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Antigen-Specific T Cell Repertoire Modification of CD4⁺CD25⁺ Regulatory T Cells¹

Yuki Hayashi^*,, Shin-ichi Tsukumo^*, Hiroshi Shiota, Kenji Kishihara^* and Koji Yasutomo^2,*

Departments of ^* Immunology and Parasitology, and Ophthalmology and Visual Neuroscience, School of Medicine, University of Tokushima, Tokushima, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4⁺ T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4⁺CD25⁺ regulatory T cells (T_reg) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T_reg. In this study, we demonstrate that after a primary Ag challenge, T_reg proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T_reg after primary Ag challenge augmented the total suppressive function of T_reg against TCR repertoire modification but not against the proliferation of memory CD4⁺ T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T_reg, which would be crucial for limiting excess primary and memory CD4⁺ T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T_reg is an ideal strategy for the clinical use of T_reg.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The immune system has evolved several mechanisms to control self-reactive T cells that escape thymic negative selection (1, 2, 3). However, T cell nonresponsiveness to self-Ags does not exclusively result from clonal deletion, T cell anergy, or T cell ignorance (4, 5). Recent accumulating evidence has suggested that CD4⁺CD25⁺ regulatory T cells (T_reg),³ which constitute 5–10% of the peripheral CD4⁺ T cells in normal naive mice, play an important role in controlling self-reactive T cells and maintaining immunologic self-tolerance (3, 6, 7). T_reg in normal naive mice are nonresponsive to Ag-specific stimulation in vitro, but upon stimulation through the TCR, they potently suppress the activation of other CD4⁺ T cells in an Ag-nonspecific manner (3, 6, 7). Indeed, T_reg can suppress autoreactive T cells that cause autoimmune gastritis in neonatal day 3 thymectomized mice (8) and also can suppress allo-MHC-responsive T cells in an allogeneic skin graft model (9). Also, depletion of total T_reg has been found to augment tumor-specific (10) or a parasite-specific T cell response (11). Therefore, the application of T_reg to clinical medicine (the reduction or increase in function of T_reg) such as treatment of tumors, autoimmune diseases, or allogeneic transplantation has been anticipated.

As for the lineage of T_reg, several recent papers have revealed that Foxp3 expression is crucial for their development (12, 13, 14). According to these results, Foxp3 is expressed in T_reg and to a lesser degree in CD25^– T cells (14). Furthermore, the ectopic expression of Foxp3 in CD4⁺CD25^– T cells allowed such cells to become regulatory T cells that have the ability to suppress the T cell response (12, 14), suggesting that Foxp3 is at least one transcription factor critical for the development of T_reg.

Conventional CD4⁺CD25^– T cells (T_conv) proliferate in response to antigenic stimulation with the selective survival of cells with a high affinity TCR against the Ag (15, 16, 17, 18, 19, 20, 21, 22, 23). This T_conv TCR repertoire contraction plays an important role in augmenting primary and secondary immune responses against Ags, thus establishing Ag-specific T cell memory (17, 19). Although the total T cell immune response is thought to be regulated by the interplay between T_conv-derived effector/memory T cells and T_reg, it remains unclear if T_reg recognize the same Ag as effector T cells seen in vivo.

Thus, in this study we evaluated changes in TCR diversity in both T_conv and T_reg during the emergence of the primary and memory responses to pigeon cytochrome c (PCC) in nontransgenic animals to address if T_reg really recognize the same Ags as effector T cells do and contract their own repertoire. We found that T_reg do recognize the same Ag as T_conv, and expand while contracting their repertoire. Furthermore, Ag-specific T_reg suppress TCR repertoire modification but not cell proliferation of memory CD4⁺ T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and immunization

The B10.A and B10.BR mice used were from The Jackson Laboratory (Bar Harbor, ME) and were crossed with each other to make F₁ mice. A primary immunization of 400 µg of PCC (Sigma-Aldrich, St. Louis, MO) in an emulsion with CFA was s.c. injected into the base of the mouse tail two days after cell transfer. PBS alone was used for the adjuvant only controls. Secondary challenge was a repeat of the primary regimen including adjuvant, also at the base of the tail, 8 wk after the initial priming.

Cell transfer and purification

The protocol of cell transfer is shown (see Fig. 1A). The bone marrow (5 x 10⁶) from 6-wk-old B10.A mice was transferred into 950 rad irradiated (B10.A x B10.BR)F₁ mice. Two months after bone marrow transplantation, lymph node cells from chimeric mice were stained with anti-B220, D^k, and CD8 mAbs and positive cells were removed by anti-rat mouse IgG-coated beads (Dynal Biotech, Oslo, Norway). The resultant cells were then stained with anti-CD25 mAb and CD4⁺CD25^– or CD4⁺CD25⁺ T cells were separated by negative or positive selection of CD25⁺ cells by anti-rat IgG-coated beads (Dynal Biotech) or anti-rat IgG-coated beads followed by LS+ magnetic column (Miltenyi Biotec, Cologne, Germany), respectively. The purity of CD4⁺CD25^– and CD4⁺CD25⁺ T cells in this preparation is >98% and D^k-positive cells are <1% and the CD4⁺CD25^– T cells contaminating the CD4⁺CD25⁺ T cell population is <1% (see Fig. 1B). Then, CD4⁺CD25^– or CD4⁺CD25⁺ purified T cells (1 x 10⁶) were transferred into nonirradiated (B10.A x B10.BR)F₁ mice. The mice were immunized with PCC (400 µg) emulsified with CFA 2 days after cell transfer and activated donor-derived cells were purified from lymph nodes several days or weeks after immunization.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Scheme of experimental system. A, Irradiated (B10.A x B10.BR)F1 mice were reconstituted with bone marrow from B10.A mice, and donor-derived Tconv or Treg collected and purified 2 mo after reconstitution as described in Materials and Methods. Treg from chimeric mice had T cell suppressive activity comparable to Treg from B10.A mice (data not shown). Each cell population was then transferred into nonirradiated (B10.A x B10.BR)F1 mice, and 2 days later mice immunized with PCC emulsified in CFA. Several days or weeks after PCC immunization, single donor-derived activated (CD4+CD8–B220–CD11b–V11+V3+Dk–CD62LlowCD44high) cells were sorted from lymph nodes by flow cytometry and CDR3 sequences evaluated by PCR followed by DNA sequencing. B, After purification of CD4+CD25– (left panel) or CD4+CD25+ (right panel) T cells as described in Materials and Methods, cells were stained by PE-conjugated anti-CD4 and FITC-conjugated anti-CD25 mAbs (7D4) and evaluated by flow cytometry.

In some experiments, PCC-activated or naive CD4⁺CD25⁺V11⁺V3⁺ T cells (1 x 10⁶) were transferred into nonirradiated (B10.A x B10.BR)F₁ mice that had been immunized by PCC 8 wk earlier. For the purification of activated and naive CD4⁺CD25⁺ T cells, lymph node and spleen cells were first purified from PCC-immunized or unimmunized (B10.A x B10.BR)F₁ mice reconstituted with CD4⁺CD25⁺ T cells from 8-wk-old chimeric mice (B10.A bone marrow into irradiated B10.A x B10.BR mice). Then cells were stained with anti-B220, D^k, and CD8 mAbs and positive cells were removed by anti-rat mouse IgG-coated beads (Dynal Biotech). These resultant cells were then stained with anti-CD25 mAb, and CD25⁺ T cells were separated by positive selection by anti-rat IgG-coated beads followed by LS+ magnetic column (Miltenyi Biotec). Then, cells were stained with anti-V11, V3, CD62L, and CD44 mAbs and V11⁺V3⁺CD62L^lowCD44^high T cells (activated CD4⁺CD25⁺ T cells) from PCC-immunized mice or V11⁺V3⁺CD62L^highCD44^low cells (naive CD4⁺CD25⁺ T cells) from PCC-unimmunized mice were sorted by a cell sorter. Each population was transferred into nonirradiated (B10.A x B10.BR)F₁ mice that had been immunized with PCC 8 wk earlier.

PCR products

Several days after primary and secondary immunization, CD8⁺D^k+-depleted single cells (TCRV11⁺V3⁺CD62L^lowCD44^high) from the lymph nodes were sorted into single cells and placed in a 5-µl cDNA reaction mixture (4 U/ml murine leukemia virus reverse transcriptase with recommended buffer, 0.5 nM spermidine, 100 µg/ml BSA, 10 ng/ml oligo(dT), 200 µM each dNTP, and 1% Triton X-100) and then immediately held at 37°C for 90 min. Aliquots (2 µl) of the cDNA reaction mixture were used for two separate 25 µl amplification reactions (2 U/ml Taq polymerase with the recommended reaction buffer, 0.1 mM each dNTP, 2 mM MgCl₂, and 1.2 µM primer), one for the TCR-V11 and one for the TCR-V3, using primers specific for both the variable and constant regions of each chain. The following combinations of primers were used: V11, 5'-ATGCAGAGGAACCTGGGAGC-3' and 5'-AATCTGCAGCGGCACATTGATTTGGGA-3'; V3, 5'-ATGGCTACAAGGCTCCTCTGGTA-3'and 5'-CACGTGGTCAGGGAAGAA-3'. The total of 1 µl of the first PCR product was used for further 25 µl amplification reactions (2 U/ml Taq polymerase with the recommended reaction buffer, 0.1 mM each of dNTP, 2 mM MgCl₂, and 0.8 µM primer) for each chain of the TCR, using nested primers for: V11, 5'-AATCTGCAGTGGGTGCAGATTTGCTGG-3' and 5'-GAGTCAAAGTCGGTGAACAGG-3'; V3, 5'-AATCTGCAGAATTCAAAAGTCATTCA-3' and 5'-AATCTGCAGCACGAGGGTAGCCTTTTG-3'. Nested PCR product (7 µl) was run on a 1.5% agarose gel to screen for positives (single bands of the right size). The PCR product was then directly sequenced using an ABI 373 sequencing system.

T cell proliferation assay

Total lymph node cells (5 x 10⁴/well) from (B10.A x B10.BR)F₁ mice 10 days after immunization with PCC were cultured with PCC (1 µM) and varying numbers of activated CD4⁺CD25⁺D^k–V11⁺V3⁺CD62L^lowCD44^high T cells from PCC-immunized (B10.A x B10.BR)F₁ mice reconstituted with CD4⁺CD25⁺ T cells from bone marrow chimeric mice or naive CD4⁺CD25⁺D^k–V11⁺V3⁺CD62L^highCD44^low T cells from (B10.A x B10.BR)F₁ mice reconstituted with CD4⁺CD25⁺ T cells from bone marrow chimeric mice (B10.A bone marrow into irradiated B10.A x B10.BR mice). The 1 µCi/well [³H]thymidine was pulsed during the final 8 of 72-h culture. [³H]Thymidine incorporation was evaluated using an automated beta liquid scintillation counter.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PCC immunization protocol

The PCC immunization protocol (24) was used to examine Ag-specific CD4⁺ T cells as this system allowed us to analyze the TCR-V and TCR-V CDR3 sequences critical for binding PCC presented by IE^k. The major PCC-responding CD4⁺ T cell population expresses TCR-V11 and TCR-V3 (24). We first examined the relative number of TCRV11⁺V3⁺ cells in T_conv and T_reg. Both T_conv and T_reg contained 1% TCRV11⁺V3⁺ cells (data not shown). Because CD25, the IL-2R -chain, can be up-regulated following T_conv stimulation (6), we established the following experimental system to clearly discriminate between T_reg and activated T_conv (Fig. 1A). Although both B10.A and B10.BR mice express the MHC class II k haplotype, B10.A and B10.BR express the MHC class I^d and I^k haplotypes, respectively. Irradiated (B10.A x B10.BR)F₁ mice were reconstituted with bone marrow from B10.A mice to remove donor-derived CD4⁺ T cells reactive against B10.BR-derived Ags (Fig. 1A). Donor-derived CD4⁺CD25⁺D^k– (T_reg) or CD4⁺CD25^–D^k– (T_conv) cells were then purified as described in Materials and Methods (Fig. 1B) and transferred into nonirradiated (B10.A x B10.BR)F₁ mice (designated CD25⁺ chimeric mice or CD25^– chimeric mice, respectively) (Fig. 1A). Using this protocol, donor- and host-derived cells could be discriminated according to MHC class I haplotype expression.

After immunization of the chimeric mice with PCC emulsified in CFA, single donor-derived activated cells (CD4⁺CD8^–B220^–CD11b^–V11⁺V3⁺D^k–CD62L^lowCD44^high) from lymph nodes were sorted by flow cytometry. The CDR3 regions of each cell were then amplified by PCR and sequenced as previously described (24). In parallel, total donor-derived activated cell (CD8^–D^k–V11⁺V3⁺D^k–CD62L^low) numbers from the spleen and lymph nodes were counted as described in Materials and Methods. Both the primary and memory responses were examined, we determined eight CDR3 sequence parameters from TCR-V11 and TCR-V3 of PCC-specific T cells (Figs. 2 and 3) (24), and measured total activated cell numbers (Fig. 4). The eight CDR3 sequence parameters from the PCC-specific T cells were CDR3 length, DNA sequence at positions 93 and 95, and J usage for TCR-V11 (Fig. 2), and CDR3 length, DNA sequence at positions 100 and 102 and J usage for TCR-V3 (Fig. 3). Figs. 2 and 3 summarize the results of one representative experiment and show CDR3 sequence information for TCR-V11 and TCR-V3 chains from T cells obtained after PCC immunization. A summary of the combined data obtained from individual animals is shown in Fig. 5.

View larger version (57K): [in this window] [in a new window]   FIGURE 2. Ag-driven modification of preferred TCR-V11 CDR3 sequence parameters from PCC-specific TCR. The three most-preferred CDR3 sequences for TCR-V11 (position 93, position 95, CDR3 length, and J usage) observed in PCC-specific TCR are shown. Each number represents the number of cells with the indicated CDR3 parameter from a single cell analysis (after day 0), with the preferred CDR3 sequence parameters (E) presented at the top of each panel and the percentage of single cells expressing this characteristic listed in the next row. Data are shown as a progression over time (left to right) before injection (day 0), after primary immunization (days 4, 8, 10, 14, and 18), and after memory immunization (days 0, 4, 8, and 10). The second immunization was done 8 wk after the primary immunization. The number of sequences used in the analysis (n) is displayed below the days shown on the x-axis. The day 0 group for primary immunization includes V11V3-expressing T cells before injection with resting CD44lowCD62Lhigh phenotypes.

View larger version (59K): [in this window] [in a new window]   FIGURE 3. Ag-driven modification of preferred TCR-V3 CDR3 sequences of PCC-specific TCR. The TCR-V3 CDR3 regions of PCC-specific TCR were sequenced as in Fig. 2. The three most-preferred TCR-V3 CDR3 (position 100, position 102, CDR3 length, and V usage) sequences are shown. Each number represents the number of cells having the indicated CDR3 characteristic after single cell analysis (from day 0), with data organized as in Fig. 1. The day 0 primary immunization group includes V11V3-expressing T cells before injection with a resting CD44lowCD62Lhigh phenotype.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Frequencies of TCR V11V3-expressing PCC-specific T cells. A, The total number of Ag-specific cells (CD8–V11+V3+CD62LlowDk–) from the lymph nodes and spleen of each animal was calculated. After live cell counts by trypan blue staining, spleen and lymph node cells were stained with PE-conjugated anti-TCRV11, biotin-conjugated anti-TCR-V3, allophycocyanin-conjugated anti-CD62L, FITC-conjugated anti-Dk, or FITC-conjugated anti-CD8 mAbs followed by streptavidin-CyChrome. The percentage of TCRV11+TCRV3+CD62Llow cells gated in CD8–Dk– were evaluated by flow cytometry. Ag-specific T cell numbers were calculated by multiplying total live cell numbers by the relative proportion of activated T cells. Total cell counts on the y-axis represents the mean of six animals ± SEM. Total cell counts of Tconv () and Treg () are shown. There was no significant difference in the adjuvant-only response across different days (data not shown). B, Relative increases in Ag-specific cell counts against day 0 for primary or secondary Ag challenge were calculated. The y-axis represents the mean of six relative cell counts per animal ± SEM. Total cell counts of Treg () and Tconv () are shown.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Ag-driven modification of preferred CDR3 sequences in both TCR chains. Each number represents the number of cells with the preferred TCR-V11 and TCR-3 CDR3 sequences after primary and secondary (8 days after secondary immunization) immunization for CD4+CD25– (A) and CD4+CD25+ (B) T cells.

T_reg and T_conv had similar frequencies of TCR-V11 and TCR-V3 CDR3 sequence parameters before PCC immunization (Figs. 2 and 3), which suggested that both populations had similar TCR repertoires in terms of recognition of PCC presented by IE^k. PCC immunization led to cell proliferation of both T_conv and T_reg after both primary and memory responses (Fig. 4A). However, the degree of proliferation of activated cells was lower and the amount of time to reach the peak day was longer for T_reg than for T_conv after primary immunization (Fig. 4B). For the memory response, peak proliferation occurred earlier for both T_reg and T_conv (Fig. 4B). These findings indicated that polyclonal T_reg proliferated in response to Ag immunization with adjuvant in vivo, although these findings are in contrast with a previous report of T_reg anergy after stimulation via TCR ligation in vitro and in vivo (25, 26).

TCR repertoire modification after primary PCC immunization

We next analyzed the changes in TCR-V11 and TCR-V3 CDR3 sequence parameters using lymph node T cells obtained after primary and memory responses. All preferred TCR-V11 or TCR-V3 CDR3 sequence parameters (TCR-V11 positions 93 and 95, CDR length, and J; TCR-V3 positions 100 and 102, CDR length, and J) from PCC-specific T_conv changed during the primary immune response (Figs. 2 and 3). The percentage of PCC-specific T_conv with preferred TCR-V11 or TCR-V3 CDR3 sequence parameters of TCR-V11 positions 93 and 95, TCR-V11 CDR length (Fig. 2), TCR-V3 positions 100 and 102, TCR-V3 CDR length and J (Fig. 3) exceeded 90% during the primary response. However, the percentage of T_conv with preferred J usage had increased to only 51% (Fig. 2). Using data from Figs. 2 and 3, the percentage of T_conv having six or more characteristic PCC-specific TCR CDR3 sequence parameters was calculated (Fig. 5A). Approximately 70% of PCC-specific T_conv had at least six preferred TCR-V11 or TCR-V3 CDR3 sequence parameters 10 days after primary immunization (Fig. 5A).

The pattern of TCR-V11 positions 93 and 95, TCR-V11 CDR length, and J usage (Fig. 2), and TCR-V3 positions 100 and 102, TCR-V3 CDR length, and J (Fig. 3) was also modified in T_reg at 18 days after the first immunization, but to a lesser extent compared with T_conv (Figs. 2 and 3), except for TCR-V3 position 102 (Fig. 3). Further analysis showed that only 30% of PCC-specific T_reg had six or more preferred of TCR-V11 or TCR-V3 CDR3 sequence parameters at day 18 (Fig. 5B).

TCR repertoire modification after secondary PCC immunization

We next examined whether the TCR repertoire altered during the memory response. PCC was administered 8 wk after the primary immunization and CDR3 sequences from T cells obtained (Figs. 2 and 3). Results showed that the PCC-specific T_conv TCR repertoire was further modified during the memory response in terms of preferred TCR-V11 or TCR-V3 CDR3 sequence parameters, including TCR-V11 positions 93 and 95, TCR-V11 CDR length, and J usage (Fig. 2), and TCR-V3 positions 100 and 102, TCR-V3 CDR length, and J usage (Fig. 3). However, the percentage of T_conv showing the preferred J usage was 60% (Fig. 2) compared with nearly 100% for the other CDR3 sequence parameters (Figs. 2 and 3). This further modification in T_conv reflected the fact that 90% of the cells had acquired six or more of the preferred PCC-specific TCR-V11 or TCR-V3 CDR3 sequences during the memory response (Fig. 5A). In contrast, whereas the second immunization increased T_reg cell numbers (Fig. 4A), further modification of TCR (Fig. 2) and TCR (Fig. 3) repertoire was not observed (Fig. 5B). Because >88% of T_reg before secondary immunization had three preferred CDR3 sequence parameters (TCR-V11 CDR length, TCR-V3 position 102, and TCR-V3 CDR length) (Figs. 2 and 3), it was difficult to determine small changes in these parameters after secondary immunization. However, significant differences in the frequencies of preferred TCR-V11 positions 93 and 95, J usage (Fig. 2), TCR-V3 position 100 and J usage (Fig. 3) were observed after secondary responses for both T_conv and T_reg. The relative percentages of these five preferred CDR3 sequence parameters did not change in T_reg after secondary immunization (Figs. 2 and 3). Similar results were observed in terms of TCR repertoire modification both of T_conv and T_reg when spleen cells were used instead of lymph node cells (data not shown).

Low level contamination of T_conv in the T_reg fraction did not greatly affect the repertoire modification of T_reg

To negate the possibility that T_reg proliferation and repertoire modification reflected the proliferation of low numbers of contaminating T_conv, we used a mixture of 99% T_reg and 1% T_conv or 95% T_reg and 5% T_conv instead of purified T_reg in our proliferation (Fig. 6) and repertoire modification (Fig. 7) assays, and compared the results with those obtained using purified T_reg (Figs. 4 and 5). Proliferation and repertoire modification assay results were similar between the 1% and 5% T_conv contamination samples, which were in turn similar to results obtained using purified T_reg (Figs. 5, 6, and 7). These results suggested that low levels of T_conv contamination in the T_reg fraction would not be expected to have a significant impact on T_reg proliferation and repertoire modification.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Frequencies of TCR V11V3-expressing PCC-specific T cells. Irradiated (B10.A x B10.BR)F1 mice were reconstituted with bone marrow from B10.A mice, and donor-derived Tconv or Treg collected and purified 2 mo after reconstitution as described in Materials and Methods. Mixtures of 1% Tconv and 99% Treg () or 5% Tconv and 95% Treg () were transferred into nonirradiated (B10.A x B10.BR)F1 mice, which were then immunized 2 days later with PCC emulsified in CFA. Several days or weeks after PCC immunization, donor-derived activated single cells (CD4+CD8–B220–CD11b–V11+V3+Dk–CD62LlowCD44high) were sorted from lymph nodes and CDR3 sequences evaluated by PCR followed by DNA sequencing. Total Ag-specific cell (CD4+CD8–V11+V3+CD44highCD62LlowDk–) numbers from the lymph nodes and spleen of each animal were calculated. B220, CD8, and Dk-positive cells were removed by anti-rat IgG-coated microbeads and counted by trypan blue staining. Relative V11+V3+CD44highCD62Llow cell numbers were evaluated by staining with anti-TCR V11, TCR-V3, CD44, and CD62L mAbs. Relative increases in Ag-specific cell counts against day 0 for primary or secondary Ag challenges were calculated. The y-axis represents the mean of six relative cell counts of animals ± SEM.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Ag-driven modification of preferred CDR3 sequences in both TCR chains. Each number represents the number of cells with the indicated CDR3 sequences from both TCR-V11 and TCR-3 in PCC-immunized mice as in Fig. 1. The y-axis represents the number (n) of preferred CDR3 sequences seen when mice were given a mixture of 1% Tconv and 99% Treg (A) or a mixture of 5% Tconv and 95% Treg (B). Cells with more than four preferred CDR3 sequence parameters were considered to have a restricted TCR, and the percentage of these cells is shown. Sequence information from memory response cells (8 days after immunization) is displayed. The number of analyzed sequences (n) is displayed on the x-axis.

To examine the impact of incomplete T_reg repertoire modification on effector T cell generation, we first evaluated the repertoire modification of lymph node CD4⁺CD25^– cells from PCC-immunized and CD25⁺-depleted (B10.A x B10.BR)F₁ mice using anti-CD25 mAb. The kinetics of TCR repertoire modification in activated CD4⁺CD25^–V11⁺V3⁺CD62L^lowCD44^high cells in response to PCC immunization was similar to that of CD4⁺CD25^– donor cells (CD4⁺CD25^+/–V11⁺V3⁺D^k–CD62L^lowCD44^high) from CD25^– chimeric mice (data not shown). For this reason we used (B10.A x B10.BR)F₁ mice instead of CD25⁺ or CD25^– chimeric mice to evaluate the impact of low T_reg repertoire modification on effector T cell activation or differentiation.

Following primary PCC immunization, (B10.A x B10.BR)F₁ mice that had been T_reg depleted using anti-CD25 mAb generated higher Ag-specific T cell numbers in the lymph nodes and spleen compared with non-T_reg depleted (B10.A x B10.BR)F₁ mice (Fig. 8A). The kinetics of lymph node PCC-activated T cell TCR repertoire modification was also faster in the absence of T_reg after the primary immune response (Fig. 8C). To evaluate the effect of T_reg on memory T cell responses, CD25-positive cells were depleted by anti-CD25 mAb treatment in PCC immunized (B10.A x B10.BR)F₁ mice 8 wk after primary immunization. Mice were then reimmunized with PCC and the TCR repertoire and total cell numbers of CD4⁺TCRV11⁺V3⁺CD44^highCD62L^low T cells evaluated. Depletion of T_reg before secondary immunization induced a faster TCR repertoire modification (Fig. 8D), but did not affect PCC-specific T cell numbers (Fig. 8B).

View larger version (36K): [in this window] [in a new window]   FIGURE 8. Regulation of TCR repertoire modification and T cell proliferation by CD25+ regulatory T cells. Total Ag-specific T cell counts and the percentage of cells with more than four specific CDR3 sequence parameters of PCC-specific TCR during primary (left panel) and memory (right panel) responses are shown. For primary immunization, (B10.A x B10.BR)F1 mice pretreated with () or without () anti-CD25 mAb were immunized with PCC in CFA and cell counts of Ag-specific T cells (CD4+CD25–V11+V3+CD62LlowCD44high) in spleen and lymph nodes (A) and lymph node TCR repertoires (C) evaluated as in Fig. 1. For the memory response (2 mo after primary immunization), cell counts of Ag-specific T cells (CD4+CD25–V11+V3+CD62LlowCD44high) in spleen and lymph nodes (B) and lymph node TCR repertoires (D) were evaluated several days after immunization. For the memory response, primary immunized (B10.A x B10.BR)F1 mice were immunized with PCC in CFA with () or without () anti-CD25 mAb pretreatment (2 days before the second immunization). In some experiments, Ag-specific () or naive CD4+CD25+V11+V3+ T cells () were transferred into mice before the second immunization. Ag-specific CD4+CD25+ T cells (CD4+CD25+Dk–V11+V3+CD62LlowCD44high) were obtained from donor-derived T cells (1 x 106) 14 days after primary immunization of CD25+ chimeric mice as in Fig. 2. *, p < 0.05; **, p < 0.01 are indicated. E, Total lymph node cells (5 x 104) from (B10.A x B10.BR)F1 mice immunized by PCC were incubated with PCC, with the indicated numbers of PCC-activated CD4+CD25+Dk–V11+V3+CD62LlowCD44high T cells purified from PCC-immunized CD25+ chimeric mice () or naive CD4+CD25+Dk–V11+V3+CD62LhighCD44low T cells from CD25+ chimeric mice () are shown. [3H]Thymidine incorporation was evaluated during the final 8 h of the 72 h culture. Results of the proliferation assays are shown as the mean of triplicate cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The adaptive immune response is dependent on the recognition of Ag peptides presented in the context of self-encoded MHC molecules by specific T cells. A broad preimmune TCR repertoire is established via processes involving positive and negative selection on developing T cells in the thymus (4). The particular T cell repertoire that responds to a given Ag is usually quite diverse in terms of TCR -chain V region usage and fine epitope specificity (18, 19). However, in some cases clonal dominance prevails and T cells with preferred TCR motifs are selectively expanded during primary responses and then appear to be selectively preserved for memory responses (16, 24). Although these studies have tended to focus on the repertoire of effector T cells, the total T cell immune response appears to be regulated by the interplay between effector and suppressor T cells. Therefore, to gain a better understanding of the total adaptive immune response, we attempted to clarify the mode of repertoire modification and proliferation of suppressor/regulatory T cells against a given Ag. Our study revealed an evolving T_reg clonal dominance during the primary immune response. Although T_conv were less affected, T_reg clonal modification was not observed during the memory response. Experiments using CD25⁺ cell depletion showed that removing T_reg against a given Ag-suppressed TCR repertoire modification of the T_conv-derived primary and memory T cells in vivo. In contrast, T_reg inhibited the proliferation of Ag-specific T_conv during the primary, but not the memory response. Nonetheless, addition of PCC-activated T_reg still inhibited memory T cell proliferation.

T_reg are thought to express high affinity TCRs against self-peptides (27) and suppress effector T cell responses independent of Ag specificity, at least in vitro (28). These findings suggest a role for self-Ag recognition in the acquisition of T_reg suppressive function. Although T_reg have very broad TCR V repertoires, similar to T_conv (data not shown), it remains unclear whether T_reg actually recognize non-self Ags to acquire suppressive functions in vivo. Recently, several papers have shown that T_reg suppress murine CD4⁺ and CD8⁺ T cell responses against parasitic or bacterial infections in vivo (29, 30). This suggests two possibilities regarding the Ag specificity of T_reg in vivo. First, T_reg may recognize parasite- or bacteria-derived broad Ags, acquire a suppressive function, and then regulate/suppress effector T cell responses. Second, infections may change the sensitivity of T_reg TCR signaling via local cytokine bursts or interaction with other activated cells, which then modifies the TCR signaling threshold of T_reg to allow sufficient response to self-Ags with resultant acquisition of suppressive functions. In this regard, our results indicated that the frequency of preferred TCR-V11 or TCR-V3 CDR3 sequence parameters of PCC-specific T cells were increased in T_reg after PCC immunization, which strongly suggested the direct recognition of PCC by T_reg. In addition, the repertoire modification of T_reg increased the total suppressive function of T_reg toward effector/memory T cell responses against PCC, which demonstrated that T_reg can suppress effector T cells via recognition of a specific common Ag in vivo. These findings suggested that the regulation of Ag-specific T_reg, rather than total T_reg, may be required for the clinical application of T_reg, in agreement with a number of groups that have tried to regulate effector T cells in an Ag-dependent manner (31).

Previous studies using TCR-transgenic mice have shown that T_reg are anergic in vitro, but have the potential to respond to lymphopenic conditions (25, 26). Recently, it was shown that T_reg proliferated in vivo (32). Under more physiologic conditions, as in our experimental protocol, polyclonal T_reg responded to PCC immunization and proliferated in vivo, although the response was less than the T_conv response. One might argue that the T_reg proliferation we observed might have at least partly reflected the proliferation of contaminating T_conv as CD25 is not an exclusive T_reg marker. However, this is unlikely because the TCR repertoire of all CD25⁺-derived cells was examined 18 days after the first immunization, which would reflect the presence of cell proliferation. We also performed experiments using a mixture of 99% T_reg and 1% T_conv or 95% T_reg and 5% T_conv instead of the purified T_reg population and obtained similar findings in terms of cell proliferation and repertoire modification. If the reduced TCR repertoire seen in T_reg is due to vigorous expansion of contaminated T_conv, we should have observed increased repertoire modification with increasing numbers of contaminating T_conv. However, the possibility that contaminating T_conv do contribute to T_reg result cannot be ruled out as we do not have cell surface markers able to discriminate between T_reg and activated T cells.

The CD25⁺ cell depletion experiments showed that during the memory response, T_reg inhibited effector T cell TCR repertoire modification, but not T cell-mediated proliferation. However, the addition of PCC-activated T_reg during the memory response inhibited T cell proliferation. This suggested the inability to inhibit effector T cell proliferation was due to limited T_reg expansion or TCR repertoire modification after primary immunization rather than the intrinsic failure of effector T cells to respond to the inhibitory activity of T_reg. Although T_reg generally inhibit T cell proliferation, the precise regulatory mechanisms of T_reg remain unknown, despite reports suggesting possible roles for TGF- or CTLA-4 (33, 34). T_reg inhibition of T cell repertoire modification, but not proliferation, suggests another regulatory role of T_reg in the T_conv response. Furthermore, our findings provide evidence for a distinct regulatory mechanism linking T cell proliferation and TCR repertoire modification, although these have generally been thought to be coordinated processes.

We observed no differences in the frequencies of CDR3 features involved in PCC recognition between T_reg and T_conv before immunization. Nonetheless, it is still possible that T_reg and T_conv exhibited different TCR repertoires in terms of PCC recognition that might underlie the different proliferation and TCR repertoire modification kinetics. However, this seems unlikely as a similar frequency of PCC/IE^k-specific TCRV11⁺V3⁺ T cells (0.06–0.07%) was present in total TCRV11⁺V3⁺ T_reg and T_conv (data not shown).

The reduced ability of T_reg to suppress the proliferation of memory/effector T cells due to reduced Ag-specific T_reg proliferation or incomplete TCR repertoire modification during primary responses would be advantageous to the overall immune response as the coordinated parallel evolution of T_reg and T_conv would not be useful for augmenting the memory T cell response. This divergence may have occurred to maximize the ability of the organism to mount a protective memory immune response against bacteria, protozoans, fungi, and viruses.

	Acknowledgments

 
We thank Dr. Y. Itoh for critically reading this manuscript.

	Footnotes

 
¹ This work was supported by Grants-in-Aid for Young Scientists (A) from the Ministry of Education, Science, Technology, Sports (15689016), and by the Yamanouchi Foundation for Research on Metabolic Disorders (to K.Y.).

² Address correspondence and reprint requests to Dr. Koji Yasutomo, Department of Immunology and Parasitology, School of Medicine, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail address: yasutomo{at}basic.med.tokushima-u.ac.jp

³ Abbreviations used in this paper: T_reg, regulatory CD4⁺CD25⁺ T cells; T_conv, conventional CD4⁺CD25^– T cells; PCC, pigeon cytochrome c; CDR3, complementarity-determining region 3.

Received for publication June 27, 2003. Accepted for publication February 20, 2004.

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