a Spectra Max 190 (Molecular Devices, Sunnyvale, CA) using SOFTmax PRO3.1.1. Graded amounts of recombinant mouse IL-2, IL-4, and IFN- were included in all experiments to construct standard curves. The concentration of cytokines in culture supernatants was estimated by extrapolation from the linear portion of the standard curve.

Measurement of cytokines by ELISPOT assay

ELISPOT was used to measure cytokines produced by lymph node T cells during Ag stimulation as described (4, 13). HA-multiscreen plates (Millipore, Bedford, MA) were coated with 100 µl/well 1 M NaHCO₃ buffer containing 2 µg/ml capture Ab. After an overnight incubation at 4°C, the plates were washed three times with sterile PBS, and free sites were saturated with DMEM containing 10% FCS for 2 h at 37°C. Subsequently, the blocking medium was removed, and 5 x 10⁵ lymph node cells/100 µl/well were added along with 100 µl Ag and incubated for 24 h at 37°C in a 7% CO₂ humidified chamber. The plates were then washed three times with PBS, followed by three washes with PBS-0.05% Tween. To each well, 100 µl biotinylated anti-IL-4, anti-IL-2 or anti-IFN- mAb were added, and the plates were incubated at 4°C overnight. After three washes with PBS-0.05% Tween, 100 µl avidin-peroxidase (2.5 µg/ml) were added. The plates were then incubated for 1 h at 37°C. Subsequently, spots were visualized by adding 200 µl substrate (3-amino-9-ethylcarbazole; Sigma, St. Louis, MO) in 50 mM acetate buffer, pH 5.0, and counted under a dissection microscope. The anti-cytokine Ab pairs used here were those described for the ELISA technique.

Flow cytometry analyses

Staining for CD4 and KJ1-26. Splenocytes were incubated with 10 µg/ml anti-FcR (2.4G2) for 20 min at 4°C. The cells were then labeled with anti-CD4-PE and purified KJ1-26 mAb (mouse IgG2a) for 30 min at 4°C and washed with FACS buffer. An additional incubation with goat anti-mouse IgG2a-FITC Abs was conducted to detect bound KJ1-26. Events (10–50 x 10³) were collected on a FACScan flow cytometer (BD Biosciences, Mountain View, CA) and analyzed using CellQuest software 3.3 (BD Biosciences).

Staining for CD40L. Staining for CD40L was conducted as previously described (28). Briefly purified CD4⁺KJ1-26⁺ T cells from neonatal DO11.10/SCID mice were cultured with neonatal or adult BALB/c APCs and stimulated with OVA peptide or soluble Ig-OVA. Biotinylated anti-CD40L Ab or hamster Ig-G (1 mg/ml) was immediately added due to rapid down-regulation of CD40L (29). After an 8-h incubation, the cells were harvested and labeled with anti-CD4-PerCP, KJ1-26-FITC, and avidin-PE for CD40L.

Staining for B7.1 and B7.2 costimulatory molecules. Splenocytes from neonatal or adult BALB/c mice were cultured with OVA peptide (10 µM) or Ig-OVA (1 µM) for 24 h, and the cells were then harvested and double-stained with either anti-B7.1-PE or anti-B7.2-PE and anti-B220-FITC, anti-CD11b-FITC, or 33D1-FITC.

	Results

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Presentation of OVA peptide on Igs

In prior studies, we demonstrated that neonatal exposure to Ig-PLP1 primes the immune system to develop an unusual organ-specific secondary response upon challenge with PLP1 peptide later in life. Indeed, the lymph node T cells were deviated to Th2, and the splenic T lymphocytes exhibited a defective growth and differentiation (7, 13, 22). One key question in this study is whether such organ-specific regulation is related to the diversity of the T cell repertoire or whether it occurs when the target neonatal T cells represent a homogeneous population expressing one type of TCR. To address this issue, we used TCR-transgenic T cells to develop a neonatal transfer system suitable for these investigations. The DO11.10 transgenic mouse carrying the OVA-specific TCR (23) and the KJ1-26 anti-clonotypic Ab specific for such a TCR (30) are well characterized and provide useful tools to establish such a neonatal T cell transfer system. However, to test for the organ-specific T cell regulation, an Ig chimera expressing the OVA peptide for which the DO11.10 T cells are specific was needed. Thus, the nucleotide sequence coding for OVA peptide was inserted within the 91A₃V_H gene (24), and sequencing analysis indicated that the D segment of complementarity determining region 3 region was deleted, and a nucleotide sequence coding for the full OVA_323–339 peptide was inserted instead (Fig. 1, top). Also, the sequences surrounding the OVA insert were identical with those flanking the D segment within the parental 91A₃V_H, confirming that the OVA sequence was inserted in the correct reading frame. The chimeric 91A3V_H-OVA gene was then ligated to a BALB/c 2b constant region to form a complete H chain gene and cotransfected along with the parental 91A₃ L chain (25) into the non-Ig-producing myeloma B cell line SP2/0. Supernatant from drug-selected transfectants incubated on anti-mouse 2b-coated microtiter plates allowed binding of ¹²⁵I-labeled anti-mouse L chain as did supernatant from transfectants expressing the parental IgG2b Ab, Ig-W (Fig. 1, bottom). These results indicate that the mutant H chain harboring the OVA peptide was able to pair with the parental L chain to form a complete Ig-OVA chimeric Ig. To assess for processing and presentation of the OVA peptide from Ig-OVA, a T cell activation assay was performed using splenic cells from DO11.10 mice that contain both the TCR-transgenic OVA-specific T cells and the APCs. As can be seen in Fig. 2, Ig-OVA-like free OVA peptide stimulated the DO11.10 T cells to proliferate, whereas the negative controls HA_110–120 and Ig-HA, an Ig carrying HA_110–120 peptide (31, 32), which are also restricted to H-2^d did not induce any proliferation. These results indicate that endocytic processing of Ig-OVA released the OVA peptide that bound to MHC molecules and the complexes (MHC class II-OVA peptide) trigger activation of the DO11.10 T cells as with other peptides expressed on Igs (20, 31, 32, 33, 34).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Expression of OVA peptide on the 91A3 Ig. The nucleotide sequence coding for OVA323–339 peptide was inserted into the 91A3 H chain variable region (91A3VH) in place of the D segment, and the resulting 91A3VH-OVA was analyzed by nucleotide sequencing and compared with the wild-type 91A3VH gene. Top, Results indicate that the nucleotide sequence encoding the full OVA peptide was inserted in the correct reading frame in place of the D segment. Bottom, Secretion of intact Ig-OVA chimera from drug-selected transfectants. Detection of complete Ig-OVA was conducted by incubation of supernatant of Ig-OVA or Ig-W transfectants on microtiter plates coated with rabbit anti-mouse -chain-specific Ab and revelation of captured Ig-chimeras with 125I-labeled rat anti-mouse L chain mAb. Values are the means ± SD of triplicates.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Presentation of Ig-OVA chimera to DO11.10 T cells. Splenocytes (4 x 105 cells/well) from DO11.10 mice were incubated with graded amounts of peptides or affinity chromatography-purified Ig chimeras; 72 h later, proliferation was measured by [3H]thymidine incorporation as described in Materials and Methods. HA110–120 and Ig-HA (31 32 ), Ags presented by H-2d, like OVA peptide and Ig-OVA, were used as negative control. Stimulation index represents the ratio of cpm obtained by incubation of the splenocytes without Ag to cpm obtained in the presence of Ag. Each point represents the mean of triplicates.

The secondary response of neonatally primed mice most likely derives from the primary response that arises upon exposure of neonatal T cells to the Ag. To ascertain that the neonatal DO11.10 T cells can develop a primary response rather than undergo cell death upon Ag presentation by neonatal APCs, we performed in vitro activation assays using Ig-OVA as Ag and neonatal splenocytes as APCs. Adult DO11.10 T cells were included to serve as reference. Also, adult splenocytes were used as APCs to discern any presentation discrepancy by the neonatal vs adult APCs. As can be seen in Fig. 3, the adult T cells upon stimulation with Ig-OVA presented on adult APCs proliferated (Fig. 3a), and produced IL-2 (Fig. 3b) and IFN- (Fig. 3c) but not IL-4 (Fig. 3d). The neonatal T cells, however, had reduced proliferation (Fig. 3a), produced lower amounts of the growth factor IL-2 (Fig. 3b), and were unable to secrete detectable levels of IFN- (Fig. 3c) or IL-4 (Fig. 3d). These results indicate that neonatal T cells develop a primary response upon exposure to Ag, but such a response displays no differentiation into the production of effector cytokines when compared with the primary response of adult DO11.10 T cells. Moreover, when neonatal APCs were used for presentation of Ig-OVA, both populations developed proliferative and IL-2 responses (Fig. 3, e and f), but neither neonatal nor adult T cells produced IFN- or IL-4 (Fig. 3g). These results indicate that the neonatal APCs are unable to support differentiation of T cells into IFN- or IL-4 production in vitro.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Neonatal T cells develop distinct primary response to Ig-OVA compared with adult T cells. CD4+KJ1-26+ T cells (5 x 104 cells/well) were purified from the spleen of 50 neonatal or 10 adult DO11.10/SCID mice and incubated with the optimal concentration (1 µM) of Ig-OVA or Ig-HA and irradiated (3000 rad) adult (a–d) or neonatal (e–h) BALB/c splenocytes as APCs (5 x 105 cells/well). After 48 h incubation, 1 µCi [3H]thymidine/well was added, and proliferation (a and e) was measured 14.5 h later. Duplicate cultures were prepared, incubated for 24 h, and used to measure IL-2 (b and f), IFN- (c and g), and IL-4 (d and h) by ELISA. Each bar represents the mean ± SD of triplicate wells. In vitro stimulation with the negative control Ig-HA was included for each mouse, and there was no significant proliferation or cytokine production.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Neonatal T cells develop distinct primary response to OVA peptide compared with adult T cells. CD4+KJ1-26+ T cells (5 x 104 cells/well) were purified from the spleen of 50 neonatal or 10 adult DO11.10/SCID mice and incubated with an optimal concentration (10 µM) of OVA or HA110–120 peptide and irradiated (3000 rad) adult (a–d) or neonatal (e–h) BALB/c splenocytes as APCs (5 x 105 cells/well). After 48 h incubation, 1 µCi [3H]thymidine/well was added and proliferation (a and e) was measured 14.5 h later. Duplicate cultures were prepared and incubated for 24 h, and the supernatant was used to measure IL-2 (b and f), IFN- (c and g), and IL-4 (d and h) by ELISA. Each bar represents the mean ± SD of triplicate wells. In vitro stimulation with the negative control HA110–120 peptide was included for each mouse, and there was no significant proliferation or cytokine production.

The neonatal DO11.10 T cells have shown the ability to proliferate and produce the growth factor IL-2 upon stimulation with Ag in vitro. Thus, the DO11.10 system would provide a suitable source of T cells to establish a neonatal T cell transfer system if appropriate homing could occur upon transfer into BALB/c neonates. To investigate this matter, varying numbers of 1-day-old DO11.10/SCID splenic cells containing defined numbers of DO11.10 T cells (CD4⁺-KJ1-26⁺) were transferred into BALB/c neonates via the facial vein, and the cells were assessed for homing to the recipients’ spleen. As can be seen in Fig. 5, 4 days after transfer, the number of CD4⁺KJ1-26⁺ T cells rose from undetectable in normal 4-day-old BALB/c mice (Fig. 5a) to 16, 22, and 25 per 1000 CD4⁺ splenic cells in the mice recipient of DO11.10/SCID splenocytes containing 2, 10, and 30 x 10³ DO11.10 cells, respectively (Fig. 5, b–d). When the homing was assessed 6 days after transfer, the frequency increased from 22 to 26 and 25 to 30 cells per 1000 CD4⁺ splenocytes in the murine recipients of 10 and 30 x 10³ DO11.10 cells, respectively (Fig. 5, e and f). These results indicate that neonatal DO11.10 T cells are able to home to and populate the spleen of neonatal BALB/c recipients to an extent similar to that of an adult transfer system (26).

View larger version (55K): [in this window] [in a new window]   FIGURE 5. Transfer of neonatal DO11.10 T cells into newborn BALB/c mice supports efficient homing to the spleen of the recipient animals. Splenic cells from neonatal (1-day-old) DO11.10/SCID mice containing a defined number of CD4+KJ1-26+ T cells were injected i.v. into the facial vein of 1-day-old BALB/c newborns. Subsequently, homing of the DO11.10/SCID T cells into the spleen of the recipient BALB/c mice was evaluated by double staining with anti-CD4 and KJ1-26 mAb. BALB/c newborns that were not given any cell transfer on the day of birth (a) and animals receiving DO11.10/SCID splenic cells containing 2 x 103 (b), 10 x 103 (c), or 30 x 103 (d) CD4+KJ1-26+ T cells were sacrificed on day 4 after transfer, and their splenic cells were assessed for the presence of CD4+KJ1-26+ double-positive DO11.10 T cells. Homing was also evaluated 6 days after transfer of 10 x 103 (e) or 30 x 103 (f) CD4+KJ1-26+ T cells. The percentages of double-positive cells among total CD4 cells are indicated in the upper right corner of each quadrant. The high background observed with KJ1-26 staining of CD4- cells reflects rather nonspecific labeling due to binding of KJ1-26 Ab to Fc receptor on cells other than CD4+ T cells. This statement emanates from the observation that a similarly high background was observed when an isotype control mouse IgG2a was used instead of KJ1-26. FL1-H and FL2-H, fluorescence intensity.

Requirement for a high neonatal T cell input for the induction of IFN--dependent splenic T cell anergy

The neonatal DO11.10 T cell transfer system was then used to ask whether or not organ-specific secondary responses develop when the responder cells represent a homogeneous population carrying a single TCR. To this aim, neonatal T cells from 1-day-old DO11.10/SCID mice were transferred into 1-day-old BALB/c neonates, and a few hours later the hosts were given a saline solution containing 100 µg of either Ig-OVA or the control Ig-W not encompassing any OVA peptide. After 7 wk, the now adult mice were immunized with 125 µg OVA peptide in CFA, and 10 days later their lymph node and splenic proliferative and cytokine responses were measured. The results were then compared with those of the Ig-W recipient mice and among the cell transfer groups. As can be seen in Fig. 6, the mice given Ig-OVA on the day of birth, like those recipients of Ig-W, developed equivalent proliferative responses in the lymph node upon immunization with OVA peptide at adult age. The number of transferred neonatal DO11.10 T cells had only a slight influence on these lymph node proliferative responses because murine recipients of 2 or 10 x 10³ cells exhibited similar proliferation and transfer of 30 x 10³ neonatal T cells slightly increased the proliferation. These responses are specific because in vitro stimulation with HA_110–120 peptide did not induce any significant proliferation (not shown). At the cytokine level, while the lymph node cells of Ig-W recipient mice produced IL-2 and IFN-, those from the Ig-OVA recipient group produced IL-4 instead (Fig. 6). Stimulation of these lymph node cells with HA_110–120 peptide did not induce any IL-2, IL-4, or IFN- (not shown). Moreover, although the Th1 response in Ig-W recipient mice was significant only when the neonatal transfer was conducted with 30 x 10³ DO11.10 T cells, the deviated responses in Ig-OVA recipient mice were significant with all the transfer numbers. Because control BALB/c mice that had not received neonatal T cell transfer but were tolerized with Ig-OVA on the day of birth developed lower but similar lymph node proliferative and IL-4 response (not shown), we believe that the IL-4 seen in the murine recipients of the T cell transfer is produced by both endogenous BALB/c and transferred DO11.10 T cells. In the spleen, although the murine recipients of Ig-W developed proliferative responses with all transfer regimens, those exposed to Ig-OVA did not (Fig. 7, top row). Moreover, the Ig-W group produced both type 1 (IL-2, and IFN-) and type 2 (IL-4) cytokines. In contrast, the Ig-OVA group although displaying proliferative unresponsiveness had significant IL-2 production when the transfer was conducted with 30 x 10³ neonatal DO11.10 cells, but IFN- was lacking and IL-4 was greatly reduced. Overall, the results indicate that a high neonatal T cell input is required to obtain nonproliferative splenic T cells that produce IL-2 but not IFN-. In the spleen, IL-2 production most likely emanates from DO11.10 T cells, because mice that did not receive any cell transfer, but were exposed to Ig-OVA, showed no measurable IL-2 or IFN- in their splenic responses. However, in the lymph node, both endogenous and DO11.10 T cells may be involved in IL-4 production as exposure to Ig-OVA in mice receiving no transfer induced IL-4 production. The finding that DO11.10 T cells are detectable in both lymphoid organs of these mice (Fig. 8) provides support for their contribution to the responses in both the spleen and lymph node. In addition, because their frequency in the Ig-OVA-treated mice (Fig. 8, c and d) is slightly increased relative to the control animals given Ig-W instead (Fig. 8, a and b), this may reflect a response of Ag experienced vs naive cells and further attest to their differential function (see Figs. 6 and 7).

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Newborn BALB/c mice receiving neonatal DO11.10 T cells and exposed to Ig-OVA develop deviated lymph node T cell responses on challenge with OVA peptide at the age of 7 wk. Groups of 1-day-old BALB/c mice (4–6/group) were given i.v. in the facial vein 2,000 (2K), 10,000 (10K), or 30,000 (30K) neonatal DO11.10/SCID T cells and injected with 100 µg Ig-W () or Ig-OVA () within 24 h. When the mice reached the age of 7 wk, they were challenged with 125 µg OVA peptide in CFA; 10 days later, the lymph node cells (5 x 105 cells/well) were stimulated with 10 µM OVA peptide. Proliferation (top row) was measured by [3H]thymidine incorporation after 3 days incubation. Production of IL-2 (second row), IFN- (third row), and IL-4 (bottom row) were measured after 24 h by ELISPOT as described in Materials and Methods. Ig-OVA was used at 100 µg/mouse on the day of birth because this amount was found to be optimal for induction of organ-specific regulation of neonatal immunity in the SJL/J/Ig-PLP1 system (13 14 ). Because both memory and naive T cells will be available in Ig-OVA-treated mice, the use of 125 instead of the usual 300 µg OVA peptide for immunization at adult age would be more effective in recalling Ag-experienced T cells rather than priming naive endogenous BALB/c cells. The in vitro stimulation used 10 µM peptide, which was defined optimal in this setting. ELISPOT was used to measure cytokines instead of ELISA because treatment with Igs induced highly proliferative T cells in the lymph node that reabsorb cytokine and can alter the significance of results (13 ). The indicated values represent the mean ± SD of four to six individually tested mice. In vitro stimulation with the negative control HA110–120 peptide was included for each mouse, and there was no significant proliferation or cytokine production.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Exposure of newborn BALB/c mice recipient of neonatal DO11.10 T cells to Ig-OVA allowed for IL-2 but suppressed IFN- secretion and proliferative responsiveness upon challenge with OVA peptide at the age of 7 wk. Groups of 1-day-old BALB/c mice (4–6/group) were given i.v. in the facial vein 2,000 (2K), 10,000 (10K), or 30,000 (30K) neonatal DO11.10/SCID T cells and injected with 100 µg Ig-W () or Ig-OVA () within 24 h. After 7 wk, the mice were challenged with 125 µg OVA peptide in CFA. Ten days later, the animals were sacrificed, and the splenic cells (1 x 106 cells/well) were stimulated with 10 µM OVA peptide. Proliferation (top row) was measured by [3H]thymidine incorporation after 3 days incubation. Production of IL-2 (second row), IFN- (third row), and IL-4 (bottom row) were measured after 24 h of stimulation by ELISA as described in Materials and Methods. The indicated values represent the mean ± SD of four to six individually tested mice. In vitro stimulation with the negative control HA110–120 peptide was included for each mouse, and there was no significant proliferation (mean cpm was 775 ± 200 for the six tested mice) or cytokine production (all cytokines tested were below detectable level). The mice used for these experiment are those described in Fig. 6.

View larger version (63K): [in this window] [in a new window]   FIGURE 8. Detection of DO11.10 T cells in both lymph node and spleen on challenge with OVA peptide of adult mice receiving T cell transfer and Ig-OVA at the neonatal stage. Groups of 1-day-old BALB/c mice (four to six per group) were given i.v. in the facial vein 30 x 103 neonatal DO11.10/SCID T cells and injected with 100 µg Ig-W (a and b) or Ig-OVA (c and d) within 24 h. After 7 wk, the mice were challenged with 125 µg OVA peptide in CFA; 10 days later, the lymph node and spleen were analyzed by staining for presence of CD4+KJ1-26+ double-positive DO11.10 T cells ex vivo. The percentages of double-positive cells among total CD4 cells are indicated in the upper right corner of each quadrant. Tol., tolerized; FL1-H and FL2-H, fluorescence intensity.

View larger version (13K): [in this window] [in a new window]   FIGURE 9. Restoration of proliferation and IFN- production by the anergic spleen T cells by stimulation with Ag in the presence with IL-12. A group of 1-day-old BALB/c mice were adoptively transferred with 30,000 neonatal DO11.10/SCID cells and injected with 100 µg Ig-OVA within 24 h. At the age of 7 wk, the mice were challenged with 125 µg OVA peptide in CFA; 10 days later, the animals were sacrificed and the splenic cells (1 x 106 cells/well) were stimulated with 10 µM OVA peptide in the presence of 1 or 10 U/ml IL-12. Proliferation (a) was measured by [3H]thymidine incorporation after 3 days of incubation. Production of IFN- (b) was measured by ELISA after 24 h incubation. The indicated values represent the mean ± SD of five individually tested mice. In vitro stimulation with OVA peptide without exogenous IL-12 and stimulation with the negative control HA110–120 peptide in the presence of IL-12 was included for control purposes. Results are those obtained with 10 U/ml IL-12.

We have shown above that both APCs and T cells contribute to the make up of the primary neonatal response. Also, we have found that neonatal exposure to Ig-OVA followed by an active immunization with OVA peptide at adult age gave rise to a response that includes IL-4 secreting lymph node T cells and IL-2-producing anergic splenic T cells whose proliferative and IFN- responsiveness could be restored with exogenous IL-12. In this section we attempted to define factors whose expression may be subject to developmental control and thereby play a critical role in the induction of neonatal immunity. Because B7 molecules on APCs provide important costimulatory functions for T cell activation and differentiation (35, 36, 37), we monitored the expression of both B7.1 and B7.2 on neonatal splenic APCs upon incubation with Ig-OVA or free OVA peptide and compared their expression pattern with adult APCs. As can be seen in Fig. 10, on stimulation with Ig-OVA, both adult and neonatal 33D1⁺ (mostly dendritic cells) and B220⁺ (mostly B cells) cells expressed B7.1 and B7.2 to the same extent. In contrast, neonatal CD11bCD18⁺ cells (mostly monocytes/macrophages) had a much lower expression of both B7.1 and B7.2 molecules than adult CD11bCD18⁺ cells. Similar results were obtained when the incubation was conducted with free OVA peptide (not shown). Evaluation of the frequency of the three types of APCs in the neonatal and adult spleen indicated that CD11bCD18-positive cells represent the majority (43%) of neonatal splenic cells (Table I). In the adult spleen, however, 50% of the cells expressed the B220 marker, 9% were CD11bCD18 positive, and 3% stained with 33D1 Ab. Based on these observations, it may be that monocytes/macrophages function as the major APCs in neonates and may contribute to the biased neonatal responses to Ag through the differential expression of B7 molecules relative to adult counterparts.

View larger version (47K): [in this window] [in a new window]   FIGURE 10. Differential expression of B7 molecules on adult and neonatal CD11bCD18-positive cells. Neonatal and adult splenocytes (1 x 106 cells) were incubated with 1 µM (150 µg/ml) Ig-OVA for 24 h. Subsequently, the cells were double stained with Abs to either B7.1 (top row) or B7.2 (bottom row) and Mac-1 (left column), 33D1 (middle column), or B220 (right column) as described in Materials and Methods. Results are histograms gated on Mac-1+, 33D1+, and B220+ cells, respectively. FL1-H and FL2-H, fluorescence intensity.

CD40L on activated T cells has been shown to ligate CD40 on APCs and trigger IL-12 production, which promotes T cell differentiation into the Th1 phenotype (38, 39). Also, prior studies indicated that the splenic anergic T cells when recalled with Ag at adult age display an inability to up-regulate CD40L and trigger IL-12 production by APCs (22). Consequently, these cells were unable to differentiate and produce IFN- (22). Because the neonatal TCR-transgenic T cell transfer system gives rise to splenic anergic T cells upon exposure to Ig-OVA and immunization with OVA peptide, it provides a practical system to investigate whether the lack of CD40L expression was an imprint by exposure of the neonatal T cells to Ig-OVA and therefore the cause of the splenic IL-12/IFN--dependent T cell anergy. To address this issue, we evaluated the expression of CD40L on neonatal DO11.10 T cells upon stimulation with Ag presented by neonatal or adult APCs. The results illustrated in Fig. 11 indicate that incubation of neonatal T cells with adult or neonatal APCs without Ag does not induce up-regulation of CD40L (Fig. 11, a and d). When OVA peptide was added to the culture, 69% of neonatal CD4⁺KJ1-26⁺ T cells, expressed CD40L when the APCs were of adult origin (Fig. 11b). However, the number of cells with significant levels of CD40L expression declined to 45% when the APCs were from neonatal source (Fig. 11e). In contrast, when Ig-OVA was used for stimulation, the expression of CD40L was at background levels whether the APCs were from neonatal or adult mice (Fig. 11, c and f). Overall, presentation of OVA peptide on neonatal APCs leads to a decline in the expression of CD40 ligand as was observed in other systems (40) while exposure to Ig-OVA imprints a defective up-regulation of CD40L expression on the T cells.

View larger version (47K): [in this window] [in a new window]   FIGURE 11. Defective up-regulation of CD40L on neonatal T cells. CD4+KJ1-26+ T cells (1 x 105 cells/well) were purified from the spleen of neonatal DO11.10/SCID mice on Miltenyi CD4-beads and incubated for 8 h with 1 µM (150 µg/ml) Ig-OVA or 10 µM OVA peptide, 1 µg/ml biotinylated anti-CD40L Ab or hamster IgG, and irradiated (3000 rad) adult (a–c) or neonatal (d–f) BALB/c splenocytes as APCs (1 x 106 cells/well). Subsequently, the cells were stained on ice with KJ1-26 and anti-CD4-PerCP for 30 min. To assess for bound KJ1-26, an additional incubation with anti-mouse IgG2a-FITC mAb was performed. Anti-CD40L Ab was added at the beginning of the culture to circumvent rapid down-regulation of CD40L (29 ). The dot plots show CD40L expression on KJ1-26-CD4-double-positive gated cells. FL1-H and FL2-H, fluorescence intensity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Given the fact that the organ-specific responses were readily inducible in SJL/J mice, where the frequency of PLP1-specific T cells is remarkably high (41), and reproducible with the homogeneous DO11.10/SCID system when a high number of T cells were transferred there must be a mechanism by which cell frequency promotes different type of responses in the lymph node vs the spleen. On a speculative basis, high frequency may sustain lymphocyte trafficking and supply of Ag helps broaden circulation within diverse organs and tissues (43). Consequently, various types of APCs subject to diverse environmental control may be involved in neonatal Ag presentation leading to specific regulation of the responses. In fact we have demonstrated that neonatal APCs play a critical role in the development of T cell responses in vitro (Fig. 3 and 4). In addition, the frequency of specific types of APCs in the spleen (Table I) and most likely within other organs is variable and expression of costimulatory molecules on these APCs is subject to differential regulation (Fig. 10). Thus, it is logical to envision a relationship between frequency, trafficking, and exposure to various types of APCs in the control of neonatal immunity. In fact, it has previously been shown that different types of APCs promote different outcomes in neonatal immunity (5). Finally, the organ-specific regulation of neonatal immunity occurs with peptide presented on Ig but not with free peptide (13, 14). The experiment presented in Fig. 11 indicated that free OVA peptide presented on neonatal APCs displayed a quantitative regulation of CD40L expression as has been observed in another antigenic system (40). However, when Ig-OVA was used for stimulation, CD40L expression on the neonatal T cells could not occur (Fig. 11). We have previously shown that the anergic splenic T cells of the secondary response in adult SJL mice also display an intrinsic defect for up-regulation of CD40L when stimulated with free PLP1 peptide (22). CD40L is required for cross-linking CD40 on APCs to trigger the production of IL-12 (38, 39) and IL-12 is a key cytokine for Th1 differentiation. Therefore, we postulate that neonatal exposure to Ig-OVA triggers activation of neonatal T cells without up-regulation of CD40L. Consequently, depending on the local environment differentiation may proceed with little or no IL-12 giving rise to cells fully differentiated to Th2 and others committed to Th1 but nondifferentiated. Alternatively, activation in the absence of CD40L may generate IL-2-producing nonpolarized T cells that are still susceptible to undergo differentiation to either Th1 or Th2 depending on the environment.

Overall, we believe that both the frequency of T cell precursors at the neonatal stage and the form of Ag contribute to the generation of organ-specific regulation of neonatal immunity.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI48541 (to H.Z.).

² Current address: Beirne B. Carter Center for Immunology Research, University of Virginia, 400 Lane Road, MR-4 Building, P.O. Box 801386, Charlottesville, VA 22908-1386.

³ Current address: Laboratory of Immunology, National Institute of Arthritis and Infectious Diseases, National Institutes of Health, Building 10, Room 11N314, Bethesda, MD 20892-1892.

⁴ Address correspondence and reprint requests to Dr. Habib Zaghouani, Department of Microbiology, University of Tennessee, M409 Walters Life Sciences Building, Knoxville, TN 37996. E-mail address: hzagh{at}utk.edu

⁵ Abbreviations used in this paper: PLP, proteolipid protein; EAE, experimental allergic encephalomyelitis; CD40L, CD40 ligand; HA, hemagglutinin.

Received for publication April 20, 2001. Accepted for publication July 3, 2001.

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