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The Journal of Immunology, 2006, 176: 3366-3373.
Copyright © 2006 by The American Association of Immunologists

Loss of the Surface Antigen 3G11 Characterizes a Distinct Population of Anergic/Regulatory T Cells in Experimental Autoimmune Encephalomyelitis1

Guang-Xian Zhang*, Shuo Yu*, Divina Calida*, Zhao Zhao*, Bruno Gran*, Malek Kamoun{dagger} and Abdolmohamad Rostami2,*

* Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107; and {dagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell anergy is an important mechanism in the induction of peripheral tolerance against autoimmune diseases, yet no surface marker unique to anergic T cells in these diseases has been identified. In this study we induced in vivo anergy by i.v. tolerance against experimental autoimmune encephalomyelitis in myelin basic protein TCR transgenic mice, and showed that the hyporesponsiveness of autoantigen-reactive T cells from tolerized mice was associated with a dramatic loss of 3G11, a cell surface molecule on the surface of CD4+ T cells. Purified 3G11–CD4+ T cells lost autoantigen-induced proliferation and IL-2 production, whereas 3G11+CD4+ T cells retained responsiveness. Furthermore, 3G11– T cells actively suppressed proliferation and Th1 cytokine production of 3G11+ T cells and splenocytes of nontolerized mice. Active suppression by 3G11– T cells was at least partially due to soluble immunoregulatory factors, including IL-10. The T regulatory property of 3G11– T cells was confirmed in vivo because the transfer of purified 3G11– T cells effectively suppressed clinical experimental autoimmune encephalomyelitis. We conclude that loss of the surface molecule 3G11 characterizes a distinct population of anergic/regulatory T cells. This is the first demonstration of the ability to identify and purify anergic T cells by a distinct cell surface marker in an autoimmune disease and paves the way for a better understanding of the mechanism of tolerance in autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunologic tolerance is a basic property of the immune system that provides for self/nonself discrimination to protect the host from external pathogens without reacting against self (1, 2). In the periphery, autoreactive T cells specific for tissue Ags may achieve tolerance in different ways: clonal deletion, active suppression, and anergy (3, 4, 5, 6). In T cell anergy, lymphocytes are functionally inactivated after an Ag encounter, but remain alive for an extended period of time in a hyporesponsive state (7, 8). Anergy can be induced by various manipulations in vitro (9, 10, 11) and in vivo (11, 12) and has often been characterized by the loss of IL-2 production in addition to nonproliferation (13, 14). However, T cell anergy was, for a long time, a functionally defined state of hyporesponsiveness. Although down-regulation of the cell surface molecule 6C10 on CD4+ T cells has been suggested to represent an anergic status induced by superantigen (15), a distinct cell surface marker for anergy in autoimmune diseases has not been identified, making it impossible to purify and to distinguish populations of anergic and nonanergic T cells.

Regulatory T cells (Tregs)3 can be defined as CD4+ T cells that inhibit immunopathology or autoimmune disease in vivo (16, 17). Naturally occurring CD4+CD25+ Tregs are generated in the thymus and represent 5–10% of the CD4+ T lymphocytes in healthy adult mice and humans. Peripherally induced Tregs can be induced by administration of Ag in vivo during anergy/tolerance (18, 19). Because the major effect of IL-10 is to inhibit APC function, including the production of proinflammatory cytokines, it is likely that the function of Tregs is dependent on IL-10 only when cells of the innate immune system are involved (20). In addition to CD4+CD25+ Tregs, IL-10 Tregs, Th2 cells, and APCs also produce IL-10 and thus can control the magnitude of an immune response and limit immunopathology (21). At present, peripherally induced Treg activity is a functionally defined state, with no specific marker.

3G11, an mAb, has been found to recognize determinants that are composed of lymphoid cell gangliosides, independent of Thy-1 expression (22, 23). The 3G11 molecule is expressed on both thymocytes and peripheral T cells of all mouse strains, predominantly on the membranes of CD4+ T cells. 3G11+ T cells stimulated by mitogen produced a large amount of IL-2, but the 3G11– population did not (22). We thus hypothesized that the level of 3G11 expression might reflect the functional state of CD4+ T cells in autoimmune diseases and could be a potential cell surface marker for the anergic state. In the present study we tested this hypothesis in an i.v. tolerance model in myelin basic protein (MBP) TCR transgenic mice with experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disease of the CNS (24, 25). Our data demonstrate that loss of the 3G11 molecule on autoantigen-reactive CD4+ T cells represents a distinct anergic/Treg population.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and reagents

Mice transgenic for a TCR specific for the Ac1–11 peptide of MBP (MBP1–11) were generated by introducing the rearranged TCR {alpha}- and beta-chain genes into the germline of C57BL/6 mice and extensively backcrossed to PL/J mice (26) (gift from Dr. R. Caspi (National Institutes of Health, Bethesda, MD) and the late Dr. C. Janeway (Yale University, New Haven, CT)). Mice were screened for the expression of MBP-specific TCR by FACS analysis with an anti-clonotypic mAb 19G (26). All mice were housed at University of Pennsylvania and Thomas Jefferson University animal care facilities.

MBP1–11 (Ac-ASQKRPSQRHG) was made using a peptide synthesizer and was purified through HPLC. Con A was purchased from Sigma-Aldrich. Mouse rIL-2, rIL-4, and rIL-10 and mAbs to these cytokines were purchased from BD Pharmingen. Anti-mouse 3G11 IgM mAb (gift from Dr. M. Greene, University of Pennsylvania, Philadelphia, PA) was prepared as previously described (15). The Ab is IgM{kappa}, with some association of {gamma}2b H chain contributed by MPC11. 19G mAb was a gift from the late Dr. C. Janeway.

Immunization and tolerance induction

Female MBP TCR transgenic mice, 8–10 wk of age, were immunized with 200 µg of MBP1–11 in 0.1 ml of PBS emulsified with the same volume of CFA containing 4 mg/ml Mycobacterium tuberculosis H37 RA (Difco). Pertussis toxin (200 ng/mouse/injection; List Biological) was given i.p. at the time of immunization and 48 h later. MBP1–11 (200 µg/mouse) was i.v. injected into 10 mice from day 0 and then every 3 days. The same volumes of PBS were injected in 10 mice in parallel as a control. EAE was scored daily in a blind fashion as follows (27): 1, limp tail or waddling gait with tail tonicity; 2, waddling gait with limp tail (ataxia); 2.5, ataxia with partial limb paralysis; 3, full paralysis of one limb; 3.5, full paralysis of one limb with partial paralysis of second limb; 4, full paralysis of two limbs; 4.5, moribund; and 5, death. Mice were examined daily in a blind fashion for signs of EAE. All work was performed in accordance with Thomas Jefferson University guidelines for animal use and care.

Cell preparation from lymphoid organs

Mice were killed when the disease reached its peak (16 days postinfection (p.i.)). Mononuclear cells (MNCs) were harvested from inguinal, popliteal, brachial, axillary, and cervical LN, spleen and thymus of MBP1–11-injected and PBS-injected EAE mice. Erythrocytes in the cell pellets from spleen were hemolyzed by added NH4Cl-Tris buffer for 5 min at room temperature, followed by washing. CD4+ T cells from spleens of i.v. MBP1–11-treated (MBP1–11-i.v.) animals were purified as previously described (15) by depleting non-CD4 cells with complement fixation and Abs directed against heat-stable Ag (J11d), I-Ek (14-4-4), and CD8 (3.155) and centrifuging over a cushion of lympholyte M (Cedarlane Laboratories). The purity of CD4+ cells was confirmed by FACScan. On the average, this preparation yielded 92.6% CD4+ purification of viable cells. To separate 3G11– and 3G11+ populations, purified CD4+ T cells were cultured with anti-3G11 Abs (IgM), washed, and then cultured with rat anti-mouse IgM microbeads. 3G11– and 3G11+ cells were collected by MACS magnetic cell sorting, using a type AS column following the manufacturer’s protocol (Miltenyi Biotec). On the average, magnetic sorting for 3G11 expression yielded CD4+ populations that were 82% 3G11– and 95.0% 3G11+.

Flow cytometry

To study 3G11 expression on T cells in different immune organs and the CNS from MBP1–11-i.v. and PBS-i.v. mice, 1 x 106 MNCs from LN, spleen, thymus, and spinal cord were stained with anti-mouse CD4, CD8, 19G, and 3G11 mAb. Anti-3G11 and anti-19G Ab staining was additionally labeled using indirect fluorescence-conjugated Abs. Lymphocytes were gated, and fluorescence was analyzed using CellQuest (BD Biosciences) software. CD4+19G+ cells that were MBP TCR transgenic T cells were gated for analysis of expression of 3G11. Data representing 10,000 events were presented as a histograph. To determine the naive/activation status of CD4+ T cells, cells were stained with anti-CD4, anti-19G, anti-3G11, and anti-CD62L mAb simultaneously. Data were analyzed using CellQuest.

Proliferative responses of 3G11– T cells separately and in coculture

First, purified 3G11– and 3G11+ CD4+ T cells were cultured separately (5 x 104/200 µl) in triplicate with MBP1–11 (10 µg/ml) and Con A (5 µg/ml) without Ag/mitogen. Cultures were also complemented with or without 50 IU/ml mouse rIL-2. Irradiated (2200 rad) syngeneic splenocytes (5 x 105/well) of naive mice were plated as APCs. After 60 h of incubation, the cells were pulsed for 12 h with 1 µCi of [3H]methylthymidine (sp. act., 42 Ci/mmol). Thymidine incorporation was measured by a scintillation counter.

For the coculture experiments, 3G11– T cells were cocultured with splenocytes of PBS-i.v. mice in individual wells (5 x 105/200 µl) in triplicate with MBP1–11 (10 µg/ml), with Con A (5 µg/ml) without Ag/mitogen. The ratios of splenocytes and 3G11– cells in the wells were 1:0, 1:0.1, and 1:0.3, respectively, and the final volume remained 200 µl/well. 3G11+ cells were added at the same ratios as in the control culture. Proliferative responses were determined.

Cytokine production by 3G11– T cells

Purified 3G11–CD4+ and 3G11+CD4+ T cells were cultured with irradiated naive splenocytes (APCs) in 24-well plates, respectively. Nonisolated splenocytes from MBP1–11-i.v. and PBS-i.v. mice in the same plate served as the control. Cells were cultured in the presence and the absence of MBP1–11 (10 µg/ml) with or without rIL-2 (50 U/ml). Forty-eight hours later, supernatants were harvested, and production of IL-2, IFN-{gamma}, and IL-10 was determined by ELISAs using paired mAbs according to the manufacturer’s instructions (Harlingen). The concentrations of the cytokines detected were automatically calculated based on the standard curves obtained from known concentrations.

Culture with supernatants of 3G11– T cells and Ab-blocking experiments

Splenocytes from EAE mice were cultured at 5 x 105/200 µl in triplicate with MBP1–11 (10 µg/ml). Supernatants from cultures of 3G11– T cells were pooled and added at 50, 25, 12.5, 6.25, 3.13, 1.56, and 0 µl, respectively, with the final volume kept at 200 µl. In Ab-blocking experiments, monoclonal anti-mouse IL-4 (10 µg/ml), anti-IL-10 (10 µg/ml), anti-IL-4 with anti-IL-10, and purified polyclonal mouse IgG (10 µg/ml) were added to the wells cultured with 50 µl of supernatants of 3G11– T cells. In parallel, supernatants from cultures of 3G11+ T cells were added to separate wells in the same amounts as supernatants of 3G11– T cells. rIL-4 at 200 U/ml, rIL-10 at 5 ng/ml, and rIL-4 with rIL-10 were added to the wells cultured with 50 µl of supernatants from 3G11+ T cells. Proliferative responses were determined.

Adoptive transfer of 3G11–CD4+ T cells in EAE suppression

To determine the potential in vivo regulatory function of 3G11– T cells, we adoptively transferred these cells to EAE mice. Due to the shortage of MBP TCR transgenic mice, in the current experiments we used myelin oligodendrocyte glycoprotein (MOG) TCR transgenic mice. We have shown that these mice are an excellent alternative to MBP TCR transgenic mice for EAE and tolerance studies. Both strains are TCR transgenic for a myelin Ag and develop chronic progressive EAE. More importantly, we have shown that MOG and MBP peptides are comparably effective in i.v. tolerance induction in EAE (28, 29). EAE was induced as previously described (29). 3G11–CD4+ T cells and 3G11+CD4+ T cells were purified from spleens of MOG TCR transgenic mice, stimulated with MOG35–55, and i.v. injected into MOG-induced EAE mice. A single dose of 3G11–CD4+ T cells at 10 or 40 million was i.v. injected into EAE mice at the time of immunization. Control mice received the same number of purified 3G11+CD4+ T cells or PBS only. Mice were examined daily in a blind fashion for signs of EAE.

Statistics

Mann-Whitney U test was used for the comparison of average clinical scores, and Student’s t test was used for comparing other parameters among different groups. All tests were two-sided.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Down-regulation of 3G11 molecule on transgenic T cells accompanied MBP1–11-induced T cell anergy

To induce autoantigen-specific tolerance, MBP1–11 was i.v. injected at the beginning of EAE induction in MBP TCR transgenic mice. Clinical EAE was completely suppressed in MBP1–11-i.v. mice (Fig. 1A). Histological study showed typical infiltrates in PBS-i.v. mice, but few infiltrates were found in MBP1–11-i.v. mice (data not shown). Similar absolute numbers of MNCs were harvested from MBP1–11-i.v. mice and PBS-i.v. mice in lymph nodes (33.0 ± 6.1 vs 36.2 ± 8.8 x 106 for each mouse; n = 5), spleen (74.4 ± 17.3 vs 76.7 ± 12.5 x 106), or thymus (9.0 ± 1.2 vs 8.2 ± 1.3). More importantly, the percentage of CD4+19G+ (a marker for MBP TCR transgenic T cells) in the periphery (spleen as a representative) and the thymus was similar in both groups (Fig. 1B). These results indicate that the tolerance is not due to clonal deletion. Splenocytes of MBP1–11-i.v. mice produced no IL-2 or IL-4, significantly lower levels of IFN-{gamma} (p < 0.001), and higher IL-10 (p < 0.01; Fig. 2A). Proliferative responses to MBP1–11 were nearly completely suppressed in MNCs of MBP1–11-i.v. animals (p < 0.001), and such hyporesponsiveness was not reversed by IL-2 (Fig. 2B). Thus, the suppression of clinical EAE was closely related to i.v. MBP-induced in vivo anergy of MBP-specific T cells according to criteria described recently (7).


Figure 1
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  		FIGURE 1. Suppression of clinical EAE by i.v. MBP1–11 is not due to clonal deletion. A, Clinical scores of EAE in MBP1–11-i.v. and PBS-i.v. mice (n = 10 in each group). Female MBP TCR transgenic mice, 8–10 wk of age, were immunized with MBP1–11 and CFA. Pertussis toxin was given i.p. at the time of immunization and 48 h later. To induce tolerance, MBP1–11 was i.v. injected into 10 mice from day 0 and then every 3 days. The same volumes of PBS were injected into 10 mice in parallel as a control. Mice were scored for EAE (27 ) daily in a blind fashion. Data were expressed as the mean clinical score on each day. Bars indicate the SD. *, p < 0.05; ***, p < 0.001. B, Mice were killed on day 16 p.i. when clinical EAE was at its peak. Similar absolute numbers of MNCs were harvested from MBP1–11-i.v. mice and PBS-i.v. mice in lymph nodes (33.0 ± 6.1 vs 36.2 ± 8.8 x 106 each mouse; n = 5), spleen (74.4 ± 17.3 vs 76.7 ± 12.5 x 106), and thymus (9.0 ± 1.2 vs 8.2 ± 1.3). MNCs from thymus and spleen of MBP1–11-i.v. and PBS-i.v. mice were labeled with CD4 mAb and 19G mAb, which is specific for MBP-reactive T cells. The data represent 10,000 events, and the percentage of different T cell populations is indicated by the numbers. All data in A and B are representative of three repeated experiments.

 

Figure 2
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  		FIGURE 2. Effect of i.v. MBP on cytokine production and Ag-specific proliferative response. EAE and i.v. tolerance were induced as described in Fig. 1. Mice were killed on day 16 p.i. when clinical EAE was at its peak (n = 5 each group), and splenocytes were isolated for cell culture. A, To determine cytokine production, 2.5 x 106/ml splenocytes were cultured with or without MBP1–11 (10 µg/ml). Supernatants were collected 72 h after culture to determine by ELISA the production of IL-2 (ng/ml), IFN-{gamma} (ng/ml), IL-4 (pg/ml), and IL-10 (pg/ml). B, To study proliferative responses, 5 x 105/200 µl splenocytes were cultured with 0, 0.1, 1, 10, and 100 µg/ml MBP1–11 in the presence or the absence of 50 IU/ml IL-2. Symbols refer to mean values, and bars to the SD. *, Comparison between PBS-i.v. mice and MBP1–11-i.v. mice; #, comparison between PBS-i.v. mice and MBP1–11-i.v. mice cultured with IL-2. ** and ##, p < 0.01; *** and ###, p < 0.001. Data are representative of three repeated experiments.

 
To define the correlation between 3G11 expression on MBP-specific T cells and tolerance induction, MNCs from lymph nodes, spleen, thymus, and spinal cord from MBP1–11-i.v. and PBS-i.v. mice were isolated and labeled with Abs against CD4, CD8, 3G11, and 19G, an mAb specific to MBP-reactive T cells. We found that the expression of 3G11 on CD4+19G+ cells was dramatically decreased in central and peripheral immune organs and the CNS. In lymph nodes, 72.2% of CD4+19G+ cells of PBS-i.v. mice expressed 3G11, whereas this percentage was 31.5% in MBP1–11-i.v. mice. Similar results were shown in spleen (52.3 vs 35.6%), thymus (56.4 vs 21.5%), and spinal cord (59.0 vs 28%; Fig. 3). In the thymus, a similar level of 3G11 expression was found at the CD4–8– double-negative stage (26.3% in PBS-i.v. mice vs 31.3% in MBP1–11-i.v. mice), and 3G11 expression was dramatically down-regulated on CD4+8+ double-positive cells (70.6% in PBS-i.v. mice vs 12.7% in MBP1–11-i.v. mice). These data indicate that the loss of 3G11 is positively correlated with in vivo anergy status, and that in the thymus, the loss of 3G11 expression after i.v. tolerance mainly occurred at the CD4+8+ double-positive stage.


Figure 3
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  		FIGURE 3. FACS analysis of 3G11 expression on T cells. MNCs (1 x 106) from LN, spleen, thymus, and spinal cords of MBP1–11-i.v. and PBS-i.v. EAE mice (n = 5 each group) on day 16 p.i. were harvested and stained with anti-CD4, anti-CD8, anti-19G, and anti-3G11 mAb. CD4+19G+ T cells were gated, and fluorescence was analyzed for 3G11+ vs 3G11– cells. The data represent 10,000 events and are presented as a histograph. The percentage of CD4+3G11+ T cells is indicated by the number above the brackets. p < 0.01 for all organs examined, MBP1–11-i.v. vs PBS-i.v. mice. The data are representative of three repeated experiments.

 
Expression of CD62L on 3G11– cells

To study the relation between the loss of 3G11 expression and T cell activation/memory status, we examined the expression of 3G11 and CD62L on CD4+19G+ T cells from draining lymph nodes of MBP1–11-i.v. mice and PBS-i.v. mice. In MBP1–11-tolerized mice, a portion of 3G11– cells was CD62Llow (26.0%), consistent with a memory/activation phenotype; 9.8% of 3G11+ cells were CD62Llow in these mice. Similar results were observed in PBS-i.v. mice (16.6% in 3G11– T cells vs 11.1% in 3G11+ cells). These results suggest that anergic T cells undergo an activation/memory process similar to that of nonanergic T cells.

Antoantigen-induced proliferation and cytokine production by 3G11– T cells

To investigate the functional state of 3G11–CD4+ cells in MBP1–11-i.v. mice, we separated 3G11–CD4+ cells from 3G11+CD4+ T cells. 3G11–CD4+ cells did not proliferate when stimulated with autoantigen MBP1–11 (Fig. 4A) and failed to produce IL-2 (Fig. 4B). This state of hyporesponsiveness could not be reversed by rIL-2 (Fig. 4). On the contrary, 3G11+ T cells retained a potent capacity to proliferate with autoantigen and produced comparable amounts of IL-2 as MNCs from PBS-i.v. mice. No response to control Ag OVA was found in either 3G11– or 3G11+ T cells (data not shown), and both 3G11– and 3G11+ T cells showed a similar high proliferation response to mitogen Con A (Fig. 4A).


Figure 4
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  		FIGURE 4. Proliferative responses and cytokine production of 3G11– T cells. 3G11– and 3G11+CD4+ T cells were purified from spleens of MBP1–11-i.v. mice as described in Materials and Methods. A, Autoantigen-induced proliferative response. Purified T cells were cultured (5 x 104/200 µl) in triplicate with MBP1–11 (10 µg/ml) and Con A (5 µg/ml) without Ag/mitogen and with or without 50 IU/ml mouse rIL-2. Irradiated (2200 rad) syngeneic splenocytes (5 x 105/well) from naive mice were plated as APCs. After 60 h of incubation, the cells were pulsed for 12 h with 1 µCi of [3H]methylthymidine. Incorporation of thymidine was measured with a scintillation counter. Symbols refer to mean values, and bars to the SD. **, p < 0.01; ***, p < 0.001. B, To examine the cytokine profile, 2.5 x 105/ml purified 3G11+CD4+and 3G11–CD4+ cells were cultured with 2.5 x 106/ml APCs with or without MBP1–11 (10 µg/ml). Supernatants were collected 72 h after culture to determine by ELISA the production of IL-2 (ng/ml), IFN-{gamma} (ng/ml), and IL-10 (pg/ml). IL-4 was not detectable in either PBS-i.v. or MBP1–11-i.v. mice (not shown). Symbols refer to mean values, and bars to the SD. The p values refer to comparisons between 3G11– and 3G11+ T cells, and between splenocytes of MBP1–11-i.v. mice and PBS-i.v. mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The data are representative of two repeated experiments.

 
To identify the phenotype of 3G11– T cells, we analyzed cytokine production from the culture supernatant of 3G11– and 3G11+ T cells. As shown in Fig. 4B, 3G11– T cells produced no detectable IL-2 and a low level of IFN-{gamma}, but high levels of IL-10 (p < 0.001) when stimulated with MBP1–11 at 10 µg/ml. In contrast, 3G11+ T cells produced high levels of IFN-{gamma} and IL-2, but not of IL-10 when stimulated with autoantigen, similar to the cytokine patterns of MNCs from PBS-i.v. mice. IL-4 was undetectable in both 3G11+ and 3G11– T cells (data not shown). Both 3G11+ and 3G11– T cells produced high levels of these cytokines, and no significant difference was found when they were stimulated with Con A (data not shown).

3G11– T cells actively suppress Ag-specific proliferative responses

Although a large number of CD4+ T cells in animals that were tolerized with MBP1–11-i.v. lost the expression of 3G11 Ag on their surfaces, a certain number of 3G11+ T cells retained 3G11 expression (as shown in Fig. 3) and responded well to autoantigen in vitro (Fig. 4). It was unclear why the presence of significant numbers of MBP-reactive T cells in these tolerized mice failed to cause EAE in vivo. We postulated that increased numbers of 3G11– T cells induced by i.v. tolerance play a role in the active suppression of MBP-reactive T cells. Fig. 5 shows that splenocytes from PBS-i.v. mice had high proliferative responses to MBP1–11, and when these cells were cocultured with 3G11– T cells at a ratio of 1:0.3, the proliferative response was dramatically suppressed (p < 0.001). In contrast, there were no changes in the proliferative responses to MBP1–11 of splenocytes that were cultured with and without adding 3G11+ T cells. Furthermore, the suppression by 3G11– T cells was Ag specific, because 3G11– T cells did not suppress proliferative responses to Con A (Fig. 5).


Figure 5
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  		FIGURE 5. 3G11– T cells actively suppress proliferative responses of MNCs. MNCs from spleen of PBS-i.v. mice were cultured in individual wells (5 x 105/200 µl) in triplicate with MBP1–11 (10 µg/ml) and Con A (5 µg/ml) without Ag/mitogen. 3G11– cells purified from spleens of MBP1–11-i.v. mice were cocultured in the wells with MNC at a ratio of 1:0.3 (final volume, 200 µl/well). 3G11+ cells were added separately as described above. The p values refer to comparisons between MNCs without extra cells and MNCs cocultured with 3G11– or 3G11+ T cells. #, Comparison between cultures with 3G11– T cells and no extra cells; *, comparison between cultures with 3G11– T cells and 3G11+ T cells; ##, p < 0.01; ### and ***, p < 0.001. The data are representative of two repeated experiments.

 
IL-4 and IL-10 in the active suppression by 3G11– cells

The crucial role of soluble factors from 3G11– T cells in the suppression of Ag-reactive cells was addressed by culturing the latter with supernatant of these T cells. As shown in Fig. 6A, supernatants from 3G11+ T cells clearly stimulated the proliferative responses of MBP-reactive T cells in a concentration-dependent manner. In contrast, supernatant from 3G11– T cells significantly suppressed the proliferative responses of MNCs to MBP1–11 in a dose-dependent manner (Fig. 6B). The stimulating effect of supernatant from 3G11+ T cells could be suppressed by exogenous rIL-4 or IL-10 (Fig. 6C). In contrast, the suppression by supernatants of 3G11– T cells could be partly reversed by anti-IL-10 (p < 0.05; Fig. 6D), confirming that the function of 3G11– T cells is at least partially mediated by immunoregulatory cytokines.


Figure 6
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  		FIGURE 6. Supernatant from 3G11– T cells suppresses proliferative responses. A and B, Splenocytes from PBS-i.v. EAE mice were cultured at 5 x 105/200 µl in triplicate with MBP1–11 (10 µg/ml). Supernatants from cultures of 3G11+ (A) and 3G11– (B) T cells were harvested 72 h after culture, pooled, and added to the wells containing splenocytes at volumes of 0, 1.56, 3.13, 6.25, 12.5, 25, and 50 µl, with the final volume kept at 200 µl/well. C, rIL-4, rIL-10, and rIL-4 plus rIL-10 were added with the supernatant of 3G11+ T cells. The p values refer to comparisons between MNCs cultured with supernatants from 3G11+ T cells and splenocytes cultured without supernatant or with supernatant and rIL-4 or rIL-10. D, For Ab-blocking experiments, monoclonal rat anti-mouse IL-4 and IL-10, purified polyclonal mouse IgG, and anti-IL-4 with anti-IL-10 were added with the supernatant of 3G11– T cells. Symbols refer to mean values, and bars to the SD. The p values refer to comparisons between splenocytes cultured with supernatants from 3G11– T cells and without supernatant, or with supernatant and anti-IL-4 or anti-IL-10 Abs. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The data are representative of two repeated experiments.

 
Transfer of 3G11–CD4+ T cells in vivo suppresses clinical EAE

To determine the in vivo regulatory function of 3G11– T cells, we transferred these cells into B6 mice immunized with MOG35–55 peptide. As shown in Fig. 7, a single injection of 3G11–CD4+ T cells significantly suppressed clinical EAE at both doses of cells. In contrast, transfer of 3G11+ T cells did not suppress EAE and, in fact, enhanced the clinical disease at a high cell number (Fig. 7). These data together with our in vitro results provide direct evidence that 3G11– T cells are a distinct subpopulation that possesses Treg properties in vitro and in vivo.


Figure 7
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  		FIGURE 7. Transfer of 3G11– T cells effectively suppresses EAE. 3G11–CD4+ T cells were purified from spleens of naive MOG TCR transgenic mice. After being stimulated with MOG35–55 peptide for 3 days, these cells were injected i.v. at 10 x 106 (A) or 40 x 106 cells/mouse (B) to wild type C57BL/6 mice on the day of immunization with MOG35–55 and CFA. Mice receiving purified 3G11+CD4+ T cells or no cells served as the control. Clinical EAE was scored according to a 0–5 scale as shown (n = 5 in each group). *, Comparison between 3G11– T cell-injected mice and mice receiving no cells; #, comparison between 3G11+ T cell-injected mice and 3G11– T cell-injected; @, comparison between 3G11+ T cell-injected mice and mice receiving no cells; *, #, and @, p < 0.05; ** and ##, p < 0.01; ***, p < 0.001. The data are representative of two repeated experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates that loss of the surface Ag 3G11 on Ag-specific CD4+ T cells represents a unique characteristic for anergic T cells, which also exhibit a Treg phenotype in vitro and effectively suppress clinical EAE in vivo.

Tolerance induction to EAE has been accomplished by administration of specific encephalitogenic Ags in a variety of tolerogenic forms and by various routes, including mucosal, i.v., i.p., and s.c (4, 30). Ag-specific immunological tolerance could be induced via different mechanisms, including clonal deletion, anergy, and active suppression, depending on the route, form of Ag, dose, and time kinetics (3, 27, 28, 31, 32). In our study clonal deletion could be excluded, because MBP1–11-i.v. and PBS-i.v. mice contained similar total numbers of MNCs in the central and peripheral immune organs and, most importantly, a similar percentage and absolute numbers of MBP-reactive T cells recognized by a clonotypic Ab 19G (26). Instead, our protocol, administration of MBP1–11 peptide at the beginning of EAE induction, induced Ag-specific T cell anergy in these mice, as shown by suppressed proliferation and IL-2 production. It has been suggested that anergy induced in adaptive tolerance is most often initiated in naive T cells in vivo by stimulation in an environment deficient in costimulation or high in coinhibition. Anergic CD4+ T cells fail to proliferate and produce IL-2 and IFN-{gamma} upon autoantigen stimulation, but produce a high level of IL-10, and the hyporesponsiveness cannot be reversed by exogenous IL-2 (7). Likewise, this type of anergic CD4+ T cell has been induced in our model. To search for a cell surface marker to identify anergic T cells, we tested the expression of the 3G11 molecule on transgenic T cells from tolerized and nontolerized mice, because 3G11+ T cells stimulated by mitogen produced a large amount of IL-2, but the 3G11– population did not (22). Our findings were directly confirmed by the culture system using purified CD4+3G11– T cells, which exhibit most properties of anergic T cells. Furthermore, the hyporesponsiveness in purified 3G11– T cells occurred after autoantigen stimulation, whereas the T cell response to the mitogen Con A remained intact, providing evidence that 3G11– T cells were specifically anergic to the autoantigen.

T cells rendered anergic have classically been characterized as cells that neither proliferate nor produce IL-2 after re-exposure to the cognate Ag or other TCR cross-linking reagents (8). The hyporesponsiveness can be reversed by exogenous IL-2, demonstrating that the anergic T cell can be activated to the extent that it bears a high affinity IL-2R (8). However, recent studies have shown that in most in vivo anergy models, the proliferative block cannot be reversed by IL-2. This block of IL-2R signaling can be achieved in vitro (33, 34) and in vivo (35) and may involve CTLA-4 signaling (36). The expression of low level CD62L on 3G11– T cells was similar to that on 3G11+ T cells, suggesting that a fraction of anergic T cells exhibited an activation/memory phenotype, and this may explain the reversal of anergy after discontinuation of tolerogen (7).

Of note is the finding that in MBP1–11-i.v. animals, a definite number of 3G11+ T cells persisted after induction of tolerance, and these cells were fully responsive to the autoantigen MBP1–11 when stimulated in vitro. However, it was unclear why the presence of a significant number of MBP-reactive T cells in tolerized mice failed to cause EAE in vivo. We postulated that the function of these autoantigen-reactive T cells may be inhibited in vivo by an active immunosuppressive mechanism. Indeed, we found that proliferative responses to autoantigen were dramatically suppressed in splenocytes of PBS-i.v. mice when these cells were cocultured with 3G11– T cells, and this suppression was cell number dependent. 3G11–CD4+ cells produced a dramatically increased level of IL-10, but no IL-2 and low IFN-{gamma}, indicating a Treg phenotype. More importantly, transfer of purified 3G11– T cells in vivo effectively suppressed clinical EAE, directly confirming the T regulatory property of these cells. The phenomenon of anergic T cells producing IL-10 has been reported in several recent studies (19, 37, 38). More recently, Mana et al. (39) showed that repeatedly injected superantigen induced a tolerant state, resulting from a combination of both clonal anergy and cytokine-mediated immunosuppression involved in the up-regulation of IL-10 and TGF-beta. Our present study shows that IL-10 plays an important role in the immunoregulation by 3G11– T cells. 3G11– T cells have also been found to produce IL-4 upon stimulation with anti-CD3 (40). Although IL-4 was not detectable in the present study, IL-4 may also play a role in the immunoregulatory process, because anti-IL-4 could also partially reverse the suppression. Furthermore, there must be other immunoregulatory factors involved, because blocking both IL-4 and IL-10 could not completely reverse the 3G11– supernatant-induced suppression of proliferative responses to autoantigen.

How T cells, after exposure to certain stimuli, become anergic and exhibit immunoregulatory characteristics remains unclear. Four mechanisms might potentially contribute to this phenomenon. First, inhibition of IL-2 production and loss of proliferation could be a consequence of an altered TCR-transduced signal when T cells are used as APCs (41). In that case, T cell Ag promotes a cohort of T cells in which IL-2 secretion has been silenced and the capacity to secrete Th2 cytokines maintained. This cohort of T cells has been produced in vitro, and in this study we have produced them in vivo by the induction of i.v. tolerance. Second, anergizing stimuli could lead to inhibition of the expression of one cytokine gene, but not another in the same cell (42), suggesting the likelihood that some functions were inhibited, but others remained with the same Ag specificity. When compared with Th1 cells, Th2 cells were more difficult to anergize, and this difference was probably due to different signals being generated after engagement of the TCR complex in these two cell types (42). As a result, these T cells induced with anergizing stimuli might result in the selective loss of Th1 characteristics and retention of a Th2 phenotype (43). Third, the function of 3G11– T cells may at least partially overlap with that of CD4+CD25+ T cells, a well-defined immunoregulatory population (44, 45, 46, 47). Indeed, we found that although CD25+ T cells consist of a small proportion (~5%) of CD4+ T cells, almost all CD4+CD25+ T cells (~93%) were 3G11–, whereas 3G11– T cells were only 30% CD4+CD25– T cells (G.-X. Zhang, S. Yu, and A. M. Rostami, unpublished observations), suggesting a close relation between CD25+ and 3G11– populations. Fourth, the increased expression of CD25 on 3G11– T cells suggests that consumption of IL-2 may also account for the in vitro suppressive activity of these cells. It has been found that competition for locally produced IL-2 is one of mechanisms underlying the immunosuppression by anergic T cells, because these cells express an increased level of IL-2R (CD25{alpha}) (48). CD4+CD25+ Treg cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production (47). Taken together, our present study shows that Ag-specific anergy to autoimmune diseases can be induced in vivo accompanied by an immunoregulatory response. Furthermore, the findings that 3G11– T cells exhibit Treg properties in vitro and effectively suppress clinical EAE in vivo suggest that 3G11– may be a new Treg marker in addition to CD4+CD25+.

In summary, the present study provides evidence that loss of the surface molecule 3G11 characterizes a distinct population of anergic/Treg T cells. This is the first demonstration of the ability to identify and purify anergic T cells by a distinct cell surface marker in an autoimmune disease and paves the way for a better understanding of the mechanism of tolerance in autoimmune diseases.


   Acknowledgments
 
We thank Dr. R. Caspi for the MBP TCR transgenic mice, the late Dr. C. Janeway for permission to use the mice and for the gift of 19G Abs, Dr. V. Kuchroo for MOG TCR transgenic mice, and Drs. M. Greene and H. Maeda for the gift of the antimouse 3G11 Ab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health and the National Multiple Sclerosis Society. Back

2 Address correspondence and reprint requests to Dr. Abdolmohamad Rostami, Department of Neurology, Thomas Jefferson University, 300 JHN Building, 900 Walnut Street, Philadelphia, PA 19107. E-mail address: a.m.rostami{at}jefferson.edu Back

3 Abbreviations used in this paper: Treg, T regulatory cell; EAE, experimental autoimmune encephalomyelitis; MBP1–11, peptide Ac1–11 of myelin basic protein; MNC, mononuclear cell; MOG, myelin oligodendrocyte glycoprotein; p.i., postinfection. Back

Received for publication February 7, 2005. Accepted for publication January 5, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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