TGF-ß signaling regulates CD8+ T cell responses to high- and low-affinity TCR interactions
Wajahat Z. Mehal1,2,
Shehzad Z. Sheikh2,
Leonid Gorelik3 and
Richard A. Flavell1,4
1 Section of Immunobiology and 2 Section of Digestive Diseases, Yale University School of Medicine, 300 Cedar Street, TAC S-569, New Haven, CT 06520, USA
3 Biogen Inc., Cambridge, MA, USA
4 Howard Hughes Medical Institute, Yale University, 300 Ceder Street, TAC S569, New Haven CT, 06520, USA
Correspondence to: W. Z. Mehal; E-mail: wajahat.mehal{at}yale.edu
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Abstract
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Absence of transforming growth factor-ß (TGF-ß) signaling to T cells in mice results in an increase in T cell numbers, an activated CD44 high, CD69, CD25 T cell phenotype and a T cell-mediated injury to many organs. It is not known if such T cell activation in the absence of TGF-ß signaling is spontaneous or due to aberrant T cell responses to a physiological stimulus. We used adoptive transfer of CD8+ T cells from mice double transgenic for the OT-1 TCR and the TGF-ß1-dominant negative transgene [OT-dominant-negative receptor (DNR)] to investigate the role of TGF-ß in regulating CD8+ T cell activation in vivo. The activation and expansion of single-transgenic OT and double-transgenic OT-DNR cells to oral antigens, high-affinity and low-affinity peptides were indistinguishable. Activation with high-affinity peptide and CFA however resulted in greater expansion of OT-DNR cells in comparison to OT cells. Low-affinity peptide and adjuvant did not result in OT cell activation or expansion but results in up-regulation of CD44 on OT-DNR cells. These data show that TGF-ß functions in vivo to limit the scale of CD8+ T cell expansion after high-affinity peptideMHC interactions. TGF-ß also limits T cell activation to the highest affinity peptideMHC interactions. The increase in T cell number and activation present in TGF-ß-deficient and TGF-ß DNR-expressing mice may be due to the loss of these two phenomena.
Keywords: antigen/peptides/epitopes, cellular activation, rodent, T lymphocytes
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Introduction
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The three isoforms of transforming growth factor-ß (TGF-ß) (TGF-ß1, 2 and 3) are widely expressed and have a multitude of functions, including suppressing T cell activation and limiting T cell apoptosis (1). TGF-ß1 is the predominant isoform in lymphoid organs and its critical role in the immune system was demonstrated by the severe phenotype of TGF-ß1 knockout (KO) mice (2, 3). These mice developed spontaneous self-targeted immune responses which leads to their death by 34 weeks of age. To more precisely identify the role of TGF-ß signaling to T cells, mice expressing a dominant-negative form of the TGF-ß RII were generated [dominant-negative receptor (DNR)] (4). These mice had a similar, but less severe, phenotype as the TGF-ß1 KO mice. In particular, there was increasing activation of T cells with age, with the activated cells having a CD44 high, CD69 and CD25 phenotype. In addition, the CD44 high, CD69 and CD25 cells spontaneously differentiated in vivo into effector cytokine-producing cells.
It has not been determined if the activation of T cells in TGF-ß DNR mice is an entirely spontaneous event or if TGF-ß is important in regulating specific T cell responses. Due to the predominant function of TGF-ß in suppressing T cell activation, we hypothesized that in normal mice TGF-ß limits T cell responses to stimuli which usually provide a sub-optimal activation signal to T cells. Limitation of T cell activation by TGF-ß could be global, and in addition, TGF-ß may have a particular role in local immune responses in organs known to have sub-optimal immune responses. To test this hypothesis we used T cells from the well-characterized OT-1 mice and also from mice doubly transgenic for the OT-1 and the TGF-ß DNR transgene (referred to as OT and OT-DNR, respectively) (4, 5). We chose to study the response of CD8+ T cells in the absence of TGF-ß signaling because TGF-ß-deficient mice crossed on an MHC class I-deficient background (ß2-microglobulin-null mice) live much longer than TGF-ß-deficient mice on a MHC class II-deficient background, suggesting that CD8+ T cells make a major contribution to the immune pathology in the absence of TGF-ß (6). In addition, there are higher numbers of CD44+ CD8+ T cells, compared with CD44+ CD4+ T cells in TGF-ß DNR mice, demonstrating that CD8+ T cells are a predominant cell type affected by lack of TGF-ß signaling (4).
Antigen presented via the oral route is well known to result in sub-optimal T cell responses (7). High-dose oral antigen induces activation markers on T cells and T cell deletion, but little or no T cell proliferation (8). This is a complex phenomenon in which the liver has been known to play an important role, and in addition, a number of cells in the liver are known to produce TGF-ß (9, 10). T cells are also frequently exposed to a different type of sub-optimal stimulus. This stimulus results from interactions with peptideMHC complexes with a low affinity for the TCR (11). These are known to be important in T cell homeostasis, and the majority of such interactions are presumed to occur with self-peptideMHC complexes (12).
By using adoptive transfer of OT and OT-DNR cells into wild-type mice, we showed identical responses of OT and OT-DNR cells to oral ovalbumin. In addition, both cell types had a very similar response to intraperitoneal (i.p.) injections of peptides of high and low affinity. There was however significantly greater expansion of OT-DNR cells in response to high-affinity peptide and CFA. In response to low-affinity peptide and CFA, there is no change in activation markers on OT cells, but a significant up-regulation of CD44, but not CD25 or CD69, on OT-DNR cells. These data show that in vivo TGF-ß signaling to T cells limits the scale of CD8+ T cell expansion after high-affinity peptideMHC interactions. It also limits T cell activation to the highest affinity peptideMHC interactions. The increase in T cell activation that is seen in TGF-ß-deficient and TGF-ß DNR-expressing mice may be due to these two phenomenon.
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Methods
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Mice
OT-1 and TGF-ß DNR mice were available from our mouse colony and had been back-crossed onto CD45.1 C57BL/6 mice for over 10 generations before intercrossing (4, 12). All animals were kept in specific pathogen-free conditions according to institutional guidelines for animal care and were fed an ovalbumin peptide (OVA)-free diet. Heterozygous OT-1 and TGF-ß DNR mice were bred to obtain OT-1-single transgenics (OT), TGF-ß DNR single transgenics (DNR) and OT-DNR double transgenics (OT-DNR). C57BL/6 mice from Jackson Laboratories (Bar Harbor, Maine) were used as the recipients for OT or OT-DNR cells. The University Animal Use and Care Committee approved all procedures.
Adoptive transfer of OT and OT-DNR cells
Peripheral (axillary, inguinal) lymph node lymphocytes were isolated from OT and OT-DNR mice in a sterile manner and washed in PBS containing 1% normal mouse serum. Between 56 million total lymph node cells from OT or OT-DNR donor mice were transferred by intravenous injection per recipient to CD45.2+ C57BL/6 mice and allowed to equilibrate for 2 days before the administration of antigen.
Oral feeding and in vivo activation
Mice transferred with OT and OT-DNR cells were divided into a control group and a number of experimental groups. For the oral feeding experiments, the control group was gavaged with 300 µl of sterile PBS and the experimental group was gavaged with 100 mg of OVA (Sigma, St. Louis, MO) in 300 µl of PBS daily for the duration of the experiment. For the high- and low-affinity peptide experiments, 25 nM of high-affinity SIINFEKL peptide, low-affinity G4 (SIIGFEKL) peptide (in 200 µl of sterile LPS-free water), or sterile LPS-free water was injected i.p. daily for the duration of the experiment (13). High-affinity and low-affinity G4 peptide were synthesized by the Keck Facility at Yale University. For in vivo activation with CFA, the same amount of high- and low-affinity peptide or sterile water was mixed with 200 µl of CFA (DIFCO Laboratories, MI, USA) and injected subcutaneously once in the back in the midline. For each experiment, there were three mice in each of the four groups (OT control, OT antigen, OT-DNR control, OT-DNR antigen), and each experiment was repeated at least four times. The mean and SD of cellular percentages in the various organs were calculated for each mouse in the four groups.
In vitro activation
Mixed spleen and lymph node cells from CD45.1 OT and OT-DNR mice were activated by irradiated splenocytes from CD45.2 wild-type C57BL/6 mice using a range of peptide concentrations in RPMI supplemented with L-glutamine, 10% FCS, ampicillin and gentamicin. Cells were cultured in 24-well plates with 0.5 million CD45.1 OT or OT-DNR and 1 million irradiated splenocytes from CD45.2 wild-type C57BL/6 cells per well. On Day 3 after co-culture, OT and OT-DNR T cells were analyzed for activation markers by flow cytometry gating on CD45.1 and CD8 double-positive cells.
Cell isolation and flow cytometric analysis
For the experiments with oral feeding, lymphocytes were isolated from the peripheral (axillary and inguinal) lymph nodes, spleen and the liver. For the experiments with injection of peptides with and without CFA, cells were isolated from the peripheral (axillary and inguinal) lymph nodes and spleen only. Hepatic lymphocyte isolation was carried using a modified version of the protocol previously described (14). The gradient was made with 21% Optiprep (Axis-Shield, Oslo, Norway) instead of metrizamide. Flow cytometric analysis was undertaken on a three-color BD FACScan after staining with combinations of the following antibodies from BD-Pharmingen (San Diego, CA): CD45.1FITC and PE, TCR
FITC and antigen-presenting cells (APC), CD8FITC and Cy, CD4FITC and APC, CD44APC, CD69FITC and CD25PE.
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Results
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Age-associated activation of OT cells expressing the TGF-ß DNR (OT-DNR)
Single-transgenic mice expressing the TGF-ß DNR have up-regulation of CD44 on T cells with age. At 20 weeks of age
90% of CD8+ T cells were CD44 high. To confirm that the same phenomenon was present when the OT-1 transgene was crossed onto the DNR transgene, splenocytes from 4- and 25-week old OT, DNR and OT-DNR mice were examined for expression of CD44 on CD8+ T cells. Figure 1 shows that at 4 weeks of age very few CD8+ T cells were CD44 high in the OT-DNR mice. By 25 weeks of age the percentage of CD8+ T cells that were CD44 high had increased significantly in the OT-DNR mice. The total splenocyte numbers also increased with age, but this effect was modest with 45.2 million (±11.2) in 4-week-old OT-DNR mice and 65 million (±15.3) in 25-week-old OT-DNR mice. Unlike other models of spontaneous T cell proliferation, the T cells, although CD44 high, are CD69, CD25 and B220 negative.

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Fig. 1. Age-associated activation of OT cells expressing the TGF-ß DNR (OT-DNR). Flow cytometry analysis of splenocytes from 4-week-old OT-DNR mice (A) and 25-week-old (B) OT-DNR mice showing significant increase in CD44 with age.
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Defect in the activation of OT-DNR cells from old, but not young mice
Prior to testing the ability of various in vivo stimuli to activate OT-DNR cells, we ensured that naive OT-DNR cells were able to undergo activation by high-affinity peptide in vitro. Figure 2(A) demonstrates that CD8+ T cells from 25-week-old OT-DNR mice had minimal up-regulation of CD69 even with high concentrations of peptide. In contrast, CD8+ T cells from 4-week-old OT-DNR mice undergo activation as judged by up-regulation of CD69 and CD44 after 2 days of incubation with high-affinity peptide and splenic APC (Fig. 2B and C). These data show that OT-DNR cells from young mice undergo activation in response to high-affinity peptide presented by splenic APC in vitro.

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Fig. 2. Defect in activation of OT-DNR cells from the old, but not young mice. (A) OT and OT-DNR lymph node and spleen cells from 25-week-old mice were activated by irradiated splenocytes and high-affinity (HA) peptide. By Day 3, there was significant up-regulation of CD69 on the OT cells with peptide concentrations as low as 1 nM. In contrast, CD69 was not up-regulated on OT-DNR cells even at the greatest HA peptide concentration. (B and C) CD69 and CD44 up-regulation on OT and OT-DNR cells from 4-week-old mice using HA peptide and irradiated splenocytes. At Day 3, there is significant up-regulation of CD69 and CD44 on both OT and OT-DNR cells.
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Minimal difference in expansion and loss of OT-DNR cells in response to oral antigens
After adoptive transfer of OT and OT-DNR cells, both populations make up a small percentage of the peripheral lymphocyte pool of the C57BL/6 recipient. Figure 3(A and B) shows that after 3 days of gavage with PBS, donor CD8+ T cells (CD45.1+ and CD8+) are easily identified and make up 0.2% (±0.1) [mean% (±SD)] for the OT and 0.24% (±0.15) for the OT-DNR of the total lymphocyte pool. After 3 days of gavage with ovalbumin there is no statistically significant increase in the OT populations at 0.24% (±0.2%) and a slight increase in the OT-DNR population to 0.8% (±0.18%) (Fig. 3C and D). By 7 days of gavage with ovalbumin loss of both populations is seen in the spleen and lymph nodes (spleen dataPBS: OT 0.18% ± 0.08, OT-DNR 0.23% ± 0.07; ovalbumin: OT 0.01% ± 0.03, OT-DNR 0.01 ± 0.02). The percentages of donor CD8+ T cells in the liver were very similar to the spleen data. In the absence of TGF-ß signaling to T cells there is minimal expansion of CD8+ T cells in the spleen or liver after high-dose oral administration of specific antigen. The loss of donor cells after 7 days of high-dose ovalbumin gavage demonstrates that antigen presentation to the OT and OT-DNR cells did occur.

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Fig. 3. Minimal difference in expansion and loss between OT and OT-DNR cells in response to oral ova antigen. Two days after adoptive transfer of OT (A, C, E) or OT-DNR (B, D, F) cells mice were gavaged PBS (A, B) or ovalbumin 100 mg perday (C, D, E, F). Data shown are from lymph nodes. (A and B) Low but detectable percentage of OT and OT-DNR cells 3 days after PBS gavage. (C) No increase in the percentage of OT cells after 3 days of ovalbumin. (E) Significant loss of OT cells after 7 days of gavage with ovalbumin. (F) Significant loss of OT-DNR cells after 7 days of gavage with ovalbumin.
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Similar increase of OT and OT-DNR cells in response to high-affinity peptide
To test the response of T cells to high-affinity antigen in vivo in the absence of TGF-ß signaling, high-affinity peptide was injected i.p., and the numbers of donor OT and OT-DNR cells were identified as in Fig. 3. There was a similar expansion of both OT and OT-DNR cells in response to high-affinity peptide at Day 3 after injection (PBS injection: OT 0.2% ± 0.18%, OT-DNR 0.18% ± 0.2; high-affinity peptide: OT 1.7% ± 0.21, OT-DNR 1.5% ± 0.19).
Low-affinity peptide results in the activation of OT and OT-DNR cells in vitro, but not in vivo
Before testing the ability of low-affinity G4 peptide to activate OT and OT-DNR cells in vivo, we tested the ability to activate both cell populations in an in vitro culture. Figure 4 shows the ability of G4 peptide presented by splenic APC to induce up-regulation of CD69 and CD44 on OT and OT-DNR cells. Activation by low-affinity G4 peptide required 100-fold higher concentration than high-affinity peptide (100 nM, compared with 1 nM) (Figs 2B and C and 4). Even at the highest concentration used (500 nM), the percentage of CD8+ T cells that were CD69 or CD44+ was less than that for the high-affinity peptide.

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Fig. 4. Low-affinity (LA) peptide results in the activation of OT and OT-DNR cells in vitro. OT and OT-DNR lymph node and spleen cells from 5-week-old mice were activated by irradiated splenocytes and LA peptide. By Day 3, there was significant up-regulation of CD69 and CD44 on OT and OT-DNR cells. The concentration of LA peptide required to activate OT and OT-DNR cells was 100-fold higher than that required for high-affinity peptide.
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Injection of G4 peptide into C57BL/6 mice adoptively transferred with OT and OT-DNR cells however did not result in up-regulation of CD44 or CD69 after 3, 5, 7 and 10 days of injection (data not shown). Consistent with this there was no increase in the numbers of OT and OT-DNR cells after 3, 5, 7 and 10 days of injection (spleen dataDay 3 PBS injected: OT 0.23% ± 0.1, OT-DNR 0.21% ± 0.18; G4 peptide: OT 0.19 ± 0.22, OT-DNR 0.14 ± 0.15).
High-affinity peptide with adjuvant results in greater expansion of OT-DNR cells compared with OT cells
To investigate the role of TGF-ß in immune responses stimulated by high-affinity peptide and adjuvant, mice adoptively transferred with OT or OT-DNR cells received a single injection of high-affinity peptide in CFA or CFA alone subcutaneously. Figure 5(A and B) shows that high-affinity peptide in CFA results in expansion of OT cells at Day 3. In addition, Fig. 5(D and E) shows that OT-DNR cells also have expansion with high-affinity peptide and CFA. A comparison of the responses of OT and OT-DNR cells to high-affinity peptide in CFA shows significantly greater expansion of OT-DNR cells in response to high-affinity peptide and CFA (OT CFA 0.16 ± 0.07, OT CFA-high affinity 3.5 ± 1.1, OT-DNR CFA 0.14 ± 0.1, OT-DNR CFA-high affinity 9.8 ± 1.4). Figure 5(G) shows a summary of the response of OT and OT-DNR cells from lymph nodes to high-affinity peptide and CFA at Day 3 after peptide injection.

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Fig. 5. Increased proliferation of OT-DNR cells in response to CFA with high-affinity (HA) and CFA with low-affinity (LA) peptide. Two days after adoptive transfer of OT or OT-DNR cells, mice received a single injection of CFA alone (A, D) or CFA with HA peptide (B, E) or CFA with LA peptide. (C, F). The data show percentages of donor OT (A, B, C) or OT-DNR (D, E, F) cells in lymph nodes on Day 3 after subcutaneous injection. CFA with HA peptide results in an increase in the percentage of OT cells compared with CFA alone (B, A). CFA with HA peptide results in a significantly greater increase in OT-DNR cells compared with OT (E, B). CFA with LA peptide does not result in an increase in the percentage of OT cells compared with CFA alone (C, A). CFA with LA peptide results in a slight increase in the percentage of OT-DNR cells compared with CFA alone (F, D).(G) Summary of data on proliferation at Day 3 of OT and OT-DNR cells in response to CFA with HA peptide.
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In contrast to T cells from lymph nodes, CFA and high-affinity peptide induced minimal expansion of OT and OT-DNR cells in the spleen. At Day 3 after CFA injection the percentages of OT and OT-DNR cells in the spleen were OT CFA 0.14 ± 0.09, OT CFA-high affinity 0.26 ± 0.1, OT-DNR CFA 0.12 ± 0.08, OT-DNR CFA-high affinity 0.38 ± 0.8.
Low-affinity peptide with adjuvant results in the activation of OT-DNR, but not OT cells in vivo
Low-affinity peptide in CFA did not result in an increase of OT cells compared with CFA alone, (Fig. 5A and C), but there was a slight increase in OT-DNR cells in response to low-affinity peptide and CFA (Fig. 5D and F). In contrast to the modest effect of low-affinity peptide and CFA on proliferation of OT-DNR cells, there was a very significant effect of low-affinity peptide and CFA on the up-regulation of CD44 on OT-DNR, but not OT CD8+ T cells (Fig. 6 shows data from CD8+ T cells 3 days after low-affinity peptide and CFA injection). Analogous to the proliferative response of CFA-high-affinity peptide being limited to the draining lymph nodes, the up-regulation of CD44 on OT-DNR cells by CFA-low-affinity peptide was also limited to the draining lymph nodes, with no up-regulation seen in the spleen up till Day 10 after injection. In addition, CFA-low-affinity peptide did not result in up-regulation of CD69 on OT or OT-DNR cells.

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Fig. 6. Low-affinity (LA) peptide with adjuvant results in the activation of OT-DNR, but not OT cells. Two days after adoptive transfer of OT (A, B, C) or OT-DNR (D, E, F) cells, mice received a single subcutaneous injection of CFA alone (A, D) or CFA with high-affinity (HA) peptide (B, E) or CFA with LA peptide (C, F). The data show percentages CD44 high of donor OT (A, B, C) or OT-DNR (D, E, F) cells in lymph nodes Day 3 after subcutaneous injection. CFA with HA peptide results in an increase in the percentage of CD44 high cells (B, E) relative to CFA injection alone (A, D). CFA with LA peptide results in an increase in the percentage of CD44 high cells for OT-DNR (F) but not OT (C).
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Discussion
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The very severe immune pathology in the TGF-ß KO mouse has made it difficult to understand the role TGF-ß in T cell activation and homeostasis using this model. Production of the T cell-specific TGF-ß RII DNR-transgenic cells demonstrated that in the absence of TGF-ß signaling to T cells there is normal T cell development, with preserved T cell proportions in the thymus (4). In young mice there is minimal T cell activation, but with age the numbers of CD44+ T cells increase. CD8+ T cells are more severely affected than CD4+ T cells as TGF-ß RII DNR mice age, with greater up-regulation of activation markers than CD4+ T cells and a greater increase in numbers of CD8+ CD44+ T cells. It has been proposed that in vivo TGF-ß regulates the response of T cells to physiological interactions with antigens, and the loss of TGF-ß regulation results in T cell activation (15). This fits well with the increase in activated T cells with age, but the exact interactions between T cells and antigens regulated by TGF-ß were not known. Interactions of T cells with antigens in specific pathogen-free mice are limited primarily to food antigens entering the body through the intestines and low-affinity interactions with self-antigens. Interactions with either of these antigens result in none or minimal T cell activation and proliferation in normal mice (7, 16).
TGF-ß has a very wide distribution. In the serum it is present at 1030 ng ml1 and concentrations as low as 3 ng ml1 are known to be active in vitro (17). In addition, TGF-ß functions in local autocrine and paracrine networks in concentrations which are probably higher than serum. The influence of these in vivo levels of TGF-ß on the response of T cells recognizing food and low-affinity antigens is not known. Antigens entering the body via the gastrointestinal tract clearly interact with specific T cells (18). This has been shown to result in the activation of T cells with some proliferation, subsequent deletion and generation of regulatory CD8+ T cells (19, 20). Presentation of food antigens is known to occur in the liver, and many cells in the liver known to present antigen also produce TGF-ß (21). Examples of these are hepatocytes, sinusoidal endothelium and Kupffer cells (22, 23). Our experimental protocol does not investigate the role of TGF-ß in the induction of oral tolerance but demonstrates that in the absence of TGF-ß signaling presentation of a specific oral antigen to CD8+ T cells resulted in minimal T cell proliferation in the liver, lymph nodes and spleen. Thus, the active suppression of T cells by TGF-ß is not the reason for the minimal proliferation of T cells to food antigens. The most likely reason is that presentation of food antigens by non-conventional APC such as endothelium and possibly Kupffer cells is inefficient at priming, and active suppression by TGF-ß plays a minimal role.
In addition to interactions with oral antigens, T cells are constantly interacting with self-MHCantigen complexes with low affinity for the TCR (24, 25). We used a very well-characterized altered peptide ligand of known low affinity for the OT TCR (G4) (13). The disassociation rate of G4/H2-Kb from the OT TCR is 3.6-fold faster than for high-affinity peptide/H2-Kb but slower than a series of OT TCR antagonistic ligands. In vitro presentation of G4 on H2-Kb by splenic APC has been shown to result in the activation of OT T cells (14). Approximately 1000-fold higher concentrations of G4 peptide, compared with high-affinity peptide, were required to obtain the same degree of activation of OT T cells. Interestingly, this was not due to TCR occupancy, as this was the same when tested by G4/H2-Kb and high-affinity peptide/H2-Kb tetramers. We also found that presentation of G4 by splenic APC results in the activation of OT and OT-DNR T cells in vitro, at concentrations which were a 100- to a 1000-fold higher than those required for high-affinity peptide (Fig. 4). The effect of high-dose G4 peptide in vivo was however very different, with no activation of OT and OT-DNR T cells based on up-regulation of CD44 and CD69. In addition, G4 peptide did not induce any detectable expansion of OT cells in vivo. Analogous to the conclusion for oral antigen presentation, this suggests that constitutive levels of TGF-ß have little or no role in limiting T cell responses to altered peptide ligands presented as soluble peptides.
Since G4 peptide is capable of activating OT and OT-DNR cells in vitro, lack of activation of OT and OT-DNR cells in vivo was unexpected. This difference may be due to a variety of reasons. It is unlikely that insufficient amount of G4 peptide was used in the in vivo protocol. Twenty five nanomoles of both high-affinity and G4 peptides was injected, and this would have initially reached millimolar concentrations in vivo. Peptide injections were repeated daily for the duration of the experiment. The half-life of peptide in serum is however very short, and it is possible that sustained concentrations of peptides may have been below the nanomolar range. Such low levels should be sufficient for activation by the high-affinity but not the G4 peptide. The other difference is that in vitro presentation occurred primarily by splenic APC. In contrast, in vivo peptide will be bound to H2-Kb on most cell types, and presentation in vivo may have occurred by a variety of non-professional APC.
To obtain more sustained concentrations of peptide, and to target the peptide more efficiently to professional APC, high-affinity and G4 peptides were injected subcutaneously in an emulsion with CFA. Similar to injection of G4 peptide alone, injection of G4 with CFA did not result in any up-regulation of activation markers on OT cells. This was true for time points of Days 2, 5, 7 and 10 after injection. Injection of G4 peptide with CFA however did result in up-regulation of CD44 but not CD69 on OT-DNR cells at Day 3 after injection. The OT-DNR cells activated by low-affinity peptide and CFA had a CD44 high and CD69, CD25 phenotype, which is identical to that of CD8+ T cells with age in TGF-ß1 KO and TGF-ßRII DNR mice (Fig. 6). The CD44 high and CD69, CD25 T cells are the best candidates for causing tissue injury in TGF-ß1 KO and TGF-ßRII DNR mice because tissue injury is known to be T cell dependent and they are the largest abnormal T cell population. In addition, CD44 high and CD69, CD25 cells spontaneously differentiate in vivo into effector cytokine-producing cells (4).
CFA is a mixture of paraffin oil, surfactant and heat-killed Mycobacterium tuberculosis (26). Intradermal injections of a peptide with CFA are known to significantly alter the presentation of peptides compared with i.p. injection of peptide alone. Firstly, the half-life of the peptide increases significantly, with greater localization to the draining lymph nodes and much less to the spleen (27). CFA also enhances dendritic cell (DC) maturation with increased expression of B7-1, B7-2 and CD40 ligand (CD40-L) and greater phagocytosis by DC (28, 29). A number of cytokines are also induced by CFA in the lymph nodes and spleen. These include tumor necrosis factor-
and particularly high amounts of TGF-ß1 (30). The up-regulation of TGF-ß1 mRNA by CFA was
100-fold and occurred in lymph nodes, spleen and PBMC. Our data demonstrate that the T cell responses to enhanced priming by CFA is in part controlled by TGF-ß. All the effects of CFA on antigen presentation discussed above are not sufficient to activate OT cells by G4 peptide. However, in the absence of TGF-ß signaling, as occurs for OT-DNR cells, there is significant T cell activation. The up-regulation of TGF-ß1 by CFA presumably provides a balance to the more efficient antigen presentation of DC by enhanced phagocytosis and maturation. Up-regulation of TGF-ß1 is not limited to immunization with CFA, but occurs during infection with Mycobacterium tuberculosis and leishmaniasis, and may function in these infections to limit the activation of T cells with low affinity for self-antigens (31, 32). This is clearly desirable to minimize autoimmunity. Inhibiting activation of T cells with a low affinity for antigens on infectious organisms is also desirable for a number of reasons. Such clones would most likely be anergised, not mount an effective protective response and would not be available to mount future immune responses. In addition, they may utilize local resources such as cytokines which are needed for maximum proliferation of the high-affinity T cell clones.
Injection of high-affinity peptide in CFA resulted in significantly greater expansion of OT-DNR cells compared with OT cells in the draining lymph nodes (Fig. 5G). This demonstrates that when priming of CD8+ T cells occurs by high-affinity peptide and adjuvant, TGF-ß has a significant role in controlling T cell expansion. A number of molecular interactions affect the clone size after T cell priming. Many of these may occur through co-stimulatory molecules and include the well-known ones such as B7.1 and B7.2 as well as B7H, CD40-L and 4-1BB ligand (33, 34). Soluble factors also have a significant effect on T cell proliferation (35). The overall effect of all the soluble factors in serum is to provide signals that are required for T cell proliferation. In the absence of serum there is up-regulation of early activation markers such as CD69 and CD25, but late activation markers such as HLA-DR, CD38 up-regulation, and clonal expansion does not occur (35). IL-2 and IL-12 are both required for maximal T cell proliferation and certainly contribute to the effect of serum. The serum levels of TGF-ß are in the range known to suppress T cell proliferation in vitro, but it could not be assumed that suppression occurred in vivo. In fact, in the absence of CFA we could not demonstrate a significant effect of TGF-ß on T cell proliferation. As mentioned above there is a dramatic increase in TGF-ß1 in lymph nodes in response to CFA alone, and our data comparing high-affinity peptide alone and high-affinity peptide with CFA demonstrate one functional consequence of this increase. The simultaneous up-regulation of TGF-ß and co-stimulatory molecules by CFA provides a regulatory mechanism to keep the size of T cell clones in check. Our studies do not identify the source of TGF-ß. In the various experimental designs the suppression may have been due to soluble TGF-ß secreted by dendritic cells, or it may have come from membrane-bound TGF-ß on CD4+ CD25+ regulatory T cells. Clarification of this important point requires cell-specific elimination of TGF-ß production.
In summary, we have shown that the absence of TGF-ß signaling to T cells has minimal consequences for CD8+ T cell activation in response to food antigens or high-affinity and low-affinity peptides alone. However, in the setting of a strong innate immune inflammatory response by CFA, lack of TGF-ß signaling to T cells results in CD8+ T cell activation but not proliferation in response to low-affinity peptide and a greater degree of proliferation in response to high-affinity peptide. These studies identify important regulatory functions of TGF-ß in vivo and suggest mechanisms responsible for the age-related increase in activated T cells in TGF-ß1 KO and TGF-ßRII DNR mice.
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Acknowledgements
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We would like to thank R. Budd for his helpful discussions. This work was supported by the American Diabetes Association Research Grant (R.A.F.) and the Yale Liver Center NIH award P30 DK34989. R.A.F. is an Investigator of the Howard Hughes Medical Institute. W.Z.M. is the recipient of NIH award KO8DK002965.
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Abbreviations
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APC | antigen-presenting cells |
CD40-L | CD40 ligand |
DC | dendritic cell |
DNR | dominant-negative receptor |
i.p. | intraperitoneal |
KO | knockout |
OVA | ovalbumin peptide |
SD | standard deviation |
TGF-ß | transforming growth factor-ß |
 |
Notes
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Transmitting editor: M. Feldmann
Received 26 July 2004,
accepted 4 February 2005.
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References
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