Cbl-b−/− mice have signaling defects that result in CD28-independent T cell activation, increased IL-2 production, hyper-reactive T cells, and increased autoimmunity. Although the increased autoimmunity in these mice is believed to result from the hyper-reactive T cells, the mechanisms leading from T cell hyper-reactivity to autoimmunity remain unclear. Specifically, the function and interaction of CD4+CD25+ regulatory T cells (Treg) and CD4+CD25− effector T cells (Teff) in Cbl-b−/− mice have not been examined. We now report that Cbl-b−/− CD4+CD25+ Treg exhibit normal regulatory function in vitro. In contrast, the in vitro response of Cbl-b−/− CD4+CD25− Teff is abnormal, in that it is not inhibited by either Cbl-b−/− or wild-type Treg. This resistance of Cbl-b−/− Teff to in vitro regulation is seen at the levels of both DNA synthesis and cell division. In addition to this resistance to CD4+CD25+ Treg, Cbl-b−/− Teff demonstrate in vitro resistance to inhibition by TGF-β. This second form of resistance in Cbl-b−/− Teff is seen despite the expression of normal levels of type II TGF-β receptors and normal levels of phosphorylated Smad3 after TGF-β stimulation. Coupled with recent reports of resistance to Treg in Teff exposed to LPS-treated dendritic cells, our present findings suggest that resistance to regulation may be a relevant mechanism in both normal immune function and autoimmunity.
Optimal T cell activation requires signaling through both the TCR and the CD28 costimulatory receptor. CD28 costimulation is believed to set the threshold for T cell activation. Cbl-b, a member of the Cbl/Sli family of molecular adaptors and a ubiquitin ligase, has been shown to negatively regulate CD28-dependent T cell activation (1, 2). Recent studies using gene-targeted mice lacking Cbl-b (Cbl-b−/− mice) have demonstrated the importance of this molecule in both T cell activation and the development of autoimmunity (1, 2). Cbl-b−/− T cells display increased proliferation after TCR stimulation and produce increased amounts of IL-2, but not IFN-γ or TNF-α (1). Cbl-b−/− mice have T cells that are independent of CD28 costimulation, in that they do not require CD28 engagement for IL-2 production and proliferation (1, 2).
Bachmaier et al. (1) have demonstrated that Cbl-b−/− mice develop spontaneous autoimmunity characterized by autoantibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Chiang et al. (2) have demonstrated that Cbl-b−/− mice are highly susceptible to experimental autoimmune encephalomyelitis. The increased autoimmunity found in Cbl-b−/− mice is believed to be a product of the signaling abnormalities, the CD28 independence, and the resultant T cell hyper-reactivity. However, the mechanisms leading from T cell hyper-reactivity to autoimmunity remain unclear. In the present study we have asked whether the signaling abnormalities in Cbl-b−/− T cells could play a role in the increased autoimmunity in Cbl-b−/− mice by affecting the interactions of Cbl-b−/− regulatory T cells (Treg)2 and Cbl-b−/− effector T cells (Teff). AlthoughCD4+CD25+ Treg have been shown to suppress CD4+CD25− Teff and CD8+ T cells in vitro and to play a role in the prevention of autoimmunity in vivo (3, 4, 5, 6, 7, 8, 9), the function and interaction of Treg and Teff have not been examined in Cbl-b−/−mice.
Tang et al. (10) have recently demonstrated that CD28 controls both the survival and proliferation of Treg in vivo. In addition, Treg derived from TCR-transgenic mice have been found to be capable of proliferating in response to specific Ags, but this proliferation is dependent on dendritic cells and B7 costimulation (11). Thus, in addition to the fact that Cbl-b−/− mice are prone to autoimmunity, the relevance of CD28-B7 interactions in the normal physiology of Treg suggested that CD4+ T cell regulatory interactions could be abnormal in CD28-independent Cbl-b−/− mice.
In the present studies we have examined for the first time the status of CD4+ T cell regulatory interactions in vitro in Cbl-b−/− mice. We now report that CD4+CD25+ Treg from Cbl-b−/− mice function normally in vitro in suppressing wild-type (WT) CD4+CD25− Teff. However, we have found that CD4+CD25− Teff from Cbl-b−/− mice are resistant to regulation by both Cbl-b−/− and WT Treg. In addition, Cbl-b−/− CD4+CD25− Teff are resistant to regulation by exogenously added TGF-β. This resistance of Teff to inhibition by Treg and TGF-β may help explain the increased autoimmunity seen in Cbl-b−/− mice. Furthermore, in light of similar findings in both aged NOD mice and Teff exposed to LPS-treated dendritic cells, our present findings suggest that resistance to regulation may be a relevant mechanism in both normal immune function and autoimmunity (12, 13).
Materials and Methods
C57BL/6 (WT) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Cbl-b−/− mice on a C57BL/6 background (a gift from Dr. H. Gu (National Institutes of Health, Bethesda, MD) were bred in our facilities under specific pathogen-free conditions. All animals were used between the ages of 6 and 10 wk.
The following Abs/reagents were used: from Miltenyi Biotec (Auburn, CA): anti-CD25-PE (clone 7D4) and anti-PE microbeads; and from BD Pharmingen (San Diego, CA): anti-CD25-PE (clone PC61), anti-CD4-FITC (clone GK1.5), anti-CD8α (clone 5H10-1), anti-CD24 (clone J11d), anti-CD3ε (clone 145-2C11), anti-CD4 PerCP (RM4-5), anti-CD45RB, and CD62L. Affinity-purified goat anti-rat IgG (Kierkegaard & Perry, Keene, NH) was used for panning. Human recombinant TGF-β1 was purchased from R&D Systems (Minneapolis, MN).
Splenocytes from WT and Cbl-b−/− mice were stained with anti-CD8α and anti-CD24 Abs and plated onto petri dishes (27 × 106/petri dish) coated with goat anti-rat IgG Ab (25 μg/ml). After 1.5 h at 4°C, CD8α+ T cells and CD24+ cells were depleted by panning, and the CD4+-enriched population was separated on magnetic columns (MS MACS separation column; Miltenyi Biotec) after staining with anti-CD25-PE, followed by anti-PE-conjugated microbeads.
In vitro proliferation assays
CD4+CD25− Teff (5 × 104), 5 × 104 irradiated (2600 rad) WT splenocytes, and various numbers of CD4+/+CD25+/+ Treg were plated in round-bottom, 96-well plates in RPMI 1640 supplemented with 10% FCS and 5 × 10−5 M 2-ME. Cultures were stimulated with varying doses of soluble anti-CD3 Ab for 48 h, and [3H]thymidine was added for the last 6 h. Cultures were harvested using a semiautomated cell harvester and assayed using a beta scintillation counter. To derive T cell-depleted splenocytes, Cbl-b−/− splenocytes were stained with anti-CD4 and anti-CD8 microbeads. This population was then negatively selected on magnetic columns, and the negatively selected cells were used as a source of T cell-depleted APCs. This population routinely consisted of <5% T cells (data not shown).
Freshly isolated CD4+CD25− WT and Cbl-b−/− Teff (10 × 106cells/ml) were labeled with 2.5 μM CFSE for 10 min at 37°C and subsequently washed once in 20% FCS-PBS and twice in RPMI 1640 with 10% FCS. CFSE-labeled Cbl-b−/− Teff and CFSE-labeled WT Teff were cultured with irradiated T cell-depleted WT splenocytes (as APCs) and 0.5 μg/ml soluble anti-CD3 Ab with or without Treg for the time periods indicated. After culture, cells were stained with PerCP-anti-CD4 (BD Pharmingen; RM4-5), washed, and fixed for subsequent FACS analysis.
Stimulation of Teff with TGF-β
Freshly isolated CD4+CD25− Teff (5 × 106) were plated in 24-well plates in 1 ml of RPMI 1640 supplemented with 10% FCS and 5 × 10−5 M 2-ME. Cultures were stimulated with or without TGF-β1 (5 ng/ml) for 30 min and harvested, and lysates were prepared for Western blots.
Samples were prepared by lysing 5 × 106 purified cell populations in lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.5% octyl glucoside, and 4 mM Pefabloc SC (Sigma-Aldrich, St. Louis, MO); for the Smad3 studies, 1 mM NaF, 1 mM NaVO4, and 50 mM β-glycerophosphate were also added), followed by centrifugation at 14,000 rpm to remove debris. The protein content of the lysates was quantified by the Bradford method using the Bio-Rad protein assay (Hercules, CA). The indicated quantities of lysates were denatured by boiling 5 min in sample buffer, resolved by reducing SDS-PAGE (10% (w/v) acrylamide), and electrophoretically transferred to an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked for 1 h in blocking buffer (PBS with 5% dehydrated milk) and probed with anti-TGF-β receptor type II (anti-TGF-βRII) Ab (sc-17799; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1/500 in blocking buffer, followed by incubation with HRP-conjugated goat anti-mouse secondary Ab (170-6516; Bio-Rad) at a dilution of 1/2000 in blocking buffer. Smad3 was detected with anti-Smad3 Ab (sc-6202; Santa Cruz Biotechnology), followed by HRP-conjugated anti-goat secondary Ab. For p-Smad3, membranes were blocked for 1 h in TBS with Tween 20 with 5% dehydrated milk, then probed with anti-phospho-Smad3 Ab (gift from Dr. E. Loef, Mayo Clinic, Rochester, MN), followed by incubation with HRP-conjugated goat anti-rabbit secondary Ab (170-6515; Bio-Rad). Alternatively, membranes were stripped and probed with anti-β-actin Ab (A-2066; Sigma-Aldrich) at a dilution of 1/1000, followed by incubation with HRP-conjugated goat anti-rabbit secondary Ab (170-6515; Bio-Rad) at a dilution of 1/2000 in blocking buffer. Bands were visualized using ECL substrate (Amersham Pharmacia Biotech, Arlington Heights, IL) detected by BioMax LS film (Eastman Kodak, Rochester, NY) and developed on an M35A X-OMAT film processor (Eastman Kodak).
Treg function in Cbl-b−/− and WT mice
Our first goal in these studies was to characterize CD4+CD25+ Treg cells derived from Cbl-b−/− mice. We isolated CD4+CD25+ Treg and CD4+CD25− Teff from the spleens of WT C57BL/6 mice and Cbl-b−/− mice. The Treg populations isolated were usually >95% pure, and the Teff populations isolated were usually 88–95% pure, as assessed by FACS analysis. To more completely characterize the Teff populations used, we analyzed the expression of CD45RB and CD62L as well as the expression of CD25 within the Teff populations derived from WT and Cbl-b−/− mice. Our WT and Cbl-b−/− Teff populations were similar not only in CD25 expression, but also in CD45RB and CD62L expression (Fig. 1, A–C). These results are consistent with those reported by Bachmaier et al. (1) and indicate that the Teff populations derived from Cbl-b−/− mice did not consist of a greater proportion of activated or memory cells than the Teff populations derived from WT mice. Also shown in Fig. 1 D is the similarity of CD25 expression of the WT and Cbl-b−/−-derived Treg populations. The number of Treg isolated from WT mice was ∼0.35–0.40 × 106/spleen, and the number of Treg isolated from Cbl-b−/− mice was ∼0.40–0.55 × 106/spleen.
WT and Cbl-b−/− CD4+CD25+ Treg and CD4+CD25− Teff were tested for their ability to respond and/or suppress after stimulation in vitro with soluble anti-CD3 Ab. In confirmation of previous reports, WT Treg showed no significant proliferation in response to anti-CD3 Ab using either 0.5 or 2 μg/ml anti-CD3 Ab. As expected, the WT Treg were capable of suppressing the anti-CD3 Ab-stimulated response of WT Teff using either 0.5 or 2 μg/ml anti-CD3 Ab (Fig. 2, A and B). Cbl-b−/− CD4+CD25+ Treg also showed no significant proliferation in response to anti-CD3 Ab using either 0.5 or 2 μg/ml anti-CD3 Ab. However, in contrast to WT cultures, we found that the Cbl-b−/− CD4+CD25+ Treg were incapable of suppressing the response of Cbl-b−/− CD4+ CD25− Teff at both 0.5 and 2 μg/ml anti-CD3 Ab stimulation (Fig. 2, C and D). On occasion, the responses of Cbl-b−/− Teff were actually increased in cocultures with Cbl-b−/− CD4+CD25+ Treg.
Treg function in mixed WT/Cbl-b−/− cultures
To further assess the lack of regulation seen in cultures ofCbl-b−/− CD4+CD25+ Treg and CD4+ CD25− Teff, we next set up mixed cultures combining WT Teff with Cbl-b−/− Treg and Cbl-b−/− Teff with WT Treg. We found that Cbl-b−/− Treg were fully capable of suppressing the anti-CD3 Ab-stimulated response of WT Teff. This was true using either 0.5 or 2 μg/ml anti-CD3 Ab (Fig. 3, A and B). This suggests that Cbl-b−/− Treg are fully capable of normal regulatory function. In contrast, WT Treg, which were capable of suppressing WT Teff, were not capable of suppressing the response of Cbl-b−/− Teff using either 0.5 or 2 μg/ml anti-CD3 Ab (Fig. 3, C and D). Again, the Cbl-b−/− Teff response was often slightly increased when cells were cocultured with WT Treg. These results suggest that although the in vitro regulatory function of Cbl-b−/− Treg is normal, the ability of Cbl-b−/− CD4+CD25− Teff to be suppressed by either WT or Cbl-b−/− Treg is abnormal.
Response of Cbl-b−/− Teff with varied Treg:Teff ratios
Having found an abnormality in the suppressibility of Cbl-b−/− Teff at what is usually considered an optimal or supraoptimal Treg:Teff ratio of 1:1, we next tested the ability of WT and Cbl-b−/− Teff to be suppressed at a lower and a higher ratio. At a ratio of 0.5:1, the response of WT Teff was suppressed 62% by WT Treg. In contrast, at this ratio, the response of Cbl-b−/− Teff was not suppressed by Cbl-b−/− Treg (Fig. 4). We then asked whether we could detect any suppression of the Cbl-b−/− Teff response using the very high Treg:Teff ratio of 2:1. At a ratio of 2:1, the response of WT Teff was suppressed 90% by WT Treg. In contrast, at a 2:1 ratio, the response of Cbl-b−/− Teff was only 34% suppressed by Cbl-b−/− Treg (Fig. 4). Thus, even at the very high Treg:Teff ratio of 2:1, there was very little suppression of Cbl-b−/− Teff.
Cbl-b−/− vs WT APCs
All of our prior studies were performed using WT-derived, irradiated, whole splenocytes as the APC population. We next wanted to examine the possibility that there might be functional differences between the WT and Cbl-b−/− APC populations that played a role in the resistance to regulation seen with Cbl-b−/− Teff. In addition, we wanted to confirm our results using an APC population that was depleted of T cells. We, therefore, repeated our studies using irradiated, T cell-depleted (Tds), Cbl-b−/− splenocytes as the APC population. Using these APCs, we found no significant difference in results from previous experiments. At a 1:1 Treg:Teff ratio and using 0.5 μg/ml anti-CD3 Ab for stimulation, the responses of WT Teff were ∼85% suppressed. This level of suppression was mediated by both WT Treg and Cbl-b−/− Treg (Fig. 5, A and B). However, the responses of Cbl-b−/− Teff were unable to be suppressed by either WT Treg or Cbl-b−/− Treg (Fig. 5, C and D). These results suggest that the resistance to regulation of Cbl-b−/− Teff is not related specifically to either WT or Cbl-b−/− APCs.
Evaluation of Treg function using CFSE staining
To further evaluate the resistance to regulation seen in Cbl-b−/− Teff, we next examined the Teff responses at the level of cell division rather than at the level of DNA synthesis (i.e., [3H]thymidine incorporation). WT and Cbl-b−/− CD4+CD25− Teff were labeled with CFSE, stimulated with anti-CD3 Ab and APCs, and cultured with or without the addition of Treg. In the FACS analyses used to evaluate these responses, we gated on CD4+ T cells. As such, in those cultures to which Treg were added, the Treg were seen as a CSFE-negative population (Fig. 6, B, D, and F). The Teff responses were examined at 48 and 60 h. In confirmation of our [3H]thymidine incorporation studies, WT Treg suppressed the anti-CD3 Ab-stimulated division of WT Teff. This was seen as a decrease in the number of waves of division in the presence of Treg. The results of a typical experiment for WT Teff, harvested at 60 h, are shown in Fig. 6, A and B (data not shown for the 48 h WT Teff responses). In contrast, Cbl-b−/− Treg did not suppress anti-CD3 Ab-stimulated division of Cbl-b−/− Teff. This was seen as either no decrease in the waves of division or even a slight increase in the progression through cell division of the CFSE-labeled Cbl-b−/− Teff. The results of typical experiments for Cbl-b−/− Teff are shown in Fig. 6, C and D (harvested at 48 h), and in Fig. 6, E and F (harvested at 60 h). These results suggest that the resistance to Treg regulation of Cbl-b−/− Teff occurs not only at the level of DNA synthesis, but also at the level of cell division.
Effect of exogenously added TGF-β on the response of Teff
TGF-β has been demonstrated to be an important regulator of immune responses in vivo (14, 15). In addition, TGF-β has been postulated to be a mechanism of suppression used by Treg (5, 16). To investigate the possibility that the resistance to regulation of Cbl-b−/− Teff is a more general characteristic of these cells, we next examined the effect of exogenously added TGF-β on the anti-CD3 Ab-stimulated responses of WT and Cbl-b−/− Teff. No Treg were used in these assays. As shown in Fig. 7, A and B, 5 and 25 ng/ml TGF-β suppressed the anti-CD3 Ab response of WT Teff by ∼55–65%. In contrast, neither 5 nor 25 ng/ml TGF-β was able to significantly suppress the response of Cbl-b−/− Teff. At 5 ng/ml TGF-β, the response of Cbl-b−/− Teff actually increased (Fig. 7,C). At 25 ng/ml TGF-β, the Cbl-b−/− Teff response decreased, but only by ∼11% (Fig. 7 D). Ab to TGF-β reversed the effects of the added cytokine, confirming the specificity of the TGF-β effect (data not shown). Thus, Cbl-b−/− Teff demonstrated resistance to regulation not only by both WT and Cbl-b−/− Treg, but also by TGF-β. Together these findings of Cbl-b−/− Teff resistance to both TGF-β-mediated suppression and regulation by Treg suggest that the autoimmunity seen in Cbl-b−/− mice might be the result of numerous regulatory abnormalities associated with Cbl-b−/− T cells.
TGF-βRII expression by CD4+CD25− Teff
To further characterize the defect in suppression of Cbl-b−/− Teff by TGF-β, we examined the expression of TGF-βRII by WT Teff and Cbl-b−/− Teff. Western blots were performed on lysatesof freshly isolated, nonstimulated WT Teff and Cbl-b−/−CD4+CD25− Teff. Using Ab specific for TGF-βRII, we examined two dilutions of lysates for both Cbl-b−/− CD4+CD25− Teff (Fig. 8,A, lanes b and c) and WT CD4+CD25− Teff (Fig. 8,A, lanes d and e). We found that the levels of expression of TGF-βRII were not significantly different in Cbl-b−/− Teff vs WT Teff (Fig. 8 A). These results suggest that the abnormality in TGF-β-mediated suppression of Cbl-b−/− Teff is not the result of a decrease in expression of TGF-βRII.
Finally, we examined Smad3 phosphorylation in WT Teff and Cbl-b−/− Teff using Western blots. The ligation of TGF-βRII by TGF-β leads to the heterodimerization of TGF-βRII with TGF-βRI. This activated receptor complex then recruits Smad2 and Smad3, which are phosphorylated by TGF-βRI. The phosphorylated Smad2 and Smad3 moieties each then heterodimerize with Smad4, and these Smad complexes can translocate to the nucleus and activate transcriptional responses (17). We incubated WT and Cbl-b−/− CD4+CD25− Teff with 5 ng/ml TGF-β for 30 min and then performed immunoblots on the cell lysates using Abs to both Smad3 and phosphorylated Smad3. We found that non-TGF-β-stimulated WT Teff and Cbl-b−/− Teff expressed equal levels of nonphosphorylated Smad3 (Fig. 8,B). Furthermore, although TGF-β suppresses WT CD4+CD25− Teff proliferation, but does not suppress (and often enhances) Cbl-b−/− CD4+CD25− Teff proliferation, we found that WT Teff and Cbl-b−/− Teff demonstrated an equal increase in phosphorylated Smad3 after incubation with TGF-β (Fig. 8 B). Thus, our results suggest that the resistance of Cbl-b−/− Teff to TGF-β-mediated suppression is the result of neither a decrease in expression of TGF-βRII nor an abnormality in the subsequent phosphorylation of Smad3, but may relate instead to abnormalities distal to these initial signaling events.
Cbl-b, a ubiquitin ligase, acts as a type of gate-keeper in that it serves as a negative influence on T cell activation unless unleashed through CD28 stimulation (18). This unleashing occurs, at least in part, by CD28’s ligation leading to Cbl-b ubiquitination and degradation (19). Two different laboratories have generated C57BL/6 mice that do not express Cbl-b (1, 2). In these mice the Cbl-b deficiency does not affect cellular development, but does lead to T cells that demonstrate increased proliferation and produce increased amounts of IL-2 after TCR stimulation (1, 2, 18). Chiang et al. (2) have shown that in Cbl-b−/− T cells, the main TCR signaling pathways are not affected, but Vav activation is significantly enhanced. Thus, mutation of Cbl-b uncouples T cell proliferation, IL-2 production, and phosphorylation of Vav1 from the requirement for CD28 costimulation, leading to CD28 independence of Cbl-b−/− T cell activation (1, 2).
Cbl-b−/− mice have been found to be prone to both spontaneous and induced autoimmune diseases (1, 2). The spontaneous autoimmunity involves the development of autoantibodies to dsDNA and massive infiltration in multiple organs (1). The induced autoimmunity involves an increased susceptibility to the induction of experimental autoimmune encephalomyelitis (2). In addition, a mutation in the Cbl-b gene has been shown to be associated with a spontaneous model of diabetes in the rat (20). Although the CD28 independence, the decreased threshold for stimulation, and the T cell hyper-reactivity are postulated to be responsible for the increased autoimmunity in Cbl-b−/− mice, the actual mechanisms underlying the increased autoimmunity are as yet unclear.
The mechanisms of suppression via CD4+CD25+ Treg have yet to be definitively identified (9). Unresolved issues include questions about the cytokines and stimulatory factors required for the development and function of these cells and the nature of their Ag specificity. Shevach et al. (7, 9) have demonstrated that Treg require stimulation through their TCR to mediate suppressive function and that Treg suppress IL-2 production by Teff. This suppression is noted at both the IL-2 mRNA and protein levels (7, 9). These CD4+CD25+ Treg appear to have significant in vivo functional relevance, as evidenced by their ability to prevent certain autoimmune syndromes and their regulatory role in normal immune responses (3, 4, 9, 21, 22). The suppressive function of Treg has also been demonstrated in vitro, where they have been found to suppress both anti-CD3 Ab-stimulated and Ag-specific responses of CD4+ and CD8+ T cells (8, 9).
CD4+CD25+ Treg have been shown to depend on CD28 for their development, homeostatic proliferation, and Ag-driven proliferation (4, 10, 11). CD28−/− NOD mice have been demonstrated to develop exacerbated diabetes because of the lack of CD4+CD25+ Treg development (4). Tang et al. (10) have recently shown that CD28 controls both the survival and the proliferation of Treg in vivo. In addition, Treg derived from TCR-transgenic mice have been found to be capable of proliferating in response to specific Ags, but this proliferation is dependent on dendritic cells and B7 costimulation (11). Thus, given the relevance of CD28-B7 interactions in the normal physiology of Treg, the CD28 independence of Cbl-b−/− T cells and the increased proclivity of Cbl-b−/− mice to autoimmunity suggested that CD4+ T cell regulatory interactions could be abnormal in these mice.
We now report that the function of Cbl-b−/− CD4+CD25+ Treg is essentially identical with that of Treg derived from WT mice. However, the ability of Cbl-b−/− CD4+CD25− Teff to be regulated is significantly different from that of Teff derived from WT mice. Specifically, in the in vitro response to soluble anti-CD3 Ab, Cbl-b−/− Teff, compared with WT Teff, are significantly less able to be suppressed by either Cbl-b−/− or WT Treg. Using [3H]thymidine incorporation proliferation assays, this resistance to regulation of Cbl-b−/− Teff is seen over various ratios of Treg:Teff. In addition, this resistance is found when the strength of activating signal is varied by stimulation with either 0.5 or 2 μg/ml anti-CD3 Ab and when either WT or Cbl-b−/− APCs are used in the assays. Finally, using CFSE labeling, we confirmed that the resistance to Treg regulation of Cbl-b−/− Teff occurs not only at the level of DNA synthesis, but also at the level of cell division.
To date, almost all studies of CD4+ regulatory function have focused on the Treg populations rather than on the regulated Teff. However, our present results of Teff resistance to regulation are suggestive of two other recently described findings (12, 13). In the first, a decrease in suppressibility of murine CD4+CD25− Teff has been found in conjunction with LPS-activated dendritic cells (13). Such LPS-activated dendritic cells have been found to mediate a decrease in CD4+CD25− Teff suppressibility through the secretion of IL-6 and an as yet unidentified cytokine (13). At a 1:1 ratio, Treg-mediated suppression has been shown to decrease from ∼85% with normal dendritic cells to ∼25% when LPS-treated dendritic cells were used (13). These results are similar to ours; at a 1:1 ratio we have found 85% suppression of the WT response and no suppression of the Cbl-b−/− Teff response. In a second relevant study, Gregori et al. (12) have reported age-related changes in both CD4+CD25+ Treg and CD4+CD25− Teff in NOD mice. These authors have found that as autoimmune diabetes progresses, the CD4+CD25− pathogenic (effector) T cells become progressively less sensitive to immunoregulation by CD4+CD25+ Treg (12). These results in NOD mice suggest that our present findings may be applicable to various autoimmune states in addition to that seen in Cbl-b−/− mice.
The mechanisms underlying our findings of Cbl-b−/− Teff resistance to regulation are as yet unclear. There is evidence suggesting that the strength of the activating signal may play a role in determining the suppressibility of Teff (23, 24). These studies have demonstrated that stronger activating signals are associated with a decrease in the suppressibility of CD4+CD25− Teff. Baecher-Allen et al. (23) have demonstrated a correlation between the in vitro strength of signal of activation and the ability of human CD4+CD25− Teff to be suppressed by Treg in vitro. In these studies the stronger activating signals are associated with a decrease in suppressibility of Teff (23). The stronger activating signals are also associated with a decrease in the suppressor function of Treg (23). Although we did not examine this issue of Treg function specifically, in general, our findings indicate that WT Treg and Cbl-b−/− Treg function with equal efficiency. We believe that it is possible that the resistance to regulation described now for Cbl-b−/− Teff results from the fact that the hyper-reactivity of Cbl-b−/− T cells is equivalent to a state of increased strength of activating signal for any given stimulus. However, the resistance to regulation ofCbl-b−/− Teff is not simply related to differences in absolute levels of proliferation between Cbl-b−/− and WT Teff. This can be seen in our results, where Cbl-b−/− Teff are not suppressible when proliferating at a level of ∼38,000 cpm of [3H]thymidine incorporation (Fig. 3,D), whereas WT Teff are highly suppressible at a similar level of proliferation (Fig. 5, A and B).
Given the finding of resistance to Treg regulation, we also examined whether Cbl-b−/− CD4+CD25− Teff are resistant to other forms of immunoregulation. It has previously been reported that Cbl-b−/− T cells are suppressed normally by CTLA-4 (2). Suppression by IL-10 has been reported to be mediated through down-regulation of B7 expression on APCs and also through alteration of the CD28 costimulation pathway in T cells (25, 26). The CD28 dependence of these mechanisms suggested that Cbl-b−/− Teff are unlikely to be suppressible by IL-10. We therefore examined the suppressibility of Cbl-b−/− Teff by TGF-β. TGF-β plays a role in immunoregulation, and there are reports suggesting that cell-bound TGF-β is important in the suppression mediated by Treg (5, 14, 15, 16). TGF-β inhibits T cell proliferation and function at many different levels, including the production of IL-2 (27). We now report that Cbl-b−/− Teff are also significantly less suppressed by exogenously added TGF-β than are WT Teff. To further understand this TGF-β resistance, we first characterized the expression of TGF-βRII by freshly isolated Teff. We found that the expression of TGF-βRII did not significantly differ between WT and Cbl-b−/− Teff. We then examined Smad3 phosphorylation in WT Teff and Cbl-b−/− Teff after incubation with TGF-β. Although TGF-β suppresses WT CD4+CD25− Teff proliferation, but does not suppress (and often enhances) Cbl-b−/− CD4+CD25− Teff proliferation, we found that WT Teff and Cbl-b−/− Teff demonstrated an equal increase in phosphorylated Smad3 after incubation with TGF-β (Fig. 8 B). This suggests that the resistance of Cbl-b−/− Teff to TGF-β-mediated suppression is the result of neither a decrease in the expression of TGF-βRII nor an abnormality in the subsequent phosphorylation of Smad3, but may relate instead to abnormalities distal to these initial signaling events. Future studies in our laboratory will involve identifying these more distal TGF-β signaling alterations in Cbl-b−/− T cells.
The findings of resistance to TGF-β-mediated suppression together with the resistance to regulation by Treg suggest that the autoimmunity seen in Cbl-b−/− mice might be the result of numerous regulatory abnormalities associated with Cbl-b−/− T cells. Given recent reports of similar resistance to Treg regulation in aged NOD mice and in Teff exposed to LPS-treated dendritic cells (12, 13), resistance to regulation may be a relevant mechanism in both normal immune function and autoimmunity. Future studies in our laboratory will attempt to confirm these immunoregulatory abnormalities in Cbl-b−/− mice using in vivo approaches.
We thank Dr. Ethan Shevach, National Institutes of Health, for his critical reading of this manuscript and his helpful comments.
Abbreviations used in this paper: Treg, regulatory T cell; Tds, T cell-depleted splenocyte; Teff, effector T cell; TGF-βRII, TGF-β receptor type II; WT, wild type.