DCs Expand CD25+ CD4+ T Cells from Autoimmune NOD Mice.
We tested the ability of autoantigen-specific CD25+
T cells to expand in response to DCs. We chose an autoreactive T cell that responds to a natural autoantigen and is diabetogenic. CD4+
T cells from BDC2.5 TCR transgenic NOD mice respond to a protein expressed by islet β cells (33
). Although the β cell autoantigen remains to be identified, a series of mimetope peptides have been uncovered, which also stimulate proliferation of BDC2.5 T cells (38
). We used one of these mimetope peptides as antigen, which will be referred to as BDC peptide. This particular mimetope peptide has a high functional affinity (low EC50
) and also is recognized by a small fraction of T cells from nontransgenic NOD mice (38
We recently found that CD25+
cells from nonautoimmune mice will grow in response to antigen-bearing DCs, particularly mature CD86+
bone marrow–derived DCs (26
). Therefore, we prepared >95% pure CD25+
BDC2.5 T cells and NOD bone marrow DCs (from male normoglycemic mice). In agreement with previous data on the DCs from NOD mice (35
), there was a lower (approximately twofold) frequency of CD86 high DCs relative to other strains like C57Bl/6. However, we used magnetic beads to enrich the smaller subset of CD86+
NOD DCs. These expressed high levels of CD86, comparable to other strains ( A and not depicted).
When we cultured BDC2.5 CD25+
T cells with NOD CD86+
DCs pulsed with BDC peptide, the T cells proliferated by day 3 ( B). Proliferation also took place in response to CD86−
DCs pulsed with BDC peptide, but it was more limited (not depicted). CD25−
cells likewise proliferated to DCs with BDC peptide, but the addition of IL-2 did not significantly change proliferative responses. CD25+
T cells cultured with DCs and IL-2 (but not with BDC peptide) also showed significant proliferation, as was evident with ovalbumin-specific CD25+
T cells (26
), but the combination of IL-2 and BDC peptide with DCs was more effective, resulting in higher [3
H]thymidine incorporation than with CD25−
cells. Previously, groups have observed some proliferation of CD25+
T cells cultured with spleen APCs, a TCR stimulus, and IL-2 (24
). We found that these conditions (BDC2.5 T cells, spleen APCs, BDC peptide, and IL-2) induced at least 3.5-fold lower proliferation than with DCs under the same conditions ( B, right). Using the condition in B, which gave the highest level of proliferation, we tested the ability of the cells to expand during a 1-wk culture. Relative to the number of cells placed into culture, there was a 5–10-fold expansion in the number of recovered T cells from cultures of CD25+
T cells, DCs, and BDC peptide with and without IL-2 at 5 d. CD25+
T cells expanded similarly up to day 5, but only the latter continued to expand up to day 7 ( C). Thus, CD25+
T cells from BDC2.5 transgenic mice can grow in response to DCs in an antigen-specific manner, in much the same way as recently reported for ovalbumin-specific T cells (26
To show that nontransgenic regulatory T cells from autoimmune NOD mice were capable of proliferation and expansion with DCs, we sorted NOD CD25+ CD4+ T cells and stimulated them with NOD CD86+ DCs and anti-CD3. With this polyclonal stimulus, DCs were able to induce DNA synthesis and expansion of NOD CD25+ CD4+ T cells (). The T cells also proliferated when cultured with DCs and IL-2 in the absence of a TCR stimulus, but IL-2, DCs, and anti-CD3 synergized to induce very high levels of DNA synthesis and expansion of cell numbers, >10-fold by 5 d. In contrast, NOD CD25+ CD4+ T cells cultured without DCs but with IL-2 with or without anti-CD3, gave only 2 × 103 or 7 × 103 cpm of DNA synthesis, respectively. Control NOD CD25− CD4+ T cells given DCs and anti-CD3 with or without IL-2, showed both proliferation and expansion. These results indicate that both DCs and T cells (either BDC2.5 or nontransgenic) from autoimmune NOD mice can interact to significantly expand CD25+ CD4+ regulatory T cells.
Phenotype of DC-expanded CD25+ CD4+ T Cells.
To identify BDC2.5-specific T cells after DC-mediated expansion, cultures were stained with an mAb specific for the BDC2.5 TCR. Approximately 80% of freshly isolated or DC plus IL-2–expanded BDC2.5 CD25+ CD4+ T cells expressed high levels of this clonotype (, left). In contrast, when BDC2.5 CD25+ CD4+ T cells were stimulated with DCs, IL-2, and anti-CD3, the level of clonotype expression was much lower than on cells expanded with DCs presenting BDC peptide. As expected, DC plus anti-CD3–stimulated NOD CD25+ CD4+ T cells did not express significant levels of BDC clonotype compared with isotype controls (, left). Because T cells expressing a transgenic TCR also can express endogenous TCR-α chains, we checked expression of two different endogenous TCR alphas (Vα2 and Vα8.3), and found similar percentages of cells expressing endogenous V alphas before and after DC-BDC peptide stimulation (, middle, and not depicted). Interestingly, the level of Vα2 expressed on CD25+ CD4+ T cells expanded with CD3 was lower than freshly isolated cells or those expanded with BDC peptide. This suggests that anti-CD3 causes a down-regulation of TCR expression that is not specific to the BDC2.5 TCR. For comparison, clonotype and Vα2 expression on CD25− CD4+ T cells expanded under the same conditions is shown, and down-regulation of either clonotype or Vα2 expression is not as severe as with CD25+ CD4+ T cells. This data indicates that in contrast to T cells expanded with DCs plus anti-CD3, BDC2.5 T cells expanded with DCs plus BDC peptide express much higher levels of TCR on their cell surfaces.
To characterize the DC-expanded regulatory cells, these cells were stained with antibodies specific for CD25, CD62L, and GITR. The cultured CD25+
T cells maintained high levels of CD25 and GITR, and many of the cultured CD25−
T cells up-regulated both CD25 and GITR (not depicted). The freshly isolated BDC2.5 CD25+
T cells contained ~40% CD62L low cells, and DC expansion did not significantly change this phenotype (, right). In contrast, the freshly isolated CD25−
T cells contained only ~10% CD62L low cells, and activation with DCs greatly increased the percentage of the CD62L low cells (, right). IL-2 was added to all cultured CD25+
T cells in addition to the indicated culture conditions shown in , but similar expression of clonotype, Vα2, and CD62L was observed for CD25+
T cells cultured without IL-2 (not depicted). Collectively, expression of these activation markers before and after DC stimulation is similar to that found in nonautoimmune strains (26
), indicating that DC activation of NOD regulatory cells occurs normally.
Expansion of CD25+ CD4+ T Cells with DCs In Vivo.
To determine if DCs also can induce proliferation of CD25+ CD4+ T cells in vivo, we purified CD25+ CD4+ T cells from BDC2.5 mice, labeled them with CFSE before injection into NOD mice, and 1 d later, we s.c. injected mature marrow–derived DCs that had been pulsed (or not pulsed as control) with BDC peptide. We assessed proliferation 3 d later by progressive halving of the amount of CFSE per T cell. The CD25+ CD4+ T cells proliferated, with up to six divisions per cell, in the draining lymph nodes of mice that received BDC peptide–pulsed DCs, but not in mice that received PBS or DCs alone ( and not depicted). We observed similar proliferative responses with control CD25− CD4+ cells, but CFSE was not diluted in either CD25+ or CD25− CD4+ cells in the distal lymph nodes of mice receiving either pulsed or unpulsed DCs (). Therefore, DCs are able to induce the proliferation of CD25+ CD4+ T cells from an autoimmune strain in vivo.
Figure 3. BDC2.5 CD25+ CD4+ T cells proliferate in vivo. CFSE-labeled BDC2.5 CD25− CD4+ (left) or CD25+ CD4+ (right) T cells were injected into NOD mice. 1 d later, either DCs without antigen (top) or BDC peptide–pulsed DCs (middle and bottom) were (more ...)
Enhanced In Vitro Suppressive Function of DC-expanded, CD25+ CD4+ T Cells.
To verify that the expanded CD25+ CD4+ T cells from BDC2.5 mice retained suppressive function, we used a standard in vitro suppression assay. We removed CD11c+ DCs from 7-d expansion cultures and added the T cells in different ratios to responder CD25− CD4+ T cells to measure the inhibition of CD25− CD4+ proliferation in response to BDC peptide presented by spleen APCs. Freshly isolated CD25+ CD4+ T cells, as well as CD25+ CD4+ T cells expanded with DCs and IL-2, were able to suppress, but only partially and at high doses, i.e., when mixed at a 1:2 ratio with CD25− CD4+ cells. In contrast, CD25+ CD4+ T cells expanded with BDC peptide (without or with IL-2) had stronger activity, showing suppression even at a ratio of 1 CD25+ CD4+ T cell for every 8 CD25− CD4+ cells ( A). We also tested the suppressive function of NOD CD25+ CD4+ T cells expanded with DCs and anti-CD3. Again, the T cells expanded with DCs and TCR stimulus suppressed proliferation by NOD CD25− CD4+ T cells approximately fourfold more efficiently than freshly isolated CD25+ CD4+ T cells ( B). Although freshly purified NOD CD25+ CD4+ T cells showed ~75% suppression at a ratio of 8 responder cells for 1 CD25+ CD4+ T cell, NOD CD25+ CD4+ T cells expanded with DCs and anti-CD3 (with or without IL-2) showed similar suppression at a ratio of 32:1. Therefore, either polyclonal or monospecific CD25+ CD4+ T cells from NOD mice can be expanded with DCs and anti-CD3 or antigen, and they show approximately fourfold enhancement in suppressive function.
Figure 4. DC-expanded CD25+ CD4+ T cells suppress proliferation better than unexpanded CD25+ CD4+ T cells. (A) CD25+ CD4+ T cells from NOD.BDC2.5 mice were expanded for 7 d with irradiated NOD DCs and BDC peptide and IL-2 as indicated. 104 freshly isolated, sorted (more ...)
DC-expanded CD25+ CD4+ T Cells Efficiently Suppress Diabetes In Vivo.
A critical in vivo function for CD25+
T cells is the prevention of autoimmunity. Therefore, we wanted to determine if BDC2.5 CD25+
T cells expanded in vitro with DCs and antigen could inhibit the development of diabetes. The first model of diabetes we used was one in which the pathogenic T cells to be suppressed were of the same BDC2.5 specificity. As expected from previous work (41
), most BDC2.5 mice on the NOD background did not develop diabetes, but when young BDC2.5 NOD mice were given one injection of cyclophosphamide, diabetes developed 4–7 d later in 100% of the mice. To suppress this diabetes induction, 3 d after cyclophosphamide treatment, we injected BDC2.5.NOD mice with DC-expanded CD25+
T cells from BDC2.5 mice. In two experiments, this resulted in a delay of diabetes onset and a reduced diabetes incidence. In contrast, injection of DC-expanded CD25−
from BDC2.5 mice had little effect on diabetes development ( A). These results show that the DC-expanded suppressor T cells are able to suppress autoimmunity even when the disease is developing rapidly.
Figure 5. Expanded CD25+ CD4+ T cells function in vivo to suppress development of diabetes. (A) 4–6-wk-old NOD.BDC2.5 mice were given cyclophosphamide i.p. 3 d later, either 5 × 105 DC-expanded CD25+ CD4+ T cells or CD25− CD4+ cells were (more ...)
We then tested a second model, injection of spleen cells from diabetic NOD mice into NOD.scid females, because this model is mediated by pathogenic T cells with a diverse repertoire of TCR specificities. We injected different doses of DC-expanded CD25+
T cells from BDC2.5 mice with 3–10 × 106
spleen cells from diabetic mice into NOD.scid females. The mice receiving diabetic spleen cells alone developed diabetes starting at 3–4 wk after injection as expected (42
). In the first dose-response study, the addition of 3 × 105
, or 3 × 104
expanded BDC2.5 CD25+
T cells to 3 × 106
diabetic spleen cells completely prevented diabetes development ( B). In contrast, when we injected 3 × 105
cells together with diabetic spleen cells, there was a marked acceleration of diabetes onset when compared with diabetic spleen cells alone. In a second dose-response experiment, we increased the number of diabetic spleen cells to 8 × 106
, and the number of expanded CD25+
T cells was titrated down further. Again 50,000 DC-expanded BDC2.5 CD25+
T cells completely prevented diabetes development up to 80 d after transfer. The addition of 5,000 of these regulatory cells also gave a large delay in diabetes, and even 500 DC-expanded BDC2.5 CD25+
T cells showed a significant delay in diabetes compared with those receiving spleen cells from diabetic mice alone ( C). In both experiments, unexpanded BDC2.5 CD25+
T cells showed a similar ability to block or delay diabetes development (not depicted). Three similar experiments with both DC-expanded and -unexpanded regulatory cells have now been performed with similar results.
The numbers of DC-expanded autoantigen-specific CD25+
T cells necessary to delay or block diabetes development here were much lower than the numbers of bulk (polyclonal) NOD CD25+
T cells used in other transfer studies, i.e., at least 2–5 × 105
cells were necessary to see a significant delay in diabetes development (22
). To establish the need for antigen-specific T cells in disease suppression, and to confirm in our system that DC stimulation alone was not sufficient for in vivo suppression, NOD CD25+
T cells expanded with DCs plus anti-CD3 were transferred to NOD.scid mice along with spleen cells from diabetic mice. Even 105
polyclonal NOD CD25+
T cells, either freshly isolated or anti-CD3/DC expanded, were unable to delay diabetes ( D). In addition, freshly isolated BDC2.5 regulatory T cells could also block diabetes development with similar cell numbers as the DC-expanded T cells (not depicted), suggesting that antigen specificity, rather than expansion with BDC peptide stimulation, is the most critical variable for suppression of diabetes in vivo. Therefore, autoantigen-specific DC-expanded CD25+
T cells function efficiently in vivo to suppress autoimmunity mediated by autoreactive T cells.
Mice Receiving Autoantigen-specific CD25+ CD4+ T Cells Can Develop Insulitis.
To determine at which stage disease was blocked in NOD.scid mice protected from diabetes by small numbers of BDC2.5-specific CD25+ CD4+ T cells, pancreata were isolated from those mice that still had normal glucose levels at the end of the experiment shown in C (80 d after transfer). Insulitis was scored from hematoxylin and eosin–stained sections. The mice from the groups that received 5,000 or 50,000 BDC2.5-specific CD25+ CD4+ T cells (the latter group were all diabetes free), had lymphocytic infiltrates in half of the islets scored ( A). A representative field from both protected groups is shown ( B). In a separate transfer experiment, pancreata were isolated from mice earlier on, at 23 or 28 d after transfer of the diabetogenic and regulatory cells. At this earlier time point, the mice that had received only the diabetogenic cells had some insulitis, but those that had also received BDC2.5 regulatory cells lacked lymphocytic infiltrate in the islets ( C). This indicates that protected mice can progress past the initiation of islet inflammation, checkpoint I, but the kinetics of insulitis is slower than in the absence of regulatory cells.
Figure 6. Protected mice have lymphocytic infiltrates in the pancreas. (A) Pancreata from mice that did not develop diabetes by day 80 after transfer in the experiment shown in C were scored for insulitis. 150 islets from 5 mice were scored from the group (more ...)
Autoantigen-specific CD25+ CD4+ T Cells Can Still Regulate When Given after Diabetogenic Cells.
One feature of the NOD.scid system is that T cells, when injected into a lymphopenic host, undergo antigen-independent, homeostatic proliferation. To lessen the effect of such proliferation on the CD25+ CD4+ T cells, the latter were injected after the diabetogenic spleen cells. Even when given 11 d after the diabetogenic cells, as few as 12,000 DC-expanded, BDC2.5 CD25+ CD4+ T cells prevented diabetes development ( A). When given 15 d after diabetogenic cells, 104 cells significantly delay diabetes ( B). At these time points, by FACS® staining for CD4+ cells, we showed that lymphocytes from the diabetic mice had repopulated the lymphoid organs, and even entered the pancreas (not depicted). Therefore, CD25+ CD4+ T cells can block diabetes even after the diabetogenic cells have been given time to occupy the lymphoid compartments, and initiate diabetes pathogenesis. Further studies will be needed to check the capacity of DC-expanded, antigen-specific CD25+ CD4+ T cells to suppress disease under nonhomeostatic conditions.
Figure 7. BDC2.5 CD25+ CD4+ T cells can still regulate diabetes when given after diabetogenic cells. (A) NOD.scid females were injected with 8 × 106 diabetic spleen cells and 11 d later were injected with either PBS or the indicated number of DC-expanded (more ...)