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The Journal of Immunology, 2004, 173: 3631-3639.
Copyright © 2004 by The American Association of Immunologists

B7-2 (CD86) Controls the Priming of Autoreactive CD4 T Cell Response against Pancreatic Islets1

Deepak Yadav, Valeria Judkowski, Malin Flodstrom-Tullberg2, Lori Sterling, William L. Redmond, Linda Sherman and Nora Sarvetnick3

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The B7-1/2-CD28 system provides the critical signal for the generation of an efficient T cell response. We investigated the role played by B7-2 in influencing pathogenic autoimmunity from islet-reactive CD4 T cells in B7-2 knockout (KO) NOD mice which are protected from type 1 diabetes. B7-2 deficiency caused a profound diminishment in the generation of spontaneously activated CD4 T cells and islet-specific CD4 T cell expansion. B7-2 does not impact the effector phase of the autoimmune response as adoptive transfer of islet Ag-specific BDC2.5 splenocytes stimulated in vitro could easily induce disease in B7-2KO mice. CD4 T cells showed some hallmarks of hyporesponsiveness because TCR/CD28-mediated stimulation led to defective activation and failure to induce disease in NODscid recipients. Furthermore, CD4 T cells exhibited enhanced death in the absence of B7-2. Interestingly, we found that B7-2 is required to achieve normal levels of CD4+CD25+CD62L+ T regulatory cells because a significant reduction of these T regulatory cells was observed in the thymus but not in the peripheral compartments of B7-2KO mice. In addition, our adoptive transfer experiments did not reveal either pathogenic or regulatory potential associated with the B7-2KO splenocytes. Finally, we found that the lack of B7-2 did not induce a compensatory increase in the B7-1 signal on APC in the PLN compartment. Taken together these results clearly indicate that B7-2 plays a critical role in priming islet-reactive CD4 T cells, suggesting a simplified, two-cell model for the impact of this costimulatory molecule in autoimmunity against islets.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells require two types of signals from APCs for their activation: the engagement of TCR by the MHC/peptide complex and the interaction between the costimulatory molecules and their corresponding receptors. The B7-1/2-CD28/CTLA-4 pathway has been shown to play a crucial role in negative selection (1), CD4/CD8 T cell homeostasis (2), induction of autoimmunity (3), and the maintenance of T regulatory cells (T-regs)4 (4). Interaction between CD28 on T lymphocytes and B7-1 (CD80) or B7-2 (CD86) on APCs provides a critical signal for activation, as well as differentiation of mature T cells into functional effectors (5).

B7-1 and B7-2 show differential expression patterns. B7-2 is constitutively expressed on most APCs and is up-regulated to a greater degree than B7-1 (6, 7). The relatively early induction of B7-2 suggests its role in the initiation of the immune response (8). A number of investigators have studied the individual roles played by B7-1 and B7-2 in autoimmunity. Studies done on models of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis have revealed that B7-1 has an important role during the effector stages of the autoimmune response (9, 10). Indeed, anti-B7-1 mAb treatments inhibit the development of EAE, while the anti-B7-2 mAb exacerbates disease (11). However, in murine Sjogren’s syndrome, anti-B7-2 mAb treatment prevents the development of autoimmune lesions. Thus these studies indicate that B7-1 and B7-2 may each have a unique and dynamic role during T cell responses (12). In NOD mice, a spontaneous model for type 1 diabetes, anti-B7-1 mAb treatment accelerates disease, while anti-B7-2 treatment prevents the development of diabetes (13). In parallel, it was observed that genetic deletion of B7-1 causes disease acceleration, whereas mice deficient in B7-2 in the NOD background have been shown to be completely protected from type 1 diabetes with the possible presence of a regulatory T cell component (14). Although immunoregulatory T cells indeed require costimulation (4), it is unclear whether engagement of other costimulatory molecules in the absence of B7-2 is optimal for their activation. The aim of the present study was to define the role of costimulation by B7-2 in the development of autoimmunity and to delineate the factors associated with the protection from type 1 diabetes in B7-2 knockout (KO) mice. Our results indicate that in the absence of B7-2/CD28-mediated costimulation, CD4 T cells fail to receive activation or expansion signals. The resulting T cell repertoire consists mainly of naive T cells that are functionally nonpathogenic and nonregulatory. Furthermore, B7-2 deficiency significantly depletes T-regs from the thymus. Taken together, our results indicate that lack of activation of CD4 T cells underlies protection from type 1 diabetes in the absence of B7-2. Our work provides a simple explanation for the contribution of this costimulatory molecule to the development of autoreactivity.


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

Female NODshi, BDC2.5 NOD, and NODscids were obtained from The Scripps Research Institute Animal Facility (La Jolla, CA) and used at 5–8 wk of age (unless otherwise mentioned). The B7-2KO mice on the NOD genetic background were originally obtained from J. Bluestone (University of California, San Francisco, CA) and maintained in the institute’s animal facility. Animals were kept in pathogen-free conditions. All experiments were conducted in accordance with institutional guidelines for animal care and use.

Abs and flow cytometric analyses

Single cell suspensions from the spleen, pancreatic lymph nodes (PLN), and thymuses were prepared and followed by RBC lysis with ammonium chloride lysis buffer and washed with PBS. Cells (5 x 105 per sample) were incubated with the appropriate amounts of mAbs (1 µg per million cells) and stained for indicated cell surface markers. They included CD4, CD8, V{beta}4, CD11b, CD11c, CD25, CD40, CD44, CD45RB, CD62L, CD69, CD80, annexin, and ICOS and were obtained from BD Pharmingen (La Jolla, CA). For flow cytometric analyses, the dead cells were gated out on the basis of forward and side scatter. Appropriate isotype controls were used to determine the background staining. All mean values quoted in the text are ± SD.

TCR/CD28-mediated stimulation of PLN cells in vitro

Four- to 5-wk-old B7-2KO and NOD mice were used for the isolation of PLN cells. For in vitro activation, 48-well tissue culture plates were first coated with 1 µg/ml anti-CD3 Ab (145-2C11 clone) and washed with PBS before the addition of PLN cells (1 x 106 cells per well) in complete RPMI 1640 (containing 10% FCS, 5 U/ml and 50 µg/ml penicillin-streptomycin, respectively, 10 mM HEPES buffer, and 2 mM L-glutamine). Soluble anti-CD28 Ab (0.5 µg/ml, 7.51 clone) was also added for costimulation. Cells were harvested at 24 h poststimulation, washed with PBS, and then proceeded for staining.

Adoptive transfers

For ex vivo activation of BDC2.5NOD (6 wk of age) splenocytes, the cells were subjected to RBC lysis and were incubated (2 x 106/ml) with agonist mimotope peptide 1040-79 (700 ng/ml, AVRPLWVRME) for 3 days as described by Judkowski et al. (15). The cells were then washed with PBS and transferred i.v. (3 x 107 cells per recipient) into 5- to 6-wk-old NOD and B7-2KO recipients. In addition, an aliquot of the activated cells (i.e., at day 3 postactivation) was also stained for activation markers such as CD25 and CD69 and analyzed by flowcytometry. More than 80% of V{beta}4+CD4+ cells were found to be positive for both the markers confirming their activation status. For CFSE labeling, BDC2.5 (6–7 wk of age) splenocytes (5 x 107) were incubated with 5 mM CFSE for 10 min at 37°C. The reaction was quenched with 10 ml of cold PBS and followed by washing twice in PBS. The 2.5 x 107 CFSE-labeled total splenocytes in 200 µl of sterile PBS were injected i.v. per recipient (7- to 8-wk-old NOD and B7-2KO mice). For monitoring the extent of cell death in the adoptively transferred cells, PLN cells (1–2 x 106) from the recipients were also stained for annexin and V{beta}4+CFSElow cells were analyzed for the cells positive for annexin. Flow cytometry staining determined that of the total splenocytes, ~20% were CD4+V{beta}4+ and also that 100% of cells were CFSE positive.

In other adoptive transfer experiments, 8- to 10-wk-old B7-2KO and NOD mice were used as splenocyte donors for NODscid (5–6 wk of age) recipients and 3 x 107 cells were i.v. injected per recipient.

For cotransfer experiments, 10- to 12-wk-old NOD and B7-2KO donors were used for 4- to 5-wk-old NODscid recipients, and 3 x 107 splenocytes in total were transferred i.v. either alone or in combination (ratio 1:1). Another group of NODscid received 1.5 x 107 NOD splenocytes, which served as an internal control.

For anti-CD3/CD28 stimulation-mediated adoptive transfers, whole splenocytes (2 x 106 cells/ml) from 9- to 10-wk-old NOD and B7-2KO donors were incubated in vitro with surface bound anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) in complete RPMI 1640 containing 10% FCS. The cells were incubated for 3 days, followed by extensive washing with PBS. These cells were then transferred i.v. into 4- to 5-wk-old NODscid (3 x 107 cells/recipient). The activation state of the cells before transfer was monitored by flow cytometry and more then 95% of the cells were CD25+CD44high.

Diabetes and immunohistochemistry

For histological assessment of islet infiltration, pancreata were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections (4 µm) were stained with either H&E or with anti-insulin Ab (10–15 ng/ml; DakoCytomation, Carpinteria, CA) and hematoxylin counterstain.

For anti-V{beta}4 (KT4 used at 5 ng/ml) staining, the pancreas of recipient mice were quick-frozen in OCT compound and 4-µm sections were cut for staining.

Blood glucose levels were monitored using Glucometer Elite strips (Bayer, Mishawaka, IN). Mice with two successive weekly blood glucose levels >250 mg/dl were considered diabetic.

Statistical analyses

The Student t test (unpaired) was used to determine the level of significance of the data using Statview software (Abacus Concepts, Berkely, CA). A value of p < 0.05 was considered as significant. In some sections of the results, the individual values corresponding to each animal from several independent experiments were pooled and statistically analyzed as a combined larger group, and the p value was calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of B7-2 inhibits the expansion of autoreactive CD4+ T cells in vivo

In the absence of B7-2, the autoimmune response in NOD mice is completely inhibited (14). Therefore, we asked whether expansion of islet-specific CD4+ T cells is affected when they encounter APCs that lack B7-2. For this purpose, BDC2.5 NOD TCR-transgenic mice, which harbor a TCR defined as V{alpha}1V{beta}4 recognizing an endogenous islet {beta} cell Ag, were used as donors in adoptive transfer studies (16, 17). Adoptively transferred CFSE-labeled naive BDC2.5 splenocytes were analyzed for their proliferation on the basis of CFSE dilution on day 4 posttransfer into NOD and B7-2KO recipients. Our results indicate that CD4+V{beta}4+ T cells show significantly lower levels of proliferation in B7-2KO recipients (M2; Fig. 1b), while NOD recipients displayed a significantly higher (p = 0.0015) percentage of proliferating BDC2.5 T cells (M2; Fig. 1a). Accordingly, B7-2KO animals also exhibited a significantly larger (p = 0.0016) fraction of undivided (M1) cells as compared with NOD controls. We also observed a lower percentage of activated CD4+V{beta}4+CFSE+CD44high cells in B7-2KO mice as compared with NOD mice (data not shown). These results present direct in vivo evidence of the critical role played by B7-2 costimulatory molecule in the expansion and activation of autoreactive CD4 T cells.



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  		FIGURE 1. Islet-specific T cells do not proliferate in B7-2KO NOD mice. CFSE-labeled splenocytes (2.5 x 107 cells) from 6- to 7-wk-old BDC2.5 mice were administered i.v. into 7- to 8-wk-old NOD and B7-2KO recipient mice. After 4 days, the PLN cells were analyzed by flow cytometry for CFSE dilution to assess for proliferation. The CFSE profile of V{beta}4+CD4+ T cells in NOD and B7-2KO recipients is represented by A and B, respectively. M1 and M2 represent mean ± SD (n = 4) of resting and dividing cells respectively. Analysis of cells before transfer indicated >80% of them were naive (i.e., CD4+CD44low). Data are representative of two independent experiments with similar results.

 
Predominance of naive CD4+ T cells in B7-2KO mice

Because B7-2 was shown to affect the activation status of CD4 T cells in autoimmune models (18), and based on the results above, we wondered whether the lack of B7-2 affects the activation state of CD4 T cells in B7-2KO mice. Consequently, the PLN, the primary site for pancreatic Ag priming and spleen, were analyzed by flow cytometry to test whether NOD and B7-2KO mice differ in the frequency of spontaneously generated activated T cells in the CD4 T cell compartment. We ascertained the frequency of CD4 T cells within the activated/memory compartment on the basis of CD45RBlow, CD44high, and CD25 staining. In the B7-2KO mice, significantly lower percentages of CD45RBlow, CD44high, and CD25+ T cells were observed in the CD4 compartment as compared with wild-type (WT) NOD (Fig. 2 and Table I). Also, these results were supported by the observation that B7-2KO mice exhibited a significantly higher percentage of naive CD4 T (CD44lowCD45RBhigh) cells when compared with WT NOD mice (Fig 2D and Table I). These findings illustrate the relatively naive phenotypic characteristic of the CD4 cells in the absence of B7-2 signaling, and point to a defect in the generation of a normal activated/memory CD4 T cell compartment.



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  		FIGURE 2. B7-2 critically contributes to the activation of CD4 T cells. To compare the activation state of CD4 T cells, PLN, and spleen cells from NOD and B7-2KO mice (6- to 7-wk-old) were stained for CD4 along with CD45RB (A), CD44 (B), and CD25 (C), and analyzed by FACS. For the determination of the naive CD4 population, the cells were gated on total CD4 and analyzed for CD45RBhighCD44low population (D, region R). The flowcytometric profiles are the dot plot representation (from PLN) of the data described in Table I.

 

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  		Table I. Comparison of activated CD4 T cell compartment in NOD and B7–2KO mice

 
B7-2KO mice demonstrate decreased CD4 T cell numbers in PLN

T cell proliferation normally leads to robust CD4 T cell numbers in the periphery. Therefore, we next set out to determine whether the severe deficiency in activation/proliferation of CD4 T cells in the absence of B7-2 would impact on the total number of CD4 T cells in the PLN and spleen. Indeed, we found a significant reduction in the total CD4 T cell numbers in the PLN of the B7-2KO mice as compared with NOD controls These observations were also associated with a concomitant reduction in the CD4/CD8 T cell ratio in the PLN. Interestingly, the spleen compartment of the B7-2KO mice did not exhibit any significant changes in terms of total CD4 T cell numbers or CD4/CD8 ratio, demonstrating that the lack of B7-2/CD28 costimulation displays a more drastic effect on the CD4 T cell profile in the PLN where local presentation with pancreatic Ag occurs (Table II).


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  		Table II. The total CD4 T cell numbers and CD4/CD8 ratio in spleen and PLN compartmentsa

 
B7-2 mediates CD4 T cell activation and stability upon TCR/CD28 engagement

We asked whether the lack of B7-2/CD28 interaction could affect the capacity of CD4 T cells to undergo activation. Consequently, PLN cells from B7-2KO and NOD mice were treated in vitro with anti-CD3 and anti-CD28 and stained for the conventional activation marker CD44 and ICOS. ICOS induction reflects ongoing T cell activation and its expression is dependent upon B7/CD28 costimulation (19). Interestingly, we observed a decreased percentage of activated (CD44high and ICOShigh) CD4 T cells in B7-2KO compared with NOD CD4 T cells (Fig. 3A). We extended our observations to in vitro stimulated, purified splenic CD4 T cells (not shown) from these mice. Following exposure to CD3/CD28, we observed defective up-regulation of CD44 and ICOS upon TCR/CD28 stimulation, indicating that a stable, activated state was not obtained.



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  		FIGURE 3. A, The absence of B7-2 is associated with TCR/CD28-mediated hyporesponsiveness and increased apoptosis of CD4 T cells. Pooled PLN cells (n = 3–4 per experiment) from B7-2KO and NOD mice were either exposed to plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) for 24 h or left unstimulated as internal controls. The activation status of B7-2KO and NOD CD4 T cells was determined on the basis of CD44high and ICOShigh expression. The events in the histograms are gated on total CD4 T cells. The numbers represent the percentage of CD4+CD44high and CD4+ICOShigh T cells and the data are representative of two independent experiments. B, For in vivo screening of apoptosis of CD4 T cells, adoptively transferred (25 million per recipient) CFSE+V{beta}4+ BDC2.5 cells were tracked in vivo (as described for Fig. 1). The histograms are gated on proliferating BDC2.5 cells, and the gating is done on cells depicted by marker M2 in Fig. 1, which are designated in the text as CFSElowV{beta}4+ T cells. These cells were then analyzed for annexin staining in the PLN of NOD and B7-2KO recipients. The numbers represent the mean ± SD of annexin-positive CFSElowV{beta}4+ T cells (pooled data from two independent experiments; n = 6–7). C and D, Both unmanipulated or TCR/CD28-engaged splenocytes from B7-2-deficient mice lack disease-causing potential in NODscid recipients. Splenocytes (3 x 107 cells per recipient) from 8- to 10-wk-old NOD and B7-2KO NOD donors were either transferred unmanipulated (C) or activated in vitro with anti-CD3/CD28 before transfer (3 days). Four- to 6-wk-old NODscid mice were used as recipients and blood glucose levels were monitored at weekly intervals following transfer. Animals with two consecutive observations of >250 mg/dl were considered diabetic.

 
It is known that the absence of costimulation can affect T cell survival. Therefore, we monitored CD4 T cell apoptosis in vivo. Adoptively transferred CFSE-labeled V{beta}4+ BDC 2.5 splenocytes were screened for annexin staining in the PLN compartment of NOD and B7-2KO recipients. We looked for hallmarks of apoptosis in cells undergoing proliferation (these proliferating cells are depicted by marker M2 in Fig. 1) and categorized them as CFSElow. We found that a significantly higher (p = 0.031) percentage of CFSElow V{beta}4+ T cells were undergoing apoptosis (annexin positive) in the B7-2KO recipients compared with NOD mice (Fig. 3B). This suggests that in the absence of B7-2, the proliferating CD4 T cells demonstrate enhanced instability.

B7-2KO donors fail to transfer diabetes

B7-2 costimulation induces the activation and expansion of CD4 T cells. However, it remains unclear whether the T cell repertoire in the B7-2KO mice was truly self-tolerant. Accordingly, we injected splenocytes from B7-2KO and NOD mice into NODscid mice and observed complete failure of B7-2KO splenocytes to transfer disease during the 17-wk observation period. Conversely, splenocytes from WT NOD efficiently induced diabetes in NODscid recipients (Fig. 3C).

Activated (anti-CD3/CD28 treated) splenocytes from neuropathic B7-2KO mice (>20 wk of age) have been reported to transfer neuropathy, but not diabetes, in the NODscid model (14). To test whether we could recover diabetogenic potential in normal B7-2KO mice, we performed adoptive transfer studies. Nine- to 10-wk-old NOD and B7-2KO mice were used as donors and their spleen cells were stimulated with anti-CD3 and anti-CD28 in vitro for 3 days. Thirty million of these activated cells were transferred i.v. into NODscid recipients and diabetes was monitored. None of the NODscid recipients transferred with activated splenocytes from B7-2KO exhibited any signs of diabetes over the 17-wk monitoring period. In contrast, activated NOD splenocytes could efficiently induce diabetes as early as 11 wk posttransfer. These results show the lack of ability of B7-2KO lymphocytes to transfer disease even upon TCR/CD28-mediated stimulation (Fig. 3D).

B7-2 does not affect the effector phase of anti-islet response

We questioned whether activated islet Ag-specific (BDC2.5) CD4 T cells might overcome the barrier of disease induction in B7-2-deficient mice. Consequently, BDC2.5 splenocytes were activated in vitro, using a BDC2.5 agonist mimotope peptide (15), and then adoptively transferred to NOD and B7-2KO mice. Interestingly, the activated BDC 2.5 cells could efficiently trigger autoimmune aggression against the islets leading to clinical diabetes in both B7-2KO as well as NOD recipients by day 6 posttransfer (Fig. 4A). Immunohistochemical analyses of the pancreatic sections by insulin staining also indicated a massive infiltration of lymphocytes into the islets of both B7-2KO and NOD mice, leading to the destruction of insulin producing {beta} cells (Fig. 4B, i and iii, respectively). V{beta}4 immunostaining confirmed the infiltration of transgenic lymphocytes into the islets of the B7-2KO and NOD recipients (Fig. 4B, ii and iv, respectively). These observations show that the requirement for B7-2 can be bypassed by in vitro activation.



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  		FIGURE 4. A, Ex vivo-activated islet-specific T cells can transfer diabetes in B7-2KO NOD mice. Splenocytes (2 x 106 cells/ml) from 6-wk-old BDC2.5 mice were cultured in vitro in the presence of 700 ng/ml BDC-specific mimetope for 3 days. After 3 days, cells (3 x 107 cells per recipient) were injected i.v. into 5- to 6-wk-old NOD and B7-2KO NOD recipients (n = 7 per group) and blood glucose levels were monitored daily following transfer. Animal(s) with two consecutive observations of more then 250 mg/dl were considered diabetic. The data represented are pooled from two independent experiments with similar observations. B, Adoptive transfer of in vitro activated BDC2.5 splenocytes causes massive islet destruction both in WT NOD and B7-2KO NOD mice. Paraffin sections (4 µm) from B7-2KO (Bi) and NOD (Biii) recipients were stained with Ab to insulin. For V{beta}4 staining, the pancreata from B7-2KO (Bii) and NOD (Biv) were quick-frozen in OCT; n = 7 mice were evaluated per group and representative data from one of the recipients from each group is shown.

 
Enhanced B7-1 expression is found in splenic, but not PLN, APCs

Previous reports have suggested that in the absence of B7-2, a concurrent increase of B7-1 is observed (8). Furthermore, a differential (enhanced neuronal B7-1 expression) role of B7-1 has been proposed as one of the factors responsible for the polyneuropathy observed in the B7-2KO mice (14). Therefore, we quantitated B7-1 expression on APCs both in spleen and PLN. We found a significantly higher percentage of B7-1+ cells in both CD11b+ (p = 0.018) and CD11c+ (p = 0.003) populations in the spleens of the B7-2KO mice (Fig. 5A), consistent with the earlier reports (14). Interestingly, in the PLN compartment, no change in B7-1 expression was observed in CD11b+ (p = 0.19), as well as CD11c+ (p = 0.15) populations (Fig. 5B). No significant change in the levels of CD40+ cells in both APC populations was observed in the PLN (data not shown). These results indicate that B7-1 and CD40 do not compensate for the lack of B7-2 costimulation in the PLN, where sensitization to islet Ag occurs (20).



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  		FIGURE 5. B7-1 expression is enhanced in the spleen, but remained unaffected in the PLN of B7-2-deficient mice. For the characterization of B7-1 (CD80) expression in various APC populations, both spleen (A) and PLN (B) cells from 7- to 10-wk-old mice were stained and analyzed by FACS. The dot plot represents the gating pattern from the cells obtained from PLN; a similar gating strategy was used for the analysis of splenocytes (not shown). The cells contained in R1 and R2 are annotated as CD11b+ and CD11c+ cells in the text, respectively. The data are representative of two to three independent experiments (n = 3–4 mice per group).

 
Reduced proportions of CD4+CD25+ T cell subsets

B7/CD28 may play an important role in the maintenance of CD4+CD25+ T cells (4). Therefore, we wondered if a specific lack of B7-2/CD28-mediated costimulation could affect the levels of these cells. Our foregoing results showed a significant reduction in the percentage of CD4+CD25+ T cells in B7-2KO mice (Fig. 2C and Table I). Because this population contains both activated as well as regulatory T cells (21), we performed further phenotypic characterization to distinguish these populations. PLN and spleen cell suspensions from B7-2KO and NOD mice were triple-stained for the activation markers CD62L, CD69, and CD45RB, in addition to CD4 and CD25, to differentiate between regulatory and activated T cells among the CD4+CD25+ T cell population. The results indicate that significantly lower percentages of CD4+CD25+CD62L–/low, CD4+CD25+CD69+, and CD4+CD25+CD45RBlow cells were observed in the PLN in B7-2KO mice compared with NOD controls (Fig. 6A and Table III). The CD4+CD25+CD62L+/high cell population, which has been considered "regulatory" in this diabetes model (22), did not exhibit a substantial reduction but instead showed a statistically marginal reduction in the PLN of B7-2KO mice compared with NOD controls (Fig. 6Ai and Table III). However, no significant difference in the levels of these cells was observed in the B7-2KO spleen compared with NOD (p = 0.098; Table III).



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  		FIGURE 6. A, T cells with a regulatory cell surface phenotype in B7-2-deficient and normal NOD mice. To define the CD4+CD25+ subset, cells (PLN) were analyzed on the basis of other activation markers such as CD62L, CD69, and CD45RB by FACS. Ai–Aiii are dot plot representation (PLN) of the data described in Table III and Aiv represent the thymic profile of CD4+CD25+CD62L+ T cells (see Table III). All dot plots represent total CD4-gated populations. B, Regulatory T cell function is not enhanced in the absence of B7-2. Cotransfer of NOD and B7-2KO splenocytes could induce diabetes in NODscid recipients. Thirty million splenocytes in total were administered i.v. either from NOD (group 1) or B7-2KO mice (group 2) into NODscid recipients. Group 3 represents a cotransfer of 15 million cells each of NOD and B7-2KO splenocytes (30 million in total). Group 4 serves as an internal control with 15 million cells transferred from NOD donors.

 

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  		Table III. Comparison of various CD4+CD25+ subsets in NOD and B7–2KO micea

 
The B7/CD28 interaction has been implicated in the maintenance of the thymic CD4+CD25+ T cells (4). Our next set of experiments was conducted to determine whether the selective loss of B7-2 could affect the levels of CD4+CD25+CD62L+ T cells in the thymus. Interestingly, we observed a significant reduction in the percentage of CD4+CD25+CD62L+ T-regs in the thymus of B7-2KO mice compared with NOD (Fig. 6Aiv and Table III). However, no defect in the levels of other thymic subpopulations such as CD3+CD4+CD8+, CD3+CD4+CD8–, CD3+CD4–CD8+, and CD3+CD4–CD8– T cells was observed in the B7-2KO mice (not shown).

These results demonstrate that it is the activated compartment of CD4 T cells that is significantly affected in the absence of B7-2. In addition, B7-2 is required for the generation of the thymic T-reg compartment.

B7-2KO splenocytes do not exhibit immunoregulatory activity and fail to inhibit disease induction in an adoptive cotransfer system

Previous reports (14) have described that the selective removal of CD4+CD25+ T cells from B7-2-deficient splenocytes can cause diabetes induction in the NODscid model, thereby indicating that T cells from B7-2KO mice display regulatory activity. As we have demonstrated above that the levels of T-regs in periphery did not differ between B7-2-deficient and -control NOD mice, we performed studies to test the regulatory capacity of the B7-2 splenocytes. We performed cotransfer of B7-2KO and NOD splenocytes (ratio 1:1 and 30 million in total per recipient, group 3) into NODscid recipients in addition to controls including transfer of B7-2KO and NOD splenocytes alone which is depicted by groups 1, 2, and 4. We found that these mixed population of cells could still induce diabetes with similar kinetics as described with cells from WT NOD mice (group 3 vs group 4; Fig. 6B). These results suggest that B7-2KO splenocytes do not have an overt regulatory component to influence disease induction in this model.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicate that B7-2 plays a critical role in the initial priming of CD4 T cells. The B7-1/2/CD28 pathway plays a crucial role in T cell costimulation and naive T cell differentiation (23). Although B7-1 (CD80) and B7-2 (CD86) share several important characteristics, these molecules show their capacity to deliver distinct intracellular signals (12) and different kinetics of expression (6, 24). Priming of autoreactive T cells by APCs in type 1 diabetes models involves physiological {beta} cell death (20), which in turn leads to destruction of the islet cells. In this study, we clearly show that Ag presentation in the absence of B7-2/CD28 costimulation have a direct negative impact on T cell activation. These observations are further complemented by the fact that a significantly higher percentage of CD4 T cells display a naive phenotype in B7-2KO mice both in the spleen and PLN (Fig. 2D and Table I). The homing potential of lymphocytes is a function of their activation state (25) and, therefore, reduced activation might explain the lack of pancreatic lymphocyte infiltration observed in the B7-2KO mice. These observations further add to the growing body of evidence that B7-2 plays a pivotal role in the initial priming of T cells. In the experimental autoimmune myasthenia gravis model, B7-2 has been implicated in the activation of acetylcholine receptor-specific T cells (26). In addition, a blockade of B7-2 has been shown to induce CD4 T cell anergy (27) and also inhibit CD4 T cell activation (28).

The decreased percentage of CD4+CD25+ T cells observed in the B7-2KO mice prompted us to further analyze this population because expression of the marker CD25 is shared not only by conventional activated/memory CD4 T cells but also by the T-regs (21). CD4+CD25+CD62L+ T cells have been proposed to have regulatory potential and shown to be protective in this NOD model (22, 29). B7/CD28 costimulation has been reported to maintain the survival of CD4+CD25+CD62L+/high T-reg cells (4). Our results show that the lack of B7-2 signaling diminishes the cellular levels of this T-reg population with a more pronounced reduction in the thymus (Fig. 6Aiv). Thus it appears that the absence of B7-2 has a differential effect on the levels of T-regs in the thymus and in periphery. Regulatory cells are reported to originate both in the thymus and in the periphery (30, 31, 32). Indeed, several factors can influence the local generation of CD4+CD25+ T cells, which may include costimulation through B7-1, and exposure to cytokines such as IL-10 and IL-2 (33, 34). We tested for the presence of the regulatory component in B7-2KO mice by the adoptive cotransfer of NOD and B7-2KO splenocytes and observed similar kinetics of diabetes induction as controls. Diabetogenic T cells have been shown to express the phenotype of activated/memory T cells: low levels of CD62L (29, 35) and CD45RB (36). Indeed a significant reduction in CD62Llow and CD45RBlow subpopulations within CD4+CD25+ T cells was observed in the B7-2KO mice. We believe that these cells represent the autoantigen-specific conventional effector subpopulations within CD4+CD25+ T cells as recently suggested by Szanya et al. (22). Our data provides a simple explanation for the requirement B7-2 for pathogenic autoimmunity that it is the absence of the activated/primed T cell response and not the presence or activity of T-regs which determines the final outcome of disease. However, more detailed analysis of T-regs may be warranted to explore relevant immunoregulatory pathways.

Blockade of B7-2 has been shown to inhibit IL-2 production (6, 37), which may be one of the reasons for reduced expansion of naive BDC2.5 CD4 T cells. Furthermore, the paucity of IL-2 would provide reduced growth signals for CD25+ T cells. In contrast, once activated ex vivo, BDC2.5 cells could efficiently transfer diabetes in B7-2KO mice. These observations provide further credence to the notion that B7-2 plays a significant role during the initiation of autoreactive T cell responses (18, 38, 39) and is dispensable during the later (effector) phase of the immune response. Accordingly, late B7-2 blockade in NOD (10 wk of age or older) is ineffective in controlling diabetes (13). Indeed, B7-2 expression has been correlated with islet-infiltrating cells in spontaneously occurring diabetes in NOD mice (40).

Interestingly, our in vivo experiments revealed both hyporesponsiveness and a propensity to death by apoptosis in the absence of B7-2. Indeed, costimulation through CD28 has been shown to promote T cell survival and activation (41, 42). Whereas the blockade of B7-2 induces CD4 T cell unresponsiveness (26, 27), reflecting the net induction of tolerance through the TCR in the absence of costimulation has been reported to induce rapid Fas-dependent, proliferation-independent death of purified naive CD4 T cells (43). It is also possible that this CD4 T cell death may be mediated through engagement of B7-1 alone or inhibitory receptors during T cell activation. Furthermore, alterations in signaling events in the absence of B7-2/CD28 costimulation may be responsible for the defective CD4 T cell response. ERK signaling has been shown to play a critical role in the processes leading to IL-2 production and activation of T cells (44). Interestingly, we observed reduced levels of phospho-ERK-positive CD4 T cells in the PLN compartment of B7-2KO mice compared with NOD controls (not shown). Cumulatively, we suggest that in the absence of B7-2-mediated costimulation, the PLN microenvironment becomes such that it does not support efficient CD4 T cell response. This may also further involve the deficiency of the compensatory support from other cytokines and costimulatory molecules necessary for full-blown CD4 T cell activation and proliferation.

B7-1 has been shown to compensate for the absence of B7-2 (8). Furthermore, B7-1 has been shown to play an important role in the induction of EAE (11) and experimental autoimmune myasthenia gravis (26). Indeed, enhanced B7-1 expression has been linked to the spontaneous autoimmune peripheral polyneuropathy in B7-2KO NOD mice. We observed an enhanced B7-1 signal associated with both in CD11b+ and CD11c+ populations in the spleens of B7-2KO mice, in agreement with the earlier observations (14). Interestingly, no significant changes in B7-1 expression levels were observed in the PLN (both in CD11b+ and CD11c+ populations), suggesting the tissue-specific expression profile of B7-1. These results also indicate that B7-1 cannot always compensate for the absence of B7-2. It is likely that local cellular interactions and the cytokine/chemokine microenvironment may play a role in this tissue specific up-regulation of B7-1. Moreover, B7-1 has been shown to play an important role in the effector phase of immune response (9, 45), whereas B7-2 regulates the initiation phase (5, 38, 46, 47). Furthermore, despite the presence of enhanced B7-1 signals in the spleen, there is an absence of pathogenic islet reactive T cells. This may reflect different threshold requirements for the activation of islet reactive T cells in the spleen. The other possibility is that tissue-specific expression of B7-1 may be the deciding factor in determining the generation of an autoimmune repertoire. Indeed, B7-2 expression on microglial cells is associated with neuroprotection (48) and B7-1 expression may lead to nerve damage (49). Therefore, the absence of B7-2 may expose nerve tissues to a B7-1-mediated immune attack. B7-2 has been shown to be present on immunogenic APCs in the islets (40). However, our studies have revealed the absence of B7-1 induction in the PLN, demonstrating that there may be a differential regulation of B7-1 and B7-2 by PLN and nerve tissue APCs.

Taken together, our results demonstrate that the primary role of B7-2 is to initiate T cell priming, directly affecting autoimmune T cell frequency and pathogenicity. Logically, if in the absence of B7-2, the T cell repertoire has been rendered benign, it is unclear what role would be required of regulatory T cells. Our study provides a direct link between the lack of B7-2 and the absence of an autoreactive response owing to defective initial T cell priming to islet Ags.


   Acknowledgments
 
We thank Lee Tucker and Mary Cleary for technical assistance, and the members of the lab for critical reviews of the manuscript.


   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 a grant from National Institutes of Health (DK 57644 with L.S. as principal investigator and N.S. as coprincipal investigator). D.Y. is a recipient of Osserman/Sosin/McClure Fellowships by Myasthenia Gravis Foundation of America for 2003. This is manuscript number 16511-IMM from The Scripps Research Institute. Back

2 Current address: Department of Medicine, Center for Infectious Medicine, Karolinska Institute, Stockholm, Sweden. Back

3 Address correspondence and reprint requests to Dr. Nora Sarvetnick, Scripps Research Institute, Department of Immunology, 10550 North Torrey Pines Road, Mail Drop IMM-23, La Jolla, CA 92037. E-mail address: noras{at}scripps.edu Back

4 Abbreviations used in this paper: T-reg, T regulatory cell; EAE, experimental autoimmune encephalomyelitis; KO, knockout; PLN, pancreatic lymph node; WT, wild type. Back

Received for publication April 27, 2004. Accepted for publication July 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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