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Neonatally Primed Lymph Node, but Not Splenic T Cells, Display a Gly-Gly Motif within the TCR -Chain Complementarity-Determining Region 3 That Controls Affinity and May Affect Lymphoid Organ Retention¹

Jacque C. Caprio-Young^2,3,, J. Jeremiah Bell^2,*,, Hyun-Hee Lee^*,, Jason Ellis^*, Danielle Nast^*, Gary Sayler, Booki Min and Habib Zaghouani^4,*,

^* Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212; Department of Microbiology, University of Tennessee, Knoxville, TN 37996; and Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Ig-proteolipid protein 1 (Ig-PLP1) is an Ig chimera expressing the encephalitogenic PLP1 peptide corresponding to amino acid residues 139–151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals are comprised of Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN- or IL-4, secrete significant amounts of IL-2. Studies were performed to determine whether the two populations display any structural differences in the TCR H chain variable region that could contribute to the differential affinity and retention in different organs. Responsive Th2 lymph node T cells and anergic splenic lymphocytes were immortalized, and the structures of their TCR V were determined. The results show that V and J usage was random, but the CDR3 regions of the lymph node cells had a conserved Gly-Gly motif. Analysis of TCR affinity/avidity correlated the Gly-Gly motif with lower affinity and retention of the Th2 cells in the lymph node. Also, it is suggested that a higher TCR affinity may be a contributing factor for the development of the neonatal Th1 response in the spleen.

	Introduction

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During the last decade, it has become clear that neonatal exposure to Ag primes, rather than inactivates, T cells (1, 2, 3). In most cases, however, the response lacks Th1 immunity, resulting in an indisputable bias toward Th2 cells (4, 5, 6, 7, 8). This phenotype may account for the susceptibility of the neonate to microbial infections and allergic reactions. Lately, a tremendous effort has been made to define regimens that could establish a balance among Th1 and Th2 responses in the neonate to overcome the limitations associated with the poor efficacy of pediatric vaccines (9, 10). The use of CpG nucleotide as an adjuvant seems to facilitate development of Th1 responses in the neonate (11, 12, 13). Also, depending on the type of microbial agent, the dose of viral load can be adjusted to mobilize Th1 immunity (3, 14). However, safe and practical regimens able to induce protective Th1 immunity remain to be defined (9). Our approach using Igs for peptide delivery to increase Ag presentation and to circumvent the use of adjuvants has revealed new insights on the induction of Th1 immunity (15, 16, 17). For instance, when the proteolipid protein 1 (PLP1)⁵ (5) peptide corresponding to amino acid residues 139–151 of PLP (18) was delivered to newborns in the form of an Ig-PLP1 chimera, the animals produced, in response to a challenge with PLP1/CFA as adults, lymph node Th2 cells producing IL-4 and IL-2-secreting splenic Th1 cells that could not proliferate or produce IFN- (15). However, these anergic splenic T cells were able to recover and develop both proliferative and IFN- responses when assisted with exogenous IL-12 or IFN- (15). Additional analysis of the mechanism underlying the defective Th1 responses indicated that both APCs and factors intrinsic to the T cells contribute to the development of the splenic anergy (19). Indeed, the T cells could not up-regulate CD40L during restimulation with Ag, leading to nonengagement of CD40 on the APCs (19). Consequently, B7 molecules remained at the basal level during the APC-T cell interaction, and activation was defective (19). In fact, engagement of B7 molecules by anti-B7 Abs triggered IL-12 production, restored costimulation, and rescued T cell reactivation (20). Although these observations shed light on the contribution of costimulation to the anergy of splenic T cells, the mechanism underlying the organ-specific localization of the two subsets of Th cells remains unclear. One postulate that could be put forth for this conundrum is that differential affinity of the TCR to the peptide contributes to retention of the T cells in different organs. To test this hypothesis, splenic anergic T cells were rescued with exogenous IL-12 from five different mice and immortalized along with five lymph node T cells by fusion with TCR-negative BW1100 tumor cells. The resulting hybridomas were then used for cloning the TCR and structural characterization of the V CDR3 region. The findings indicated that although V, J, and D usage was random among lymph node and anergic splenic T cells, the CDR3 of the lymph node T cells contained a conserved Gly-Gly motif that was not present in any of the splenic T cells. Additionally, three-dimensional computer modeling indicated that the lymph node TCRs display fewer planar contact surfaces, suggesting lower affinity to the Ag. Furthermore, competitive inactivation of both subsets with PLP-LR antagonistic peptide (21, 22) revealed that lymph node T cells display lower affinity/avidity to PLP1 peptide than their anergic splenic counterparts. Thus, it is suggested that structural variability in the TCR CDR3 contributes to the variable affinity that dictates organ-specific localization of neonatal Th subsets.

	Materials and Methods

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Mice

SJL/J mice (H-2^s) were purchased from Harlan Sprague Dawley and maintained in our animal facility for the duration of the experiments. For the generation of newborn mice, breeding sets of one adult male and three females were caged together. When pregnancy was visible, the females were separated and caged individually. Offspring were weaned when they reached 3 wk of age. All experimental procedures were conducted according to the guidelines of the institutional animal care committee.

Peptides

All peptides used in this study were purchased from Research Genetics and purified by HPLC to >90% purity. PLP1 peptide (HSLGKWLGHPDKF) encompasses an encephalitogenic sequence corresponding to aa 139–151 of PLP (18). PLP-LR (HSLGKLLGRPDKF) is derived from PLP1 peptide by mutating the TCR contact residues W144 and H147 to L144 and R147, respectively (21). PLP-LR binds to I-A^s MHC class II molecules equally as well as PLP1 peptide, but functions as an antagonist for PLP1-specific T cells (21, 22). PLP2 peptide (NTWTTCQSIAFPSK) encompasses an encephalitogenic sequence corresponding to aa 178–191 of PLP (23). PLP2, like PLP1, is presented by I-A^s MHC class II molecules and induces experimental allergic encephalomyelitis in SJL/J mice (23).

Ig chimeras

Ig-PLP1 and Ig-W chimeras have been described previously (22). Briefly, construction of the chimeras used genes coding for the L and H chains of the anti-arsonate Ab, 91A3. The H chain CDR3 region was deleted and replaced with nucleotide sequences coding for PLP1 to generate the Ig-PLP1 chimera (22). Ig-W is the parental Ig (IgG2b ) that does not encompass any peptide (22). Both chimeras were expressed in the non-Ig-secreting myeloma B cell line SP2/0. Transfected cells were grown in large-scale cultures of DMEM containing 10% iron-enriched calf serum (Serologicals). The chimeras were purified from culture supernatant on affinity chromatography columns made of rat anti-mouse -chain coupled to cyanogen bromide-activated Sepharose 4B (Amersham Biosciences). To avoid cross-contamination, separate columns were used to purify each chimera.

Neonatal injections of Ig-PLP1 and adult immunizations with PLP1 peptide

Neonatal injections of 100 µg of either Ig-PLP1 or Ig-W were performed i.p. in 100 µl of saline within 24 h after birth. When the mice reached 7 wk of age, they were immunized s.c. in the foot pads and base of the limbs with 100 µg of PLP1 peptide emulsified in 200 µl of PBS/CFA (v/v). Ten days later, the mice were killed, and their spleens and lymph nodes (axillary, lateral axillary, inguinal, and popliteal) were removed for the generation of T cell lines.

Generation of T cell hybridomas

Separate single-cell suspensions were made from the lymph nodes and spleens of mice given Ig-PLP1 on the day of birth and PLP1 peptide in CFA at 7 wk of age. The culture medium contained DMEM supplemented with 10% FCS (HyClone), 0.05 mM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µg/ml gentamicin sulfate (Fisher Scientific). The T cell lines were stimulated in vitro with 15 µg/ml PLP1 peptide and irradiated, syngenic, splenic APCs. In addition, the splenic T cell lines were given 10 U/ml rIL-12 and 10 µg/ml anti-IL-4 upon stimulation. The cells were subsequently rested in culture medium supplemented with 3% T-stim (Fisher Scientific). The lymph node and splenic T cell lines were fused separately with the TCR-negative thymoma BW1100 (American Type Culture Collection) using polyethylene glycol 4000 (Sigma-Aldrich). Hybrids were selected by supplementing the culture medium with hypoxanthine-azaserine (Sigma-Aldrich). Hybridomas were then cloned by limiting dilution.

Screening of hybridoma clones by ELISA

Lymph node hybridoma clones were screened for reactivity to PLP1 peptide by testing for the production of IL-4, whereas the splenic hybridoma clones were screened for the production of IFN-. Hybridoma clones (5 x 10⁴/100 µl/well) were incubated in 96-well plates with irradiated, syngenic APCs (5 x 10⁵/50 µl/well) and either the stimulator peptide, PLP1, or the control peptide, PLP2 (15 µg/ml), for 24 h. Cytokine production was measured by ELISA using 100 µl of culture supernatant. All Abs were purchased from BD Pharmingen, and all ELISAs were performed according to their instructions. Capture Abs were rat anti-mouse IL-4 11B11, and rat anti-mouse IFN- R4-6A2. Biotinylated anti-cytokine Abs were rat anti-mouse IL-4, BVD6-24G2, and rat anti-mouse IFN-, XMG1.2. The OD₄₀₅ was measured on a SpectraMax 340 counter using SoftMAX PRO 1.2.0 software (Molecular Devices). Graded amounts of mouse rIL-4 and rIFN- were included in all experiments to construct standard curves. The concentrations of cytokines in the culture supernatants were estimated by extrapolation from the linear portion of the standard curves.

Flow cytometric analyses

Hybridoma clones were stained with the mouse V TCR screening panel (BD Pharmingen), which includes FITC-labeled Abs specific for V2, -3, -4, -5.1/5.2, -6, -7, -8.1/8.2, -8.3, -9, -10, -11, -12, -13, -14, and -17. Cells (1 x 10⁶) were incubated with one V mAb along with PE-labeled anti-CD4 (BD Pharmingen) for 30 min at 4°C. Positive controls consisted of PE-labeled anti-CD3 (clone 145-2C11) along with FITC-labeled anti-TCR -chain (clone H57-597; BD Pharmingen). Cells were then washed, fixed, and analyzed on a FACScan flow cytometer using CellQuest software 3.3 (BD Biosciences).

Measurement of cytokines by ELISPOT assay

ELISPOT assay was used to measure cytokines produced by lymph node and splenic T cells during Ag stimulation as previously described (15). Hemagglutinin multiscreen plates (Millipore) were coated with 100 µl/well 1 M NaHCO₃ buffer containing 2 µg/ml capture Ab. After 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 cells/50 µl/well were added along with 50 µl of PLP1 peptide (1 µg/ml) and 50 µl of varying concentrations of PLP-LR peptide. After 24-h incubation at 37°C in a 7% CO₂ humidified chamber, the plates were washed, and 100 µl of biotinylated anti-cytokine Ab (1 µg/ml) was added. The anti-cytokine Ab pairs used in this study were those described for the ELISA technique. After overnight incubation at 4°C, the plates were washed, and 100 µl of avidin-peroxidase (2.5 µg/ml) was added. The plates were then incubated for 1 h at 37°C. Subsequently, spots were visualized by adding 100 µl of substrate (3-amino-9-ethylcarbazole; Sigma-Aldrich) in 50 mM acetate buffer (pH 5.0) and were counted on an Immunospot series 3B analyzer using Immunospot version 3.2 software (CTL Analyzers).

Molecular biology

mRNA was extracted from the hybridoma clones using the µMACS mRNA isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. cDNA was synthesized using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies) according to the manufacturer’s protocol and using a primer for the TCR C-chain (5'-GCCAAGCACACGAGGGTAGCC-3') (20). PCR amplification was conducted using the C primer along with one of 19 V primers. The primers used for cDNA synthesis and PCR are illustrated below. PCR conditions were denaturation at 95°C for 2 min, annealing at 55–65°C for 1 min, and extension at 72°C for 1 min for 25 cycles, followed by a final extension step at 72°C for 7 min. The primer sequences are as follows: primer sequence (Tm): V1, 5'-ATC TAA TCC TGG GAA GAG CAA AT-3' (47); V2, 5'-GGC GTC TGG TAC CAC GTG GTC AA-3' (56); V3, 5'-GTG AAA GGG CAA GGA CAA AAA GC-3' (50); V4, 5'-GAT ATG CGA ACA GTA TCT AGG C-3' (48); V5.1, 5'-ACA TAA CA AAG GAA AGG GAG AA-3' (45); V6, 5'-TCC TGA TTG GTC AGG AAG GGC AA-3' (52); V7, 5'-TAC CTG ATC AAA AGA ATG GGA GA-3' (47); V8, 5'-GTA CTG GTA TCG GCA GGA CAC-3' (51); V9, 5'-AGC TTG CAA GAG TTG GAA AAC CA-3' (48); V10, 5'-GAT TAT GTT TAG CTA CAA TAA TA-3' (39); V11, 5'-ACA AGG TGA CAG GGA AGG GAC AA-3' (52); V12, 5'-ACC TAC AGA ACC CAA GGA CTC AG-3' (52); V13, 5'-CAG TTG CCC TCG GAT CGA TTT TC-3' (52); V14, 5'-GCC GAG ATC AAG GCT GTG GGC AG-3' (57); V15, 5'-AGA ACC ATC TGT AAG AGT GGA AC-3' (48); V16, 5'-CAT CAA ATA ATA CAT ATG GGG CA-3' (45); V17, 5'-GTA GTC CTG AAA AGG GCA CAC T-3' (50); V18, 5'-CAT CTG TCA AAG TGG CAC TTC A-3' (48); V19, 5'-AGA CAT CTG GTC AAA GGA AAA G-3' (46); and C, 5'-GCC AAG CAC ACG AGG GTA GCC-3' (55).

Nucleotide sequencing and analysis

Purified PCR products were sequenced on an ABI 3100 DNA sequencer (Applied Biosystems) by the UTK Molecular Biology Resource Facility using the C primer and the ABI Big Dye DNA Sequencing kit (Applied Biosystems). Nucleotide sequences were entered into the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) search database (www.ncbi.nlm.nih.gov) as well as the Immunogenetics V-Query and Standardization databases to align the sequences with known TCR sequences; to identify the V, D, and J regions; and to analyze the CDR3 regions (http://imgt.cines.fr:8104).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Immortalization of normal Th2 and anergic Th1 neonatal lymphocytes

Recently, we have shown that priming with Ig-PLP1 on the day of birth, followed by a challenge with PLP1 peptide at 7 wk of age, leads to secondary responses characterized by Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen (15, 19). The studies presented in this paper attempted to immortalize both type of cells to determine the structures of their TCR and assess whether the subsets display differential affinity to Ag. As indicated in Fig. 1, newborn SJL/J mice were given 100 µg of Ig-PLP1 in saline on the day of birth and were challenged with 100 µg of PLP1 peptide in CFA at the age of 7 wk. Ten days later, lymph node and splenic T cells were harvested and stimulated with PLP1. The anergic splenic T cells were also given exogenous IL-12 in vitro. A few cycles of stimulation followed by resting were performed to generate lines with sufficient numbers of cells for fusion with the BW1100 TCR-negative thymoma cells (American Type Culture Collection). Ten hybridomas, each from a different mouse, were generated, including five splenic and five lymph node T cell hybridomas. The next step was to determine whether these hybridomas display a cytokine production profile similar to the polyclonal responses. Accordingly, the T cells were incubated with irradiated SJL/J splenic APCs loaded with peptide, and cytokine production was measured. Fig. 2 shows that lymph node-derived T cell hybridomas produce IL-4 (Fig. 2a), but not IFN- (Fig. 2b), upon stimulation with PLP1 peptide. In contrast, the splenic T cells did not produce IL-4 (Fig. 2c), but secreted IFN- upon stimulation with PLP1 peptide (Fig. 2d). Cytokine production by the lymph node or splenic T cell hybridomas is Ag specific, because stimulation with the negative control PLP2, which is restricted to I-A^s MHC molecules like PLP1 peptide, did not induce any cytokine production. Overall, the T cell hybridomas derived from the lymph nodes of mice neonatally tolerized with Ig-PLP1 and immunized with PLP1 at 7 wk of age produced IL-4, whereas those generated from the spleen secreted IFN-. These results are in good standing with previous reports showing a similar organ-specific bias in polyclonal secondary neonatal responses (15, 16, 17, 19).

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Generation of lymph node (LN) and splenic (SP) T cell hybridomas from neonatally tolerized mice. One-day-old SJL/J mice were given i.p. 100 µg of Ig-PLP1 in 100 µl of saline solution and 7 wk later were challenged with 100 µg of free PLP1 peptide in CFA as described in Materials and Methods. Ten days later, the lymph nodes and spleens were harvested, and single-cell suspensions were prepared and stimulated with free PLP1 peptide. Because of their anergic state, the splenic T cells were stimulated in the presence of 10 U/ml rIL-12 to restore proliferation. The cells were then rested for 7–10 days and restimulated again with PLP1 peptide presented on fresh splenic APCs. After three or four stimulation/resting cycles, the now T cell lines were fused with the TCR-negative thymoma BW1100. Wells displaying cell growth were tested for the production of either IL-4 or IFN- upon stimulation with PLP1 peptide and splenic APCs. Positives were cloned by limiting dilution and were used for cytokine and TCR structure analysis. Attempts were made with >15 mice for the generation of T cell clones, but due to the anergic state of the splenic cells, five splenic hybridomas were generated, each from a different mouse. Also, the five lymph node T cells are each from a different mouse.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Lymph node and splenic hybridomas from neonatally tolerized mice produce distinct lineage-specific cytokines. Hybridoma T cells (5.0 x 104 cells/well) generated from lymph node (a and b) and the spleen (c and d) of Ig-PLP1-tolerized and PLP1 peptide-immunized SJL/J mice were incubated with irradiated (3000 rad) splenic APCs (5.0 x 105 cells/well) and 15 µg/ml of either PLP1 () or PLP2 peptide (). After 24 h, IL-4 (a and c) and IFN- (b and d) were measured by ELISA using 100 µl of culture supernatant. The concentration of cytokines was estimated by extrapolation from a standard curve constructed with recombinant cytokines. Each bar represents the mean ± SD of triplicate wells. This experiment is representative of a least three separate experiments.

Secondary lymph node and splenic T cells from neonatally tolerized mice use diverse TCR -chain variable region segments

The T cell hybridomas were initially assessed for TCR V using a panel of commercially available, specific anti-TCR V Abs. However, when the cells stained negatively with all the available Abs, we resorted to RT-PCR for V typing, using specific primers for all defined mouse TCR V genes. As shown in Fig. 3, all lymph node T cells stained positively for CD3 and TCR -chain, indicating the expression of a TCR on the surface of the cells. Moreover, three of the five lymph node hybridomas (3E6G8, 31G5.B12, and 20F7.A3) stained with anti-V4 Ab, one (6F6.A1) stained with anti-V14, and the fifth (22B6.G7) did not stain with any Ab. Upon testing by RT-PCR using specific primers for the 19 TCR V variable region genes, the 22B6.G7 generated a band of appropriate m.w., only with V16 primers (Fig. 3, lower panel, left gel picture). Given that an anti-V16 Ab was not available, the cells did not stain with any other Ab in the panel, and no band was observed with other primers, it is likely that 22B6.G7 uses a V16 TCR variable region gene. Overall, the V usage among lymph node T cell hybridomas includes V4, -14, and -16.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. TCR V usage among lymph node (LN) T cell hybridomas derived from neonatally tolerized mice. Lymph node hybridomas (1 x 106 cells/tube) were double stained with PE-labeled anti-CD4 mAb and each one of the FITC-labeled panel of mAbs to V2, -3, -4, -5.1/5.2, -6, -7, -8.1/8.2, -8.3, -9, -10, -11, -12, -13, -14, and -17. In parallel, each clone (1 x 106 cells/tube) was double stained with PE-labeled anti-CD3 and FITC-labeled anti-TCR-chain to ensure TCR expression. Also, mRNA was extracted from each clone and used for TCRV typing by RT-PCR using a panel of primers representing 19 different TCRV genes (see Materials and Methods). The left panel shows dot plots for CD4 and TCRV expression; the right panel shows CD3 and TCR-chain expression on each clone. Note that hybridoma LN22B6.G7 did not stain positive with any of the available anti-TCRV mAbs, and the indicated dot plot represents a typical pattern seen with all 15 anti-V mAb tested. The gel illustration to the right of the corresponding dot plot shows DNA amplification by primers specific for TCR V16, indicating that this hybridoma uses the V16 gene that could not be detected by cell surface staining because no Ab is available for TCR V16.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. TCR V usage among splenic (SP) T cell hybridomas derived from neonatally tolerized mice. Splenic hybridomas (1 x 106 cells/tube) were double stained with PE-labeled anti-CD4 mAb and each one of the FITC-labeled panel of mAbs to V2, -3, -4, -5.1/5.2, -6, -7, -8.1/8.2, -8.3, -9, -10, -11, -12, -13, -14, and -17. In parallel, each clone (1 x 106 cells/tube) was double stained with PE-labeled anti-CD3 and FITC-labeled anti-TCR -chain to ensure TCR expression. mRNA was also extracted from each clone and used for TCR V typing by RT-PCR using a panel of primers representing 19 different TCR V genes (see Materials and Methods). The left panel shows dot plots for CD4 and TCRV expression, and the right panel shows CD3 and TCR-chain expression on each clone. Note that hybridomas SP4D1#3, SP3A1#11, and SP10E5#6 did not stain positively with any of the available anti-TCRV mAbs, and the indicated plots represent a prototype pattern from staining with all 15 anti-V mAb tested. The gel illustrations to the right of the bottom three plots show DNA amplification by primers specific for TCR V16 and TCR V1, indicating that the hybridomas SP4D1#3, SP3A1#11, and SP10E5#6 use V16, V1, and V16 genes, respectively. Abs to these Vs are not available and therefore could not be detected by cell surface staining.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Lymph node (LN), but not splenic (SP), T cell hybridomas from neonatally tolerized mice display biased D TCR usage. The rearranged V genes of lymph node and splenic T cell hybridomas were cloned, and their nucleotide sequences were determined. The sequences were then entered into the NCBI BLAST Search Database (www.ncbi.nlm.nih.gov) and matched with defined V genes. Subsequently, the nucleotide sequences were entered into the Immunogenetics V-Query and standardization (IMGT/VQuest) search engine (http://imgt.cines.fr.8104) for alignment analysis, and the V, D, and J sequences were determined. Shown are the D regions of the lymph node and splenic hybridomas compared with the germline D1 (top panel) and D2 (bottom panel) nucleotide sequences. The alignment score indicates a degree of homology between the sample D segment and the germline D segment. A score of 20 or higher is considered significant.

View this table: [in this window] [in a new window]   Table I. TCR-chain variable region gene segments used by lymph node and splenic T cell hybridomas

The CDR3 has been defined as the most influential component in the formation of the Ag binding site and, thereby, in specificity as well as affinity of the TCR to Ag (24). Thus, for comparison of the Ag binding region between the splenic and lymph node clones, the nucleotide sequences were translated to the corresponding amino acid sequences, and the CDR3 regions were examined. The -chain CDR3 region was identified according to the IMGT/V Quest database (http://imgt.cines.fr.8104). As shown in Fig. 6, all five lymph node clones showed a CDR3 length of 11–13 aa. Similarly, the splenic clones had a 10- to 13-aa-long CDR3, with the exception of SP3A1#11, which was 18 aa long. Remarkably, however, all five lymph node clones had a glycine-glycine (G-G) motif that was not present in any of the splenic clones.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Restricted expression of a glycine-glycine motif within the CDR3 region of lymph node T cell hybridomas of neonatally tolerized mice. The nucleotide as well as the amino acid sequences of the TCR-chain CDR3 region are shown. The upper panel shows the sequences from lymph node (LN)-derived T cell hybridomas, and the lower panel illustrates the sequences of spleen (SP)-derived T cell hybridomas. The highlighted box within the CDR3 of lymph node T cells shows the glycine-glycine motif.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. TCR CDR3 of lymph node T cells displays a flexible three-dimensional structure. Modeling of a splenic (a) and a lymph node (b) TCR V-chain was generated using the SWISS-MODEL (www.expasy.ch/swissmod/SWISS-MODEL.html). The blue region indicates the CDR1 loop, the purple region indicates the CDR2 loop, and the yellow region indicates the CDR3 loop. The illustrated splenic model was derived from TCRV of SP10E5#6 clone, whereas the lymph node model is from the TCRV LN3E6.G8 T cell clone. These were chosen as representative clones, because other spleen and lymph node clones were modeled and showed no significant differences.

To determine whether structural differences among the CDR3 of neonatally induced Th1 and Th2 cells contribute to the overall affinity of the T cells to PLP1 ligand, we used PLP-LR antagonist peptide and performed competitive inhibition of T cell activation to measure the strength of interaction between the TCR and its ligand. PLP-LR is an antagonist that was derived by mutating the TCR contact residues 144W and 147H of PLP1 into 144L and 147R (21). PLP-LR binds to I-A^s MHC class II molecules to the same extent as the parental agonist peptide, PLP1 (21), but acts as an antagonist for PLP1-specific T cells (21, 22). This function provides a readout system to estimate the relative affinity/avidity of neonatal Th1 and Th2 cells to PLP1 peptide. Accordingly, the amount of PLP-LR/I-A^s complexes required to out-compete 50% of PLP1/I-A^s-mediated T cell activation would be indicative of the strength of the TCR-ligand interactions for both Th1 and Th2 populations. To this end, SJL mice were given Ig-PLP1 on the day of birth and were challenged with PLP1/CFA at the age of 7 wk. Ten days later, the splenic and lymph node cells (containing both T cells and APCs) were stimulated with 1 µg/ml PLP1 peptide in the presence of graded concentrations of PLP-LR peptide. Reductions in IL-4 and IFN- production by lymph node and splenic T cells, respectively, were measured by ELISPOT assay. Measurement of cytokines used ELISPOT instead of ELISA to overcome quantitative variability among individual cells. Also, the choice of 1 µg of PLP1 for stimulation was based on preliminary analysis and represents a suboptimal concentration that does not saturate the APCs or the T cells in either organ. The results presented in Fig. 8 show that 50% T cell inactivation was achieved with 2.4 µg/ml PLP-LR for IL-4-producing lymph node Th2 cells, but with 24 µg/ml for IFN--producing splenic Th1 cells. The control PLP2 peptide, which is also presented by I-A^s MHC class II molecules, did not produce significant inhibition of T cell activation, indicating that PLP-LR is not displacing PLP1 from class II MHC molecules, but, rather, competing for engagement of PLP1-specific TCR. Overall, a 10-fold higher amount of PLP-LR was required to inhibit activation of splenic Th1 cells compared with lymph node Th2 cells. This probably implies that splenic Th1 cells have a higher affinity for PLP1 than lymph node Th2 cells.

View larger version (15K): [in this window] [in a new window]   FIGURE 8. A higher amount of antagonist is required by Th1 cells for inhibition of activation. Lymph node (LN) and splenic (SP) cells (5 x 105/well) from Ig-PLP1-tolerized and PLP1-immunized mice were stimulated with 1 µg/ml PLP1 agonist in the presence of graded amounts of PLP-LR antagonist, and cytokine production was measured by ELISPOT after 24-h incubation as described in Materials and Methods. Because the splenic cells are anergic, IL-12 was added at 2 ng/ml to restore IFN- responses. Lymph node and splenic T cell antagonism was assessed by measuring IL-4 (•) and IFN- spots (), respectively. IL-4 spots obtained with lymph node T cells using PLP2 instead of PLP-LR were included for control purposes. Each point represents the mean of triplicate wells. The amount of cytokine produced in the absence of PLP-LR (100%) was 43 ± 6 spots for IL-4 and 103 ± 4 spots for IFN-. The percent residual cytokine production was estimated using the following formula: number of spots obtained with the test sample divided by the number of spots obtained in the absence of PLP-LR antagonist. Each point represents the mean ± SE of triplicate determinations. Dotted projections show the amounts of PLP-LR antagonist required for 50% residual cytokine production, which was 2.4 µg/ml for IL-4 and 24 µg/ml for IFN-.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants RO1AI48541 and R21AI062796 (to H.Z.) from the National Institutes of Health. J.J.B. was supported by Predoctoral Training Grant T32GM08396-13 from National Institute of General Medical Sciences. D.N. was supported by a life science fellowship from the University of Missouri, Columbia.

² J.C.C.-Y. and J.J.B. contributed equally to this work.

³ Current address: Atmospheric Glow Technologies, 924 Corridor Park Boulevard, Knoxville, TN 37932.

⁴ Address correspondence and reprint request to Dr, Habib Zaghouani, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, M616 Medical Sciences Building, Columbia, MO 65212. E-mail address: zaghouanih{at}health.missouri.edu

⁵ Abbreviations used in this paper: PLP, proteolipid protein; BLAST, Basic Local Alignment Search Tool.

Received for publication July 26, 2005. Accepted for publication October 26, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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