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* Division of Immunology, Beckman Research Institute, City of Hope, Duarte, CA 91010; and
Torrey Pines Institute for Molecular Studies, San Diego, CA 92121
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, they were biased toward being IL-10-producing cells. Additionally, <3% of these cells expressed TCR V
4. In vivo adoptive transfer experiments showed that NOD/scid recipient mice cotransferred with tetAg7/p79+ T cells and NOD spleen cells, like mice transferred with NOD spleen cells only, developed diabetes. Therefore, we have generated Ag-specific tetramers that could detect a heterogeneous population of T cells, and a very small number of NOD mouse T cells may represent BDC2.5-like cells. |
Introduction |
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The cells of the diabetogenic CD4+ T cell clone, BDC2.5, can rapidly induce insulitis and hyperglycemia in NOD or NOD/scid mice within 2 wk after birth. However, they cannot transfer the disease to NOD mice older than 3 wk of age or to adult NOD/scid mice (8, 9, 10). NOD mice expressing the BDC2.5 TCR transgene have been a very useful model for studying the role of these cells in the pathogenesis of IDDM, while T cells derived from BDC2.5 mice may behave differently from the original T cell clone (11, 12, 13). Although BDC2.5 TCR transgenic NOD mice (BDC2.5 mice) can develop an aggressive form of IDDM, only 
10–20% of BDC2.5 mice develop the disease (12). This may be due to the fact that regulatory T cells, such as those that express the surface marker DX5, are present in these animals, and they can prevent islet cells from being destroyed (14). However, activated T cells from these animals can transfer disease to NOD/scid recipients. Additionally, all NOD/scid mice that express the BDC2.5 TCR transgene are diabetic by 4 wk of age (15).
Previous studies have shown that the BDC2.5 T cells are specific for a islet granule membrane Ag presented by the NOD class II MHC I-Ag7 (16). However, the identity of this Ag remains unknown. While searching for the Ag recognized by BDC2.5 T cells using a positional scanning combinatorial peptide library, a series of peptide analogs were identified that stimulate T cells from BDC2.5 mice (17). Some of these peptides include sequences similar to the GAD528–539 peptide or the longer GAD526–541 peptide, suggesting that the GAD peptide is a candidate autoantigen for BDC2.5 T cells. After being activated by these synthetic peptide analogs, BDC2.5 transgenic T cells can adoptively transfer the disease to adult NOD/scid recipients. In addition to T cells from transgenic mice, T cells derived from NOD mice also respond spontaneously to these peptides in culture.
To better understand the development of BDC2.5 T cells in the animals and their role in the pathogenesis leading to IDDM, we have generated Ag-specific I-Ag7 tetramers to detect, isolate, and characterize BDC2.5 T cells or T cells from NOD mice that were stimulated by the peptides. In a previous report we described the generation of an I-Ag7 tetramer specific for the GAD524–543 peptide (which includes both the 528–539 and 526–541 epitopes that can weakly stimulate BDC2.5 T cells) (18). However, the tetramer stained GAD524–543 peptide-specific hybridoma cells weakly and failed to detect a significant number of T cells in NOD spleens and islets (18). This may be due to the fact that the autoreactive T cells specific for GAD epitopes are present at a low frequency in NOD mice and that their TCRs have low affinities for their ligands. Therefore, the binding affinity of the tetAg7/GAD524–543 tetramer for its cognate TCRs could be too low for the T cells to be detected. In this study we have generated new I-Ag7 tetramers specific for the previously identified peptide analogs that could stimulate BDC2.5 T cells (17). Among these peptides, the 1040-79 peptide (p79), one of the most active peptides identified in the previous report, stimulates BDC2.5 T cells with an EC50 of 0.5–0.7 nM. Because the p79 peptide can stimulate BDC2.5 T cells at a very low concentration, we hypothesize that the tetramer specific for the p79 peptide, tetAg7/p79, is a better reagent to detect T cells that share the same Ag specificity as that of BDC2.5 T cells in NOD mice. We also generated an additional tetramer specific for another peptide, 1136-17 (p17), which, like p79, stimulates BDC2.5 T cells at low concentrations. We report here the use of these tetramers to stain and characterize T cells from BDC2.5 and NOD mice.
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Materials and Methods |
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NOD and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Biozzi/ABH (ABH) mice, which also express the class II MHC I-Ag7, were purchased from Harlan Co. (Indianapolis, IN). BDC2.5 transgenic mice were a gift from Drs. D. Mathis and C. Benoit (11). Less than 10% of mice in the NOD background and 100% of mice in the NOD/scid background in our colony develop diabetes by 6 mo and 4 wk of age, respectively. All the animals used were housed in a specific pathogen-free environment in the animal facility at Beckman Research Institute, City of Hope.
Peptides and production of class II MHC tetramers
The p79 and p17 peptides were synthesized at Beckman Research Institute, City of Hope, and purified to at least 90% using reverse phase HPLC. The p79 peptide has been described as 1040-79 (17). A detailed description of the p17 peptide will be reported by Judkowski et al. (unpublished observations). The method of class II MHC tetramer production has been described previously (18, 19, 20).
Isolation of pancreatic islet-infiltrating cells
Pancreases from BDC2.5 or NOD mice were perfused with collagenase P (Roche, Indianapolis, IN), removed from the animal, and incubated for 16–18 min in a water bath at 37°C. Pancreatic tissues were washed several times with HBSS plus 10% FCS, then filtered through a strainer, and islets were isolated after Histopaque density-gradient purification (Sigma-Aldrich, St. Louis, MO). Islets were dissociated into single cells by trypsin/EDTA treatment. The islet cell suspension was then either directly stained with tetramers and Abs or cultured with peptides and irradiated APC for 7 days before staining.
Flow cytometry and confocal microscopy
Staining of cells using I-Ag7 tetramers has been described previously (18). Briefly, islet or spleen cells were stained with PE-labeled tetramers plus unconjugated H57 Ab at 37°C for 3 h. Anti-CD4 Abs (BD PharMingen, San Diego, CA) were added during the last 30 min of incubation. Cells were washed and either analyzed by FACS using FACSCalibur (BD Biosciences, San Jose, CA), or fixed with 4% paraformaldehyde overnight for confocal microscopic analysis. All other Abs and annexin V-FITC were purchased from BD PharMingen, except for F4/80 Ab which was a gift from Dr. S. Kovats (Duarte, CA).
Tetramer binding kinetics
The association and dissociation kinetics measured using the tetramers were determined using a previously described method (21), with minor modifications. For analysis of tetramer staining at equilibrium, BDC2.5 spleen cells were stained for 1 h at room temperature with increasing concentrations of tetAg7/p79 (0–35 nM) and with anti-CD4 Ab (BD PharMingen) in RPMI medium. Apparent Kd values were derived from the negative reciprocal of the slope of the regression line fit to Scatchard plots of bound tetramer/free tetramer (fluorescence units per nanomolar concentration of tetramer) vs bound tetramer (fluorescence units). The median tetramer staining intensity was used as the measurement of bound tetramer.
To measure the half-life of tetramer binding to cells, cells were stained for 1 h at room temperature with a suboptimal concentration of the tetAg7/p79 tetramer plus anti-CD4 Ab in RPMI medium. The blocking anti-I-Ag7 Ab was used at 30 µg/ml (American Type Culture Collection, Manassas, VA). Aliquots were taken at appropriate time points and analyzed by flow cytometry. The binding half-life (t1/2) is equal to ln (2) divided by the slope value of the natural logarithm (ln) of the normalized fluorescence plotted vs time. The normalized fluorescence was determined by calculating the percentage of total fluorescence (sum of fluorescence intensity of tetramer+ CD4+ T cells normalized per CD4+ cell at each data point) with respect to the initial time point.
Cytokine assay
The IL-2 production bioassay has been previously described (18). For ELISA, cell culture supernatant was harvested after incubating cells with peptides or tetAg7/p79 and irradiated APC for 24 h. Mouse IFN-, IL-4, and IL-10 OptEIA ELISA assay kit sets (BD PharMingen) were used to measure the amount of cytokines according to the manufacturer’s instruction.
CFSE labeling and in vitro T cell stimulation
CD4+ T cells from NOD mouse spleens were purified using magnetic beads (Miltenyi Biotec, Auburn, CA) and labeled with CFSE as previously described (22, 23). Briefly, CD4+ T cells were resuspended at a concentration of 10 x 106 cells/ml in serum-free HBSS or PBS and incubated with CFSE (0.8 µM, final concentration) for 10 min a 37°C. CFSE labeling was stopped by adding an equal volume of heat-inactivated FCS. After washes, the cells were cultured with peptides (50 µg/ml) and irradiated NOD mouse CD4+ T cell-depleted APC in RPMI medium containing 10% FCS for 3 days. Live cells were further incubated and maintained in medium supplemented with IL-2. After 10–13 days in culture, cells were stained with tetAg7/p79 and anti-CD4 Ab. Following activation with peptides and expansion in vitro, CD4+, tetramer+ T cells were isolated using FACS and magnetic beads (Miltenyi Biotec).
Intracellular cytokine staining
Intracellular cytokine staining was performed according to a previously described method (24). Briefly, T cells were incubated with PMA plus ionomycin and monensin, stained with surface Abs, fixed with paraformaldehyde, and resuspended in 0.1% saponin buffer (w/v; Sigma-Aldrich, St. Louis, MO). The cells were then intracellularly stained using Abs against cytokines or negative isotype control Abs.
Adoptive transfer of T cell into NOD/scid mice
Four- to 5-wk-old NOD/scid mice received a single i.v. injection of tetramer+ T cells, tetramer+ T cells plus NOD splenocytes, or NOD splenocytes alone. Recipient mice were monitored up to 26 wk of age and were considered diabetic after 2 consecutive wk of glycosurea >2% and blood glucose level >250 mg/dl.
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Results |
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-chain is covalently linked to the previously identified p79 peptide (17). This tetramer was first used to stain T cells from islets and spleens of BDC2.5 mice. Initial studies showed that this tetramer could detect a majority of CD4+ T cells in spleens (82.5%) and pancreatic islets (90.5%) of the BDC2.5 mice (Fig. 1A). The tetramer was specific for BDC2.5 T cells because it did not detect a population of T cells in the spleens of control ABH mice (Fig. 1A). ABH mice also express the same class II MHC I-Ag7 as that expressed in NOD mice, but they do not develop diabetes (25). The BDC2.5 TCR bears a V4 fragment in its -chain (26). The percentage of CD4+, tetramer+ T cells correlates with that of CD4+ T cells bearing TCR V4, although some V4+ T cells (15%) were not stained by the tetramer. This is consistent with previous fndings that some V4+ T cells bear a different TCR 
-chain (11). The tetramer could also detect essentially all CD4+ T cells from NOD/scid mice expressing the BDC2.5 TCR transgene (data not shown). Additionally, the tetramer did not stain NOD mouse T cells specifically for the pGAD206 (p206) peptide isolated using the tetAg7/p206 tetramer (data not shown) (18). In comparison, the tetAg7/p206 tetramer did not stain T cells derived from the BDC2.5 TCR transgenic mice (Fig. 1B). TetAg7/p206 is considered as a good control tetramer because it contains an irrelevant GAD peptide, p206, whose amino acid sequence is not related to that of p79. The advantages of having p206 as an irrelevant peptide in the tetramer are that it is a mouse self-peptide, but its amino acid sequences are different from those of p79. Furthermore, p206 does not stimulate BDC2.5 T cells, nor does it stimulate p79-specific NOD mouse T cells (data not shown, also refer to Fig. 9). Besides the GAD p206 peptide, we have also performed additional control experiments using another tetramer, tetAg7/pcOVA, to stain T cells from BDC2.5 TCR transgenic mice. This control tetAg7/pcOVA tetramer contained a non-self peptide, cOVA323–339, as an irrelevant peptide and it also did not stain BDC2.5 T cells (see Fig. 5B). Therefore, it appears that tetAg7/p79 tetramer is a good staining reagent to detect essentially all BDC2.5 T cells from transgenic mice. In addition to using FACS to analyze T cells stained with the tetramer, we also used a confocal microscope to observe CD4+, tetramer+ T cells from spleens and islets (Fig. 2, A and B). Similar to the FACS analyses, the majority of T cells in the spleens and islets of BDC2.5 mice were positively stained by both FITC-conjugated anti-CD4 Ab and PE-labeled tetAg7/p79 tetramer and appeared as yellow colored cells (Fig. 2A). In comparison, the green CD4+, BDC2.5 T cells were not stained by the tetAg7/p206 tetramer (Fig. 2B).
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72 and 68% of the CD4+ T cells from the islets and spleens, respectively, of 4-wk-old BDC2.5 mice (Table I). The percentage of CD4+, tetAg7/p79+ T cells increases with age and can reach an average of 93% in the islets and 82% in the spleen of 9-wk-old mice. These results suggest that while other T cells are present in islets and spleens of BDC2.5 mice, BDC2.5 T cells eventually accumulate or expand in both organs as the mice age.
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Ab, H57 (32). The results showed that there was no significant difference in the TCR expression level found on T cells from 4- or 9-wk-old mice (data not shown).
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and, to a lesser extent, IL-4, and IL-10. However, it seems that the tetramer stimulated the cells to produce lower amounts of the cytokines, especially IL-4 and IL-10, than in cells stimulated with the p79 peptide. The difference may be due to the presence of a lower concentration of the antigenic peptide in the recombinant protein compared with that of the synthetic peptide used in the assays. To determine whether this is the case, we compared the IL-2 production response to equal molarities of the synthetic peptide and the recombinant peptide present in the tetramer used in the assays. The results shown in the bar chart in Fig. 4 demonstrated that after being adjusted to the concentration of the peptide rather than the amount of the protein used in the assays, the tetramer could actually induce a stronger (2- to 5-fold) IL-2 production response than did synthetic peptide (Fig. 4). Therefore, it seems that the tetramer is a more active reagent than the synthetic peptide in stimulating BDC2.5 T cells.
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0.2%; data not shown; also refer to Figs. 1A and 5B). Although 0.27% of BALB/c mouse cells were stained by tetAg7/p79, they did not appear as a distinct population compared with that in NOD spleen cells. Additionally, 0.2% of ABH mouse CD4+ cells were stained by tetAg7/p79. Therefore, compared with control cells, 0.2–0.3 and 0.4–0.5% of NOD mouse spleen and islet CD4+ T cells, respectively, were stained by tetAg7/p79. In addition to control cells, we performed experiments using control tetramers containing irrelevant peptides. As a control tetramer, consistent with our previous findings (18), the tetAg7/p206 tetramer did not detect a significant population of NOD mouse T cells in islets and spleens (Fig. 5A). The reason for this is not clear, but it could be that the TCR affinity of p206-specific T cells for their ligands or the frequency of such cells in NOD mice was too low to be detected by tetAg7/p206. We have performed additional control experiments using the tetAg7/pcOVA tetramer to stain NOD mouse T cells to further confirm that the tetAg7/p79+ cell population detected in NOD mice was a small, but real, population. The results shown in Fig. 5B demonstrated that the percentage of NOD mouse CD4+ T cells stained by the tetAg7/pcOVA control tetramer (0.21%) was at least 2-fold less than the percentage of T cells stained by the tetAg7/p79 tetramer (0.52%).
It is conceivable that autoreactive T cells could have been activated previously by self-Ags and became larger blast cells, which could be distinguished from other naive T cells. To determine whether this is true for T cells detected by the tetramer, we reanalyzed the results by electronically gating on the cells of a larger size according to their forward and side scatter profiles. Interestingly, the percentage of CD4+ T cells positively stained by the tetramer increased 2- to 3 fold to 1.9 and 1.8% in islets and spleens, respectively (Fig. 5A, lower panel). Furthermore, the frequency or number of tetramer+ cells did not increase in older animals or diabetic animals (data not shown). Additionally, we have performed further control experiments to determine whether the CD4+ T cells detected by tetAg7/p79 were not T cells but were macrophages, dendritic cells, or apoptotic cells that may be nonspecifically stained by the tetramer. The results showed that tetAg7/p79 did not detect a population of CD4+ T cells that were positively stained by annexin V, which stains apoptotic cells; by anti-CD11C Ab, which stains dendritic cells; or by F4/80, an Ab which stains macrophages (Fig. 5C) (33). Together, these results suggest that tetAg7/p79 can detect a small population of T cells in NOD mice, and that the tetramer+ T cells are activated in vivo, perhaps by endogenous peptides homologous to p79.
To further confirm the results obtained from FACS analyses, we used a confocal microscope to determine whether T cells from NOD mice were stained by the tetAg7/p79 tetramer. The results obtained from confocal microscope analyses were consistent with those of FACS analyses (Fig. 2, C and D). The estimated percentage of tetramer+ T cells stained by tetAg7/p79 was comparable to that obtained using FACS, as shown in Fig. 5.
We then wanted to know whether the p79 peptide was able to stimulate and expand splenic T cells from NOD mice, and whether the stimulated and expanded T cell population could be detected more easily using the tetramer. First, we labeled purified splenic CD4+ T cells with CFSE and then incubated the cells with the p79 peptide plus irradiated CD4-depleted APC. Ten days later, we stained the cells with anti-CD4 Ab plus the tetramer and analyzed the cells using FACS. Interestingly, some of the cells underwent at least six rounds of cell division after the first 10-day culture period (Fig. 6, A and B). The results show that the percentage of T cells detected by the tetramer increased in cells that went through more rounds of cell division (Fig. 6, C and D). The percentage of tetramer+ T cells increased almost 20-fold, from 0.19% in cells that did not undergo division to 3.32% in cells that divided more than six times (Fig. 6D). As a control we also stained p79-stimulated cells with tetAg7/p206 tetramer, and the results showed that even cells that underwent more rounds of cell division were not stained by this tetramer (Fig. 6, C and D). It is not clear why tetAg7/p79 did not stain all dividing cells. It could be that other T cells proliferated in response to non-p79 peptides because the purity of the p79 peptide we used in the studies was 90%, and that the culture medium contained FBS. Some T cells may respond to the 10% non-p79 peptides or FBS-derived peptides and further divide in cell culture containing IL-2. Autoreactive T cells specific for other autoantigens may also proliferate in the presence of APC and IL-2. Additionally, it is likely that many p79-stimulated T cells may bear low affinity TCRs and are not stained by tetAg7/p79. However, following initial stimulation, further culture of the cells with additional antigenic stimulation could result in more significant expansion of tetramer+ T cells and increase the total percentage of the cells to at least 20% (data not shown). In our previous report we showed that Ag-specific NOD mouse T cells express higher levels of CD4 after they are activated by their Ags (18). Consistent with those studies, the activated tetAg7/p79 tetramer+ T cells also expressed higher levels of CD4 on their surface. Therefore, the tetAg7/p79 tetramer+ T cells responded to the p79 peptide and were enriched in the more divided cell population.
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in response to the p79 peptide (Fig. 9). However, unlike BDC2.5 T cells, tetramer+ T cells also secreted a large amount of IL-10 and, to a lesser extent, IL-4 (Fig. 9). To study in more detail the cells secreting IL-4, IL-10, and IFN-, we performed intracellular cytokine staining using Abs against IL-4, IL-10, or IFN- (Fig. 10). The results showed that 17–18% of the tetramer+ cells produced IL-10, 4–5% of the cells produced IFN-, and 1% of the cells produced both cytokines (Fig. 10B). In addition, although the cells secreted some IL-4, essentially no cell that produced IL-4 was detected using this method compared with the isotype control staining. Therefore, the tetramer+ cells seem to be biased toward being IL-10-producing cells, and a smaller population produced IFN-, while very few, if any, cells produced IL-4. Furthermore, we also analyzed the TCR V usage of tetramer+ T cells, and the results showed that tetramer+ T cells contained a heterogeneous population of T cells that express different TCRs. There were <3% of the cells that bore TCR V4, whereas 30% of the cells expressed TCR V7 (data not shown). Therefore, the population of splenic T cells that responded to the p79 peptide in vitro is not biased toward the BDC2.5-like, TCR V4+ T cells that secreted a large amount of IFN- and much less IL-4 and IL-10. The results suggest that although the tetAg7/p79 tetramer can detect BDC2.5 T cells, only a small portion of the p79 peptide-stimulated T cells from NOD mice represent BDC2.5 T cells. To determine the role of isolated tetAg7/p79+ T cells in diabetes, we adoptively transferred the cells into NOD/scid mice with or without NOD spleen cells (Fig. 11). The results showed that mice receiving only tetramer+ T cells did not develop diabetes. However, all mice that received both tetramer+ T cells and NOD spleen cells (cotransfer), like mice receiving only NOD spleen cells (single transfer), developed diabetes by the age of 25 wk. Although cotransferred mice showed slightly delayed (by 2 wk) onset and relatively synchronized development of diabetes compared with single-transferred mice, the difference in disease development between single-transferred and cotransferred mice was not statistically significant. Therefore, although the isolated tetAg7/p79+ T cells secreted some IL-10, they did not significantly inhibit diabetes development in recipient mice.
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Discussion |
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68% of CD4+ splenic T cells were detected by the tetramer. In addition, the percentage of CD4+, tetAg7/p79+ T cells increases with the age of the mice, suggesting that these cells gradually expand and accumulate in both spleens and islets. However, diabetes occurs spontaneously in <10% of the BDC2.5 mice in our colony, as reported by others (12). The BDC2.5 mouse-derived T cells were functional, because after stimulation with the p79 peptide or the tetAg7/p79 tetramer, the BDC2.5 T cells produced cytokines such as IL-2 and IFN- (Fig. 4). Therefore, the low incidence of diabetes shown in previous reports could not be due to the fact that BDC2.5 T cells were anergic to stimulation, because they respond to the p79 peptide and the tetAg7/p79 tetramer. This could be due to a recently identified CD4+, DX5+ T cell that can regulate the development of diabetes in BDC2.5 mice by protecting islet cells from damage (14).
Our results also have shown that the avidity of BDC2.5 TCR for the tetramer slightly increases with age. The reason for the increase is not clear. Because the BDC2.5 TCR is expressed as a rearranged TCR transgene in these mice, its binding affinity for its ligand should not change with age. Therefore, the increase in the avidity of BDC2.5 TCRs for their ligands may not be due to the avidity maturation of autoreactive T cells proposed previously for studies of CD8+ T cells in NOD mice (35). An explanation for the increased avidity is that it is due to the increased BDC2.5 TCR levels in T cells in older animals. Because the avidity of TCR represents the sum of affinities of individual TCR for their ligands, an increased TCR expression level on cells would therefore increase both the number of binding sites and their avidity for the ligands. An increased tetramer staining intensity on T cells correlates with an increase in the total affinity or avidity of TCR for their ligands (20). However, staining of T cells with the anti-TCR C Ab, H57, or the tetramer showed that total TCR or the BDC2.5 TCR expression level on T cells did not change significantly when mice became older (data not shown). Another possible explanation may be that the BDC2.5 T cells in transgenic mice may bear different levels of a second TCR. It has been shown that >25% of CD4+ T cells in BDC2.5 mice may express an endogenous TCR -chain (11). Consistent with the previous studies, the tetAg7/p79 tetramer staining results showed that not all CD4+ T cells bearing the TCR V4 gene fragment were detected by the tetramer. Therefore, it is likely that more T cells of younger mice than older mice bear a second TCR other than the BDC2.5 TCR by expressing different TCR -chains. In older animals, T cells bearing more or only the BDC2.5 TCR may expand faster and eventually predominate in the periphery, especially in the islets. This may be due to encounter of BDC2.5 T cells with self-Ags in the older animals. Alternatively, rearrangement and assembly of a second TCR -chain gene may gradually shut down in older BDC2.5 animals.
It has been suggested that autoreactive T cells bear TCRs with low avidity for their ligands (27, 28). It is interesting that the avidity of BDC TCR for the tetAg7/p79 ligand was relatively higher than that of some of the CD4+ T cell TCRs studied previously (20, 29, 30, 31). This may be because p79 is not the natural agonist peptide, but a superagonist peptide for BDC TCR, which results in a stronger binding affinity for the TCR. It is likely that the avidity of BDC TCR for its unknown natural ligand would be lower than its avidity for tetAg7/p79. It could also be that we used a tetramer to determine the avidity of TCR, whereas the previous studies used MHC/peptide monomers. The tetrameric ligand would have a much stronger binding affinity for the TCR than did the monomeric ligand. In addition, our avidity measurement studies showed that the avidity of TCR expressed on the isolated tetAg7/p79+ T cells was about one-half that of BDC2.5 TCR. The reason for this is currently unknown. One possibility could be that the superagonist p79 peptide induced apoptosis of T cells that bear TCRs with higher avidity for I-Ag7/p79 during in vitro expansion. This would result in the expansion of T cells bearing TCR of lower affinity.
NOD splenic T cells responded spontaneously to the p79 peptide; however, the response was not very strong. This suggests that the frequency of splenic T cells specific for the peptide was not high, as shown in the FACS and confocal microscopy studies. These studies show that, compared with the controls, the tetAg7/p79 tetramer stained a small, but real, population of CD4+ T cells present in both spleens (0.2–0.3%) and pancreatic islets (0.4–0.5%) of NOD mice. The percentage of tetramer+ T cells did not increase in older or diabetic NOD mice. Although we were very careful in handling and staining cells, the cells that were positively stained by the tetramer may still include a small number of macrophages, dendritic cells, and apoptotic cells that may be nonspecifically stained by the tetramer. If that is the case, then the percentage of tetramer+ cells may be even lower than those shown in Fig. 5. In addition, as shown in Fig. 5C, tetAg7/p79 did not stain annexin V+ cells, CD11C+ cells, and F4/80+ cells. These results indicate that the cells detected by tetAg7/p79 were not apoptotic cells, dendritic cells, or macrophages. In addition, the very small percentage of cells stained by the tetramer probably represents a real population of live cells, because we could further expand and isolate such cells in vitro. It is conceivable that the tetAg7/p79 tetramer may detect more than one T cell population in NOD mice that include BDC2.5 T cells and other T cells that could also respond to p79
Indeed, our studies on characterizing the p79 peptide-stimulated T cells that were isolated with the tetAg7/p79 tetramer are consistent with this view. Studies of the isolated tetramer+ T cells showed that although the T cells secreted a large quantity of IFN- in response to the p79 peptide, they also secreted a larger amount of IL-10 and, to a lesser extent, IL-4 than did BDC2.5 T cells. Further intracellular cytokine staining studies showed that these cells were biased toward being IL-10-producing cells rather than IFN-- or IL-4-producing cells. Additionally, <3% of the cells expressed TCR V4. Therefore, considering the small percentage of CD4+ T cells detected by the tetAg7/p79 tetramer, it seems that BDC2.5 T cells are present at very low numbers in NOD mice. The BDC2.5 T cells are probably present in NOD mice at a frequency even lower than 0.5% of the total CD4+ T cells detected by the tetAg7/p79 tetramer in spleens or islets, as shown in Fig. 5. Alternatively, because the cells were cultured in vitro before analyses, it is likely that BDC2.5 T cells proliferate more slowly than other cells (such as those that bear TCR V7) and are eventually present as a minor population in tetramer+ T cells. Interestingly, the isolated p79-responsive T cells did not respond to other BDC2.5 T cell-stimulating peptides, such as p17 and pGAD526 peptides, although tetAg7/p79 tetramer could detect T cells stimulated by the p17 peptide. This suggests that the tetramer may be able to detect a more heterogeneous T cell population than T cells stimulated in vitro by the peptide linked to the tetramer. Therefore, the p17 and other peptides, such as the p79 peptide, may also stimulate a unique population of T cells derived from NOD mice. Based on these results, it seems that T cells specific for more than one self-Ag may play critical roles in the pathogenesis of autoimmune diabetes. It is likely that these diabetogenic T cells, like BDC2.5 T cells, may also be present in NOD mice at a very low frequency. Alternatively, it is likely that T cells specific for different self-antigenic peptides may infiltrate the islets in NOD mice that are at different stages of the disease.
Although the isolated tetAg7/p79+ T cells secreted a large quantity of IL-10, the tetramer+ T cells did not inhibit the development of diabetes when they were cotransferred with NOD mouse spleen cells into NOD/scid mice. The in vivo adoptive transfer experiments showed that both cotransferred (receiving both tetramer+ cells and NOD splenocytes) and single-transferred (receiving NOD splenocytes alone) NOD/scid mice developed diabetes by the age of 25 wk. The difference of diabetes development between cotransferred animals and single-transferred animals was not statistically significant. It is not clear why the tetAg7/p79+ T cells did not enhance diabetes development in recipient animals. Several possibilities may explain these results. One possibility is that the number of BDC-like T cells present in the tetAg7/p79+ population was too small to induce a faster development of diabetes. These BDC-like cells may also expand in vivo at a rate slower than the other cotransferred T cells, or they may undergo apoptosis and eventually constitute an even smaller portion of the cells in the recipient mice. In addition, the slightly delayed diabetes onset in cotransferred animals suggests that T cells specific for other self-Ags may play a role during early diabetes development. It is also possible that IL-10 produced by some tetAg7/p79+ cells resulted in the delay.
Both p79 and p17 peptides could stimulate BDC2.5 T cells, but p79 was identified as a peptide analog using a positional scanning combinatorial peptide library. Interestingly, p17 could stimulate a smaller population of NOD splenic T cells than that stimulated by p79. Additionally, p17-stimulated T cells proliferated more slowly than those cells that responded to p79. However, T cells from NOD mice can be stimulated spontaneously by the p17 peptide in vitro, suggesting that a peptide(s) with a sequence similar to or identical with that of the p17 epitope is present in these mice. Therefore, the p17 peptide or its homologues in NOD mice may be involved in diabetes. Future studies on T cells specific for p17 and other peptides, such as the GAD peptides, that can stimulate BDC2.5 T cells should provide more helpful information on the role of these cells in the pathogenesis of diabetes and show whether they are related to BDC2.5 T cells.
In summary, although it has been relatively easy to detect different populations of Ag-specific CD8+ T cells using class I MHC tetramers, it has been difficult to detect and isolate autoreactive CD4+ T cells. A major contribution of these studies is that we were able to generate class II MHC tetramers to detect and isolate a heterogeneous population of NOD mouse T cells specific for peptides that stimulate diabetogenic BDC2.5 T cells. The knowledge obtained from these studies and the use of similar reagents and approaches should facilitate further studies to determine the roles of T cells and different autoantigens in the development of autoimmune diabetes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chih-Pin Liu, Division of Immunology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3000. E-mail address: cliu{at}coh.org
3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; GAD, glutamic acid decarboxylase; NOD, nonobese diabetes.
Received for publication March 13, 2002. Accepted for publication February 6, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell apoptosis in T cell mediated autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94:213.-granule antigen. Diabetes 43:197.[Abstract]
, IL-4 and IL-10 in CD3+4+ T-cells from rat spleen. J. Immunol. Methods 244:29.[Medline]
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