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CD4⁺ T Cell Expressed CD80 Regulates Central Nervous System Effector Function and Survival during Experimental Autoimmune Encephalomyelitis¹

Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD80 expressed on the surface of APCs provides a positive costimulatory signal to naive CD4⁺ T cells during activation. Therefore, it was hypothesized that treatment of SJL mice with various forms of anti-CD80 mAb during remission from the acute phase of relapsing experimental autoimmune encephalomyelitis (R-EAE) would ameliorate disease progression. We previously reported that treatment of SJL mice with anti-CD80 Fab during R-EAE remission blocked activation of T cells specific for endogenous myelin epitopes, inhibiting epitope spreading and clinical disease progression; however, treatment with the native form of the same anti-CD80 mAb exacerbated disease progression. The current data show that intact anti-CD80 mAb binds both CNS-infiltrating CD4⁺ T cells and CD11c⁺ dendritic cells and that exacerbation of R-EAE directly correlates with increased survival and activity of myelin-specific CD4⁺ T cells, while the percentage of CD11c⁺ dendritic cells in the CNS and their APC activity was not altered. In vitro data show that cross-linking CD80 on the surface of CD4⁺ T cells activated in the presence of Th1-promoting cytokines increases the level of T cell activation, effector function, and survival by directly up-regulating the expression levels of transcripts for T-bet, IFN-, and Bcl-x_L. These findings indicate a novel regulatory role for CD80-mediated intracellular signals in CD4⁺ T cells and have important implications for using anti-costimulatory molecule mAb therapy in established autoimmune disease.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Blockade of B7-CD28 interactions during initial CD4⁺ T cell activation leads to long-term unresponsiveness of CD4⁺ T cells undergoing activation at the time of treatment (1, 2). This blockade of naive CD4⁺ T cell activation is based on the two-signal requirement for a T cell to become fully activated. The first signal necessary for naive CD4⁺ T cell activation is specific TCR binding of the MHC II-Ag complex on an APC (2). The second "costimulatory" signal is largely provided via CD28 expressed on the CD4⁺ T cell surface binding one of the B7 family members of APC-expressed costimulatory molecules, CD80 or CD86 (3, 4). Due to the role that the B7-CD28 costimulatory pathway plays in the activation of CD4⁺ T cells, it has been hypothesized that blockade of these costimulatory signals may serve as a therapy for autoimmune disease (5, 6, 7). Although the signaling events following CD28-B7 interaction has been studied for the CD28 signaling cascade in CD4⁺ T cells, the ability of CD80 stimulation to induce an intracellular signal has been investigated only recently in "classic" CD80-expressing cell types, such as B cells and dendritic cells (DCs)³ (8, 9). Besides APCs, activated CD4⁺ T cells also express CD80 and CD86, and CD4⁺ T cells have been shown to be the primary CD80-expressing cell type in the CNS during relapsing experimental autoimmune encephalomyelitis (R-EAE) (10). Furthermore, the potential of CD80-CD28/CTLA4 involvement in T cell-T cell interactions during graft-vs-host disease has been reported (11). Therefore, it is possible that these costimulatory molecules potentially directly signal to CD4⁺ T cells following cross-linking with a mAb (12, 13), thereby affecting T cell effector function.

The role of B7 costimulatory molecules in chronic autoimmune diseases, such as multiple sclerosis (MS), is suggested by the increased levels of CD80 detected in MS lesion sites (14), as well as in cerebrospinal fluid from MS patients (15). Thus, CD80 appears to be the predominant costimulatory molecule in MS and the experimental model of disease in SJL mice, i.e., R-EAE. It has been hypothesized that blockade of CD80/CD28 costimulation may specifically inhibit the activation of T cells to endogenously presented myelin epitopes, which play a role in disease progression. However, studies using anti-CD80 mAb to "block" CD80/CD28 interactions have led to conflicting results regarding the role of CD80 in the induction and progression of disease. These published findings suggest that there is a time dependency for treatment effect as determined by disease outcome. Furthermore, there is a differential effect on disease outcome depending on which form of the Ab is used, i.e., intact anti-CD80 mAb vs anti-CD80 Fab (6, 7, 12, 16, 17, 18, 19, 20). This is supported by our previous demonstration that treatment of SJL mice during remission from the acute clinical episode of R-EAE with anti-CD80 Fab blocked the activation of T cells specific for endogenous myelin epitopes, thus inhibiting epitope spreading and consequently clinical disease progression. In contrast, treatment with the native form of anti-CD80 mAb exacerbated disease progression (17). These findings suggest that intact anti-CD80 mAb may directly signal through CD80 expressed on activated CD4⁺ T cells (21) and/or APCs to influence cellular function (9).

The current data show that intact anti-CD80 mAb is bound to both CD11c⁺ DCs and CD4⁺ cells in the CNS following treatment. While published data have begun to determine the effect of CD80 cross-linking on DC function (9), our study focused on the consequence of cross-linking CD80 on the surface of CD4⁺ T cells. We show that CD80 cross-linking and signaling in CD4⁺ T cells activated in the presence of Th1-promoting conditions increases both the level of IFN- production and cell survival. Accordingly, CD80 stimulation also increased the levels of IFN- and T-bet mRNA and Bcl-x_L protein. The in vivo relevance of these in vitro findings is suggested by the finding that treatment with the intact form of anti-CD80 mAb enhanced the survival of PLP_139–151-specific T cell in the CNS, whereas treatment with anti-CD80 Fab decreased survival. Collectively, these findings suggest that CD80 signaling in CD4⁺ T cells enhances effector function, whereas CD80 blockade with anti-CD80 Fab interferes with pathogenic mechanisms of autoimmune disease progression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice, cell isolation, peptides, and reagents

Female SJL mice were purchased from Harlan Sprague Dawley, and 5B6 TCR transgenic (Tg) (PLP_139–151/I-A^s-specific) either on wild-type or Thy1.1⁺ background are currently bred in the Northwestern University Center for Comparative Medicine. Naive CD4⁺ T cells were purified using mouse naive CD4⁺ T cell AutoMacs Magnetic Bead isolation kit (Miltenyi Biotec) and found to be >98% CD4⁺,CD25^–,CD62L^high via flow cytometry. Peptides (PLP_139–151, PLP_178–191, and MBP_84–104) were purchased from Peptides International and purified by HPLC (purity of 96–99%).

PLP_178–191 EAE, 5B6 Tg CD4⁺ T cell transfer, and Ab treatment

Six- to 7-wk-old female SJL mice were immunized s.c. with 100 µl of an emulsion containing 200 µg of Mycobacterium tuberculosis H37Ra (BD Biosciences) and 100 µg of PLP_178–191 distributed over three sites on the flank. In experiments where PLP_139–151-specific 5B6 CD4⁺ Tg T cells were transferred to PLP_178–191-primed mice, 5 x 10⁶ naive transgenic CD4⁺ T cells were transferred i.v. at the time of onset of acute disease. At the onset of remission (approximately day +15 for most animals), mice received five daily i.p. injections of either 50 µg of hamster control Ig, anti-CD80 mAb (clone 16-10A1 (BioExpress)), or anti-CD80 Fab (BioExpress). Individual animals were observed at the indicated time points, and clinical scores were assessed in a blinded fashion on a 0–5 scale: 0, no abnormality; 1, limp tail; 2, limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelimb weakness; and 5, moribund. The data are reported as the mean daily clinical score; the cumulative mean disease score (summation of the daily mean clinical scores for each day over the duration of the experiment) and the relapse frequency (mean number of relapses per mouse per treatment group). A relapse is defined as worsening of at least one grade of clinical disease after stabilization for at least 2 days.

CD4⁺ Th cell-promoting culture conditions

Naive T cells were either untreated or labeled with CFSE at a final concentration of 0.8 µM for 8 min at room temperature, and the reaction was quenched with an equal volume of FCS for 5 min, and cells were activated in the presence of polystyrene beads coated with 1 µg of anti-CD3 and/or 1 µg of anti-CD28 at a ratio of five beads: one naive CD4⁺ T cell in neutral (200 U/ml IL-2), Th1 (200 U/ml IL-2, 40 U/ml IL-12, and 10 µg/ml anti-IL-4), or Th2 (200 U/ml IL-2, 500 U/ml IL-4, and 10 µg/ml anti-IFN-) driving conditions in the presence or absence of either soluble control Ab (Armenian hamster IgG (eBioscience)) or intact anti-CD80 Ab. After 3–7 days of culture, the T effector cells are isolated and culture supernatants collected. The percentage of viable cells was analyzed using the Guava ViaCount system (Guava Technologies).

CD11c⁺ DC isolation and coculture with CD4⁺ T cells

CNS leukocytes isolated from cerebellum and spinal cords of 16–20 PBS-perfused mice at peak of PLP_178–191-induced R-EAE were pooled, and signal cell suspensions of infiltrating cells were prepared as described previously (22). CD11⁺ DCs were purified using AutoMacs Magnetic Bead cell separation technology (Miltenyi Biotec). Purity of the CD11c⁺ DC isolation was found to be 90% CD45^high,CD11c⁺ by flow cytometry. Sorted DCs were then cocultured with naive 5B6 TCR Tg CD4⁺ T cells in the absence or presence of PLP_139–151. At either 48 or 72 h following T cell activation, all samples were pulsed with 1 µCi/well [³H]TdR and then cultured for an additional 24 h. [³H]TdR uptake was detected using a Topcount Microplate Scintillation Counter (Packard Instruments).

Flow cytometry

On days 0, 3, and 7 following the final in vivo treatment of mice with either a control Ig, anti-CD80 mAb, or anti-CD80 Fab, total CNS leukocytes were isolated from cerebellum and spinal cord, and spleens and axillary lymph nodes were collected. Flow cytometric analysis was performed on cells from individual animals (six to eight mice per group). Cells were stained with anti-CD4-allophycocyanin (RM4-5), anti-Fas-biotin (clone 15A7), anti-CD11c-APC (clone N418), anti-hamster IgG-FITC, or annexin V (eBioscience). Samples were analyzed using a BD LSR II cytometer (BD Biosciences), and 5 x 10⁴ viable cells were analyzed per individual sample. Compensation for analysis was performed by singly stained samples from a combined pooled sample of remaining cells from the individual CNS samples, using autocompensation on the BD LSR II, verified using Rainbow Calibration Particles (Sherotech), and the data were analyzed using CellQuest Pro software (BD Biosciences).

IFN- ELISA

T cell culture supernatants were collected on days 3–7 after the initial activation. Supernatant was frozen immediately at –80°C until analyzed by ELISA. Ninety-six-well microtiter plates (Nalge Nunc International) were coated with purified rat anti-mouse IFN- Ab (eBioscience) overnight, washed with PBS, and blocked with PBS/20% FCS. Cytokine containing supernatants or standards were added to each well, and plates were incubated for 1 h at 37°C in a humidified atmosphere. Plates were washed with PBS/0.02% Tween 20 in deionized H₂O, and a biotinylated rat anti-mouse cytokine-detecting Ab (eBioscience) was added to each well. A standard curve for IFN- was prepared using known quantities of recombinant mouse IFN- (eBioscience). Color development was determined on a Spectramax Plus microplate reader (Molecular Devices) at a wavelength of 450 nm. The lower limit of detection for IFN- was <0.5 U/ml.

Real-time PCR

Total mRNA was isolated with TRIzol Reagent (Invitrogen Life Technologies) and was reversed transcribed into cDNA using random hexamer primers. Briefly, a common master mix (LightCycler-FastStartDNA SYBR Green I (Roche), 2 mM MgCl₂, and 0.5 µM gene-specific primer) and 1.5 µl of cDNA for a final reaction volume of 15 µl were used. Each transcript was quantified used the following cycling protocol, 95°C for 10 min, followed by 35 cycles of 95°C denaturing for 15 s, gene-specific annealing temperature for 2 s, and 72°C extension for 20 s. The concentration of gene-specific cDNA was quantified by comparison to a standard curve of gene-specific PCR product diluted 1/10 for concentrations ranging from 1 ng/ml to 1 fg/ml. After each real-time reaction, a melting curve was generated, and samples were run on a 1.2% agarose gel to ensure that only one gene-specific PCR product was generated. Real-time PCR was preformed using the Rotor-gene 2000 Real-time Cycler (Phenix Research Products). The following primers were used: -actin 5'-TACAGCTTCACCACCACAGC-3' and 5'-AAGGAAGGCTGGAAAAGAGC-3' (annealing temperature 60°C, 206-bp product); IFN- 5'-CACGGCACAGTCATTGAAAG-3' and 5'-GCTGATGGCCTGATTGTCTT-3' (annealing temperature 60°C, 198-bp product); and T-bet 5'-CGGAGAATGGACTCCAGAGA-3' and 5'-CTGTTTGGCTGGCTGTTGTA-3' (annealing temperature 60°C, 201-bp products).

Western blot analysis

Naive CD4⁺ T cells (5 x 10⁶) were activated as described above. For total cellular protein, cells were collected, washed three times with PBS, lysed with 500 µl of 1x lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin, 10 nM okadaic acid, and 10 nM tautomycin), and frozen at –80°C until analysis. Protein samples (5–10 µg) were run on a denaturing 7.5% polyacrylamide gel and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore). Membranes were blocked with TBST (28 ml of 5 M NaCl, 25 ml of 1 M Tris-HCl (pH 7.5), 0.2 g of KCl, and 500 ml of Tween) + 5% dried milk for 1 h at room temperature, probed with primary Abs diluted in TBST + 5% dried milk for 2 h at room temperature, and washed three times with TBST for 5 min at room temperature. Membranes were probed with HRP-labeled secondary Abs diluted in TBST + 5% dried milk at room temperature for 1 h and washed three times in TBST. Following the last wash, HRP-labeled Abs were detected using the LumiGlo Detection kit (Cell Signaling Technology), and specific bands were visualized on Kodak Biomax MS film using an intensifying screen enabled film cassette. Abs used were anti-CD80 (16-10A1 (Santa Cruz Biotechnology)), anti-actin (C-11), anti-Bcl-x_L (Cell Signaling Technology), and anti-Bax Ab.

Statistical analyses

Comparisons of the percentage of animals showing clinical disease were analyzed by ² using Fisher’s exact probability, and two-way ANOVA with a Bonferroni posttest was used to determine statistical differences between mean clinical disease scores. The statistical significance of cytokine and percentage of viable or apoptotic cells was analyzed using a one-tailed ANOVA with group means compared using the Scheffé multiple comparison test. Single comparisons of two means were analyzed by Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Intact anti-CD80 mAb exacerbates PLP_178–191-induced R-EAE and increases CD4⁺ T cells survival in the CNS

Treatment of mice with an intact anti-CD80 mAb has varying effects on EAE progression depending on the time of Ab treatment, the inducing encephalitogenic peptide, the strain of mouse, and the form of the anti-CD80 mAb used (6, 7, 17, 18, 19, 20). To determine a possible mechanism by which treatment with an intact anti-CD80 mAb during disease remission exacerbates R-EAE (12), SJL mice were primed with PLP_178–191 and followed for disease progression. At the onset of disease remission, mice were treated with five daily i.p. injections (50 µg per injection) of either an anti-CD80 mAb, anti-CD80 Fab, or control Ig. As shown in Fig. 1, A and B, treatment with an intact anti-CD80 mAb during disease remission exacerbates both the mean clinical score and the cumulative mean clinical disease score (cumulative mean disease score: control Ig = 19.1 ± 0.6, anti-CD80 mAb = 35.6 ± 1.2, anti-CD80 Fab = 7.8 ± 0.3, respectively), in PLP_178–191-induced disease. In contrast, treatment of mice with anti-CD80 Fab inhibited disease progression, but these mice did eventually relapse on day 41 postdisease induction (five of seven mice relapsed), possibly due to the persistent release of PLP_178–191 peptide from the priming sites on the hind flanks of the mice draining to the lymph nodes where PLP_178–191-specific CD4⁺ T cells could be activated. This finding is remarkable given that the half-life of anti-CD80 Fab in serum is 3–3.5 h (23, 24), although the half-life/turnover of anti-CD80 Fab is not known when abound to the cell surface. Therefore, treatment of PLP_178–191-primed mice with an intact anti-CD80 mAb during disease remission exacerbates disease, whereas treatment of mice with anti-CD80 Fab ameliorates disease.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Differential effect of intact vs Fab of anti-CD80 mAb on the development of clinical relapses and epitope spreading in PLP178–191-induced R-EAE. Active R-EAE was induced in groups of six to eight SJL mice with PLP178–191 in CFA on day 0. At the onset of remission (day +15), groups of six to eight SJL/J mice were treated with either a control Ig (), anti-CD80 mAb (), or anti-CD80 Fab (•) on 5 consecutive days (50 µg per injection) and monitored for clinical disease until day 42. Clinical results were analyzed at the indicated times and are expressed as: the mean clinical score (A); the cumulative mean clinical score (summation of the daily mean clinical scores for each day over the duration of the experiment) (B); and as the relapse frequency (mean number of relapses/total number of mice in each treatment group) (C). An asterisk (*) indicates a p value < 0.05 in comparison to control Ig-treated mice. On day 42 following disease induction, total splenocytes were collected from each treatment group and activated in the presence of increasing concentrations of (D) PLP178–191, (E) PLP139–151, or (F) MBP84–104 for 48 h, and supernatants were collected and the level of IFN- produced per well was determined via ELISA. One of three representative experiments is shown.

The severity of R-EAE positively correlates with the number of CD4⁺ T cells in the CNS, and the elimination of encephalitogenic CD4⁺ T cells from the CNS is largely regulated by apoptosis of the autoreactive cells (25, 26). To determine whether the anti-CD80 mAb-induced disease exacerbation correlated with an increased number of CD4⁺ T cells in the CNS, spinal cords were collected and analyzed on days 0, 3, and 7 following the last Ab treatment (day +19 postdisease induction). As shown in Fig. 2, the number of CD4⁺ T cells in the CNS is equivalent on day 0 following each of the Ab treatments (control Ig = 47.1 ± 9.6 x 10⁴, anti-CD80 mAb = 44.3 ± 5.9 x 10⁴, and anti-CD80 Fab = 43.2 ± 3.2 x 10⁴), but on day 3, the number of CD4⁺ T cells in the CNS of anti-CD80 Fab-treated mice (13.0 ± 4.6 x 10⁴; p = 0.02) is significantly decreased as compared with mice that received either anti-CD80 mAb (40.7 ± 9.9 x 10⁴) or control Ig (29.8 ± 6.8 x 10⁴). By day 7, following Ab treatment, the number of CD4⁺ T cells in the CNS of mice that received control Ig treatment decreased to 19.2 ± 4.3 x 10⁴, which is significantly lower than mice that received anti-CD80 mAb treatment (40.7 ± 6.3 x 10⁴; p = 0.01). The decrease in the overall number of CD4⁺ T cells in the CNS correlated with the level of Fas expression and positive staining for annexin V. Immediately following anti-CD80 Fab treatment, 36.3 ± 1.3% of the CD4⁺ T cells were double positive for Fas and annexin V correlating with decreased clinical disease at this time, whereas CD4⁺ T cells from control Ig and anti-CD80 mAb treatment groups were not double positive for Fas and annexin V until day 3 (38.15 ± 1.1%) and day 7 (25.2 ± 2.1%), respectively. Therefore, treatment of PLP_178–191-primed mice with an intact anti-CD80 mAb inhibited the induction of apoptosis leading to an increased number of CD4⁺ T cells in the CNS, suggesting that anti-CD80 mAb may induce a survival signal in CD4⁺ T cells.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Anti-CD80 mAb promotes CD4+ T cell survival while anti-CD80 Fab therapy inhibits CD4+ T cell survival in the CNS during PLP178–191-induced active R-EAE. Active R-EAE was induced in groups of six to eight SJL mice with PLP178–191 in CFA on day 0. Following recovery from acute disease episode (day +15), groups of six to eight SJL/J mice were treated with five daily treatments of 50 µg (a total of 250 µg) of either control Ig, anti-CD80 mAb, or anti-CD80 Fab. Immediately following the final mAb treatment (day 0) and on days 3 and 7 following the last Ab treatment, spinal cords were isolated, and the total number of CD4+ T cells, as well as the percentage of CD4+ T cells expressing Fas and annexin V, was analyzed. The average number of CD4+ T cells isolated per mouse from the CNS is listed above the respective dot plots. One of three representative experiments is shown.

The above findings suggest that anti-CD80 mAb treatment induces an increase in disease severity and the survival of CD4⁺ T cells in the CNS. We therefore asked if anti-CD80 mAb-induced exacerbation of EAE progression in vivo is due to an increase in spread epitope-specific CD4⁺ T cell activation and survival. 5B6 PLP_139–151-specific transgenic CD4⁺Thy1.1⁺ Tg T cells (5 x 10⁶) were transferred to SJL mice at the onset of acute PLP_178–191-induced EAE (day 12 postdisease induction). Mice were treated with five daily i.p. injections of 50 µg of control Ig, intact anti-CD80 mAb or anti-CD80 Fab during disease remission (beginning day 15 postdisease induction). As expected, anti-CD80 mAb treatment resulted in a significant increase in the mean clinical score, whereas treatment with anti-CD80 Fab decreased the mean clinical score (Fig. 3A). Starting on the last day of Ab treatment (day 0), the numbers of PLP_139–151-specific CD4⁺Thy1.1⁺ Tg T cells in the CNS were analyzed by flow cytometry. As shown in Fig. 3B, treatment of mice with either control Ig (47.1 ± 9.6 x 10⁴), anti-CD80 mAb (44.3 ± 5.9 x 10⁴), or anti-CD80 Fab (43.2 ± 3.2 x 10⁴) did not affect the number of PLP_139–151-specific CD4⁺Thy1.1⁺ Tg T cells in the CNS on day 0. However, the number of PLP_139–151-specific CD4⁺ Tg T cells in the CNS of control Ab-treated mice steadily decreased over the 7-day time course, which was further decreased by treatment with anti-CD80 Fab. In contrast, the number of PLP_139–151-specific T cells over the 7-day period in mice treated with the intact anti-CD80 mAb (40.7 ± 6.4 x 10⁴) remained equivalent to the levels determined on day 0. To determine whether the decrease in the percentage of PLP_139–151-specific CD4⁺ T cells in the CNS was due to the induction of apoptosis, the percentage of annexin V-positive PLP_139–151-specific donor CD4⁺ T cells was analyzed. The data (Fig. 3, C–E) show that the decrease in the percentage of CD4⁺ T cells specific for the PLP_139–151 spread epitope in the CNS correlated with the percentage of annexin V-positive cells in the CNS, indicating that stimulation of CD80 on Ag-specific CD4⁺ T cells during R-EAE promotes the survival and/or the retention of the encephalitogenic T cells in the CNS.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Effect of anti-CD80 mAb on development of clinical disease and activation of PLP139–151-specific Tg T cells in PLP178–191-induced R-EAE. Active R-EAE was induced in groups of six to eight SJL mice with PLP178–191 in CFA on day 0. On day 11, the mice were injected with 5 x 106 naive 5B6 Thy1.1+ PLP139–151-specific Tg T cells. At the onset of remission (day +15), groups of six to eight SJL/J mice were treated with either a control Ig (), anti-CD80 mAb (), or anti-CD80 Fab (•) on 5 consecutive days (50 µg per injection) and monitored for clinical disease at the indicated time points until day 29 (A). On days 0, 3, and 7 following the last Ab treatment, spinal cords were isolated, and the number of PLP139–151-specific CD4+Thy1.1+ cells present in the CNS was analyzed by flow cytometry for individual mice (B). An asterisk (*) indicates a p value < 0.05 in comparison to control Ig-treated mice. The level of annexin V was also analyzed on the PLP139–151-specific CD4+Thy1.1+ cells isolated from the CNS on days 0 (C), 3 (D), and 7 (E) following Ab treatment to determine the percentage of cells undergoing apoptosis. One of three representative experiments is shown.

Published data show that the primary site of spread epitope-specific CD4⁺ T cell activation occurs in the CNS and that CD45^highCD11c⁺ DCs are the major APC population involved in the activation of naive spread epitope-specific CD4⁺ T cells (22). Since activated CD11c⁺ DCs express CD80 and cross-linking of B7 on the surface of CD11c⁺ DCs has been shown to have both positive and negative effects, i.e., an increase in the production of the negative regulatory molecule IDO and an increase in the production of IL-6 and IFN- (9), respectively, the possibility existed that anti-CD80 mAb treatment in vivo affected CD11c⁺ DC function. Of the total CNS mononuclear cell infiltrate recovered, 31.9 ± 5.4% of the cells were CD4⁺ and 26.4 ± 5.1% of the cells were CD11c⁺. To detect anti-CD80 mAb bound to the cell surface during in vivo treatment, cells were stained with anti-hamster IgG-FITC for analysis. As shown in Fig. 4A, less than 1% of the CD4⁺ T cells isolated from the CNS of control Ig-treated mice stained positive for anti-hamster IgG-FITC. In contrast, the data show that intact anti-CD80 mAb is bound to both CD4⁺ T cells (30.1 ± 1.1%) and CD11c⁺ cells (36.3 ± 3.9%) in the CNS of PLP_178–191-primed mice (Fig. 4A), allowing for two possible mechanisms by anti-CD80 mAb treatment may increase the severity of disease. Interestingly, no appreciable anti-CD80 mAb was found to be associated with the CD4^–,CD11c^–CNS population (representing primarily the resident microglia and infiltrating macrophages). Based on the presence of anti-CD80 mAb binding to both CD4⁺ T cells and CD11c⁺ DCs in the CNS, we determined if treatment of mice with various forms of anti-CD80 mAb altered the percentage and/or the activity of CD11c⁺ cells in the CNS. To determine whether the anti-CD80 mAb-induced disease exacerbation correlated with an increased number of CD11c⁺ cells in the CNS, spinal cords were collected from SJL mice primed with PLP_178–191 in CFA on day 3 following the last Ab treatment (day +21 postdisease induction). This time point was chosen due to the significant difference in the percentage and number of spread epitope-specific CD4⁺ T cells shown in Fig. 3. Neither the percentage of CD11c⁺ cells in the CNS (Fig. 4A: control Ig = 24.2 ± 2.3%, anti-CD80 mAb = 27.1 ± 4.1%, and anti-CD80 Fab = 23.8 ± 2.1%), nor the total number of CD11c⁺ cells in the CNS (Fig. 4B) were significantly affected by anti-CD80 mAb or Fab treatment as compared with control Ig-treated mice.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Injected anti-CD80 mAb binds both CD4+ and CD11c+ cells in the CNS and does not alter CD11c+ cell function. Active R-EAE was induced in groups of six to eight SJL mice with PLP178–191 in CFA on day 0. Following recovery from the acute disease episode (day +15), groups of six to eight SJL/J mice were treated with either a control Ig, anti-CD80 mAb, or anti-CD80 Fab. On day 3 following the last Ab treatment, the mice were perfused, and spinal cords, spleens, and lymph nodes were isolated. The percentage of CD4+ and CD11c+ cells that bound anti-CD80 mAb in vivo was determined via the use of mouse anti-hamster IgG-FITC (A). Based on the total number of cells isolated from the CNS and the percentage of CD11c+ DCs, the number of CD11+ DCs is presented as the mean number of cells (x104) ± SEM from three separate experiments (B). Sorted CD11c+ DCs from the CNS of the three treatment groups were cocultured at various ratios with a constant number of naive 5B6 TCR Tg CD4+ T cells in the presence (control Ig (), anti-CD80 mAb (), or anti-CD80 Fab (•)) of PLP139–151 (4 x 105 T cells/well). The data are presented as the mean net cpm ± SEM from one representative experiment of three (C).

CD80 stimulation increases the level of IFN- and cell survival of CD4⁺ T cells

Activation of a naive CD4⁺ T cell requires both the stimulation of the TCR with MHC II Ag on the APC and costimulatory signals derived from the interaction of CD28 with a B7 molecule, i.e., CD80 or CD86 (3, 4). Although it is classically held that CD80 and CD86 are expressed on APCs, these molecules are also expressed on the surface of CD4⁺ T cells following activation (27, 28, 29), a situation consistent with the above data. In light of the finding that intact anti-CD80 mAb appears to exacerbate R-EAE by increasing the survival and level of IFN- produced by myelin epitope-specific CD4⁺ T cells, the possibility exists that cross-linking surface CD80 on CD4⁺ T cells regulates the differentiation/survival of Th1 cells. Alternatively, anti-CD80 mAb could block the negative regulatory signal induced via CTLA4-CD80 interaction.

To determine whether these effects were due to direct cross-linking of CD80 on the CD4⁺ T cell, we analyzed the level of CD80 expressed on the surface of purified naive CD4⁺ T cells activated by anti-CD3 and/or anti-CD28 mAbs bound to polystyrene beads in the absence of APCs. In confirmation of published results (27, 28, 30), naive CD4⁺ T cells activated in the presence of various stimuli induce the expression of CD80 in an activation- and time-dependent manner, as determined by both flow cytometry and Western blot analysis (Fig. 5, A and B). To determine whether cross-linking CD80 on the surface of a CD4⁺ T cell alters effector function, the levels of IFN- produced in cultures of naive CD4⁺ T cells activated in the presence of anti-CD3/anti-CD28 mAb-coated polystyrene beads in Th1-promoting conditions containing increasing concentrations of soluble anti-CD80 mAb were determined. As shown in Fig. 5, C and D, intact anti-CD80 mAb induced a concentration-dependent increase in the level of IFN- produced. This effect was blocked in cultures to which anti-CD80 Fab (Fig. 5C) or CTLA4 Ig (Fig. 5D) was co-added, demonstrating the specificity of the anti-CD80 mAb-induced increase in the level of IFN- and eliminating the possibility that the effect anti-CD80 mAb treatment was due to blockade of a negative regulatory signal to the effector T cell. Likewise, cross-linking of CD80 on CD4⁺ T cells activated in the presence of Th1-promoting conditions induced a concentration-dependent increase in the percentage of viable CD4⁺ T cells present on day 3 of culture (see Fig. 5, E and F). Therefore, these findings indicate that the anti-CD80 mAb-induced increase in IFN- production and cell viability are induced by a direct effect of cross-linking CD80 on CD4⁺ T cells.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Anti-CD80 mAb transduces a direct stimulatory signal to CD4+ T cells. CD4+ T cells were isolated from naive wild-type SJL mice and cultured (1 x 106 cells) with IL-2 (1 ng/ml) alone; anti-CD3 (1 µg/ml)-coated polystyrene beads; anti-CD3 beads + IL-2; anti-CD3 + anti-CD28 (1 µg/ml)-coated beads + IL-2 (Th0-promoting conditions); or anti-CD3 + anti-CD28-coated beads + IL-2 and IL-12 (4 ng/ml) (Th1-promoting conditions). Cells were collected over a 48-h time course, and the percentage of CD4+ T cells expressing CD80 was determined by flow cytometry (A) and Western blot analysis (B). One of three representative experiments is presented. Naive CD4+ T cells were activated in the presence of Th1-promoting conditions and increasing concentrations of intact anti-CD80 mAb (C–F) plus increasing concentrations of anti-CD80 Fab (0–100 µg/ml) (C and E) or CTLA4-Ig (0–100 µg/ml) (D and F). On day 3 following the initial activation, the levels of IFN- were determined by ELISA (C and D), and data were presented as the mean units of IFN-/ml produced from three replicate wells. One of three representative experiments is presented. The percentage of viable cells was also assessed by the use of Guava ViaCount (E and F) and the data presented as the mean percent viable cells from three replicate wells. One of three representative experiments is presented.

Cross-linking of CD4⁺ T cell-expressed CD80 decreases cell division while increasing the amount of IFN- produced per cell

Taking the above data, showing that the anti-CD80 mAb induces an increase in the level of IFN- secreted and induces a slight trend to increase T cell proliferation (31), we examined if direct stimulation of CD80 on a CD4⁺ T cells affected cell division. Naive CD4⁺ T cells were labeled with CFSE, and the cells were activated (anti-CD3- + anti-CD28-coated beads) in the absence or presence of anti-CD80 mAb. As shown in Fig. 6A, cross-linking of CD80 inhibited the cells from progressing beyond the first cell division. Based on published data showing that CD4⁺ T cells activated in Th1-promoting conditions do not irreversibly maintain a Th1 cell phenotype until the third cell division (32, 33), therefore, we wished to determine whether stimulation of CD80 affected the number of IFN--secreting cells. Naive CD4⁺ T cells were activated in the presence of Th1-promoting conditions for 2 days, and an equal number of viable cells were plated for IFN- ELISPOT and corresponding ELISA. The data show that an equal number of IFN--secreting cells are detected regardless of the addition of anti-CD80 mAb, anti-CD80 Fab, or control Ig (Fig. 6B). This is in contrast to the amount of IFN- secreted by that same population of CD4⁺ T cells in that CD80 cross-linking induced a 3-fold increase in the level of IFN- produced per viable cell plated (Fig. 6C). Stimulation of CD80 on CD4⁺ T cells activated in the presence of Th1-promoting conditions thus decreased the number of cell divisions while increasing the number of viable cells and the amount of IFN- produced per cell. Therefore, we hypothesized that anti-CD80 cross-linking should decrease the overall number of IFN--secreting cells while increasing the amount of IFN- produced per cell. To test this possibility, equal numbers of naive CD4⁺ T cells were activated directly in ELISPOT plates, and the number of resultant IFN--producing cells determined. As shown in Fig. 6D, there is a concentration-dependent decrease in the number of IFN- secreting cells, and this decrease is largely blocked by the addition of either excess soluble anti-CD80 Fab or CTLA4-Ig. In contrast, the amount of IFN- secreted per culture is increased in a concentration-dependent manner following the addition of anti-CD80 mAb, which is also blocked by the addition of either soluble anti-CD80 Fab or CTLA4-Ig (Fig. 6E). Therefore, stimulation of CD80 on CD4⁺ T cells activated in the presence of Th1-promoting conditions appears to switch the T cell from a proliferative phenotype to an effector IFN--secreting phenotype.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Anti-CD80 mAb cross-linking induces increased levels of IFN- produced per resultant Th1 cell. Naive CD4+ CFSE-labeled T cells were purified from unprimed wild-type SJL mice and cultured (1 x 106 cells) in the absence or presence of anti-CD3 (1 µg/ml)- and anti-CD28 (1 µg/ml)-coated polystyrene beads (5 x 106) + IL-2 (1 ng/ml) ± Th1-promoting conditions (IL-12 (4 ng/ml) and anti-CD4 mAb (1 µg/ml)). On day 3 of culture, the number of cell divisions were assayed via flow cytometry (A). Cells from parallel cultures were collected on day 2 following activation, and an equal number of viable cells were plated in IFN- ELISPOT plates or in cultures for ELISA (B and C). The number of IFN- secreting cells (B) and the amount of IFN- secreted (C) was analyzed after an additional 24 h in culture. Naive CD4+ T cells were activated directly in IFN- ELISPOT plates for 3 days in the presence of increasing concentrations of anti-CD80 mAb (0–10 µg/ml; ) (D) in the absence or presence of either anti-CD80 Fab (), or CTLA4-Ig () at a concentration of 10 µg/ml. Corresponding cultures were set up to determine the amount of IFN- secreted, as determined by IFN- ELISA (E). One of three representative experiments is shown.

Cross-linking of CD4 T cell-expressed CD80 increases the expression of IFN- and T-bet transcripts and Bcl-x_L protein

Based on the above data, the ability of CD80 cross-linking on the surface of a CD4⁺ T cell activated in the presence of Th1-promoting conditions to regulate the level of IFN- and T-bet transcripts was determined. As shown in Fig. 7A, the level of IFN- transcript produced increases in a concentration-dependent manner in naive CD4⁺ T cells activated in Th1-promoting conditions following the addition of anti-CD80 mAb but not when the cells are activated in either neutral or Th2-promoting conditions. Since the differentiation of Th1 cells is dependent upon T-bet expression (32, 33), we also determined the level of T-bet expressed. As shown in Fig. 7B, the level of T-bet expression is coordinately increased following CD80 stimulation in Th1-promotoing conditions. In contrast, little or no T-bet transcript is detected when CD4⁺ T cells are activated under neutral or Th2-promoting conditions. Taken together, these findings suggest that cross-linking of CD80 on CD4⁺ T cells activated in the presence of Th1-promoting conditions directly promotes Th1 differentiation.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Anti-CD80 mAb induces IFN- and T-bet transcript and Bcl-xL expression in activated CD4+ T cells. CD4+ T cells were isolated from naive wild-type SJL mice and cultured (1 x 106 cells) in the absence or presence of Th0 or Th1-promoting conditions as indicated in Fig. 5. To control for baseline/activation-induced levels of transcription, naive CD4+ T cells were activated in the presence of neutral or Th2-promoting conditions, i.e., anti-CD3 (1 µg/ml)- + anti-CD28 (1 µg/ml)-coated polystyrene beads (5 x 106) + IL-2 (200 U/ml), IL-4 (11.1 ng/ml), anti-IL-12 (10 µg/ml), and anti-IFN- (10 µg/ml). Total cellular RNA was isolated at 48 h following activation, and the level of IFN- (A) and T-bet (B) transcripts were analyzed by real-time PCR. The data are presented as the mean concentration of transcript (fg/ml) from triplicate wells. One of two representative experiments is shown. Total cellular protein was also collected, and the level of the proapoptotic protein (Bax) and the antiapoptotic protein (Bcl-xL) was analyzed via Western blot (C). The level of actin protein present in each sample was used as a loading control. One of three representative experiments is shown.

	Discussion

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Classically,CD80 has been studied for its ability to interact with CD28 on a CD4⁺ T cell, thereby enhancing T cell activation. For this reason, treatment of R-EAE in SJL mice with anti-CD80 mAb has been hypothesized to have its effects by altering CD80-induced APC function. The present data show that intact anti-CD80 mAb binds to both CNS-infiltrating CD11c⁺ DCs and CD4⁺ T cells following treatment. Furthermore, treatment of mice with anti-CD80 mAb during disease remission does not appear to affect CD11c⁺ DC Ag-presenting function. However, previous data from our laboratory showed that the addition of intact anti-CD80 mAb to naive CD4⁺CD62L⁺ T cells activated in the presence of Th1-promoting conditions induced an increase in the level of IFN- produced (31). Therefore, an alternative hypothesis to explain exacerbation of clinical R-EAE disease following anti-CD80 mAb injection in SJL mice is that the Ab cross-links CD80 on activated autoreactive CD4⁺ T cells, thereby increasing their effector function and/or survival. The present findings illustrate that cross-linking CD80 on CD4⁺ T cells activated in Th1-promoting conditions increases the amount of IFN- produced per cell by increasing the levels of both IFN- and T-bet transcripts. To our knowledge, these results are the first to identify a potential mechanism by which treatment of R-EAE in SJL mice with anti-CD80 mAb during disease remission increases the severity of disease and epitope spreading. Furthermore, the current data are the first to show that the addition of anti-CD80 mAb to purified naive CD4⁺ T cells activated in Th1-promoting conditions in the presence of anti-CD3/28 induces a positive regulatory signal in the CD4⁺ T cell, which is due to direct signaling of CD80 and not the blockade of a negative regulatory signal to the effector CD4⁺ T cells via blockade of CTLA4/CD80 interactions.

Although limited data are available to support a functional role of CD80 expression by CD4⁺ T cells during an immune response (35, 36), data presented herein support the hypothesis that stimulation via CD80 on CD4⁺ T cells positively regulates effector cell function. For example, in vivo down-regulation of T cell effector function is controlled by T cell-expressed B7 following ligation with CTLA4 during T cell-T cell interactions, which underscores the potential importance of T cell-T cell interactions as an important immune regulatory control mechanism (35). It should be noted that CD80 and CD86 have been shown to have differential roles in T cell activation and differentiation (16, 37, 38). To illustrate this point, conflicting results have been obtained using anti-CD80 and anti-CD86 mAbs to regulate autoimmune disease. Treatment with anti-CD80 mAb surrounding autoantigen priming has been shown to block EAE development induced with suboptimal concentrations of PLP_139–151 or MBP_84–104 in SJL mice, whereas anti-CD86 mAb treatment has been reported to either exacerbate disease (39) or have no effect (40). In contrast, treatment with intact anti-CD86 mAb initiated during the remission following acute disease does not affect disease progression (relapses) in PLP_139–151-induced R-EAE (17). In the case of anti-CD80 mAb, treatment with monovalent, non-cross-linking anti-CD80 Fab blocked clinical relapses and epitope spreading to the PLP_178–191 epitope, whereas treatment with intact anti-CD80 mAb led to a profound exacerbation of disease relapses concomitant with accelerated epitope spreading (12, 17). Likewise, treatment of mice with a small molecule inhibitor of CD28 during disease decreased disease severity, decreased proliferation of CD4⁺ T cells upon activation ex vivo, and increased CD4⁺ T cell apoptosis (41). These findings suggest that intact anti-CD80 mAb may cross-link and induce a direct signal through CD80 expressed on either APCs and/or activated T cells, whereas anti-CD80 Fab may block CD80/CD28 costimulation necessary for the activation of spread epitope-specific CD4⁺ T cell. The relative importance of CD80-mediated costimulation in R-EAE progression is supported by studies analyzing the temporal expression of B7 costimulatory molecules in R-EAE. Active immunization of SJL mice with PLP_139–151 in CFA resulted in a temporal up-regulation of surface CD80 expression, relative to CD86, on B cells, T cells, and macrophages in the spleen (42). Furthermore, a CD80-dominant expression pattern is seen on all peripheral inflammatory cell types in the CNS-infiltrating population (17). Thus, CD80 appears to be the predominant B7 costimulatory molecule in SJL mice with established disease and blockade of CD80/CD28 costimulation specifically inhibited the activation of T cells to endogenously presented myelin epitopes. Since the B7-CD28/CTLA4 costimulatory system plays a critical role in determining the fate of immune responses, it serves as a promising therapeutic target for regulating autoimmune diseases (5, 6, 7).

Considering the current in vitro data along with the aforementioned in vivo data, a putative mechanism is suggested to explain why the timing of anti-CD80 mAb treatment has differing effects on disease outcome. Both the in vitro and in vivo data support the hypothesis that the initial steps of activation of naive CD4⁺ T cells is inhibited by anti-CD80 due to a lack of costimulation through CD28 interacting with CD80 on the APC. For example, the addition of either an intact anti-CD80 mAb, anti-CD80 Fab, or CTLA4-Ig at the time of naive CD4⁺ T cell activation in the presence of antigenic peptide and APCs blocks T cell activation (43, 44). These culture conditions represent the treatment regimen in which anti-CD80 mAb is administered to the mice at the time of priming with encephalitogenic CD4⁺ T cell epitopes. In contrast, the culture conditions used in this report more closely mimic the in vivo treatment regimen wherein anti-CD80 treatment is initiated during remission following the peak of acute disease. Therefore, these results support the conclusion that cross-linking CD80 on effector CD4⁺ T cells induces the production of IFN-, leading to enhanced tissue destruction and increased epitope spreading by increasing CD4⁺ T cell effector function.

The relevance of CD80 expression by CD4⁺ T cells is not limited to the mouse system. CD80 is also expressed by human peripheral blood CD4⁺ T cells, and the ability of CD80 to initiate an intracellular signal has also been shown to exist in human immune cells (8, 21). Analysis of lesions from MS patients has identified an increase in the level of CD80 expressed at the lesion site (14). Genetic analyses of MS patients support the hypothesis that CD80 expressed by autoreactive CD4⁺ T cells may contribute to the disease exacerbation; in MS patients, there is a positive correlation between the expression of allelic variants of CTLA4 that have an increased affinity for binding to CD80 (45). Similarly, susceptibility to development of spontaneous diabetes in the NOD mouse has also been linked to a splice variant of CTLA4 (46). Based on the data presented herein showing that stimulation of CD80 on a CD4⁺ T cell increases the production of IFN- and increases cell survival, the possibility exists that autoreactive CD4⁺ T cells in patients with MS are stimulated to produce heightened levels of IFN- and display increased cell survival if the patient also has the genetic predisposition of expressing the high-affinity allelic variant form of CTLA4. In this scenario, the expression of CTLA-4 might have the opposite effect of the "classic" negative regulatory role CTLA4 has on CD4⁺ T cell activity (47, 48, 49). Instead, CTLA4, with its increased affinity for CD80, might act as a cross-linker for CD80, sending a positive regulatory signal to the autoreactive T cell during a T cell-T cell interaction. The present findings also strongly suggest that extreme caution must be taken when designing treatment regimens for autoimmune diseases such as MS based on Ab-mediated blockade of costimulatory molecules. Any treatment that has the potential to cross-link CD80 must be analyzed with the utmost care in that not only can cross-linking of CD80 on an APC affect cellular activity (8, 31), but these same regimens may also positively regulate autoreactive CD4⁺ T cell effector functions, thereby exacerbating disease.

	Disclosures

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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 in part by U.S. Public Health Service, National Institutes of Health Grants NS-034819 and NS-026543 and by support from the Myelin Repair Foundation. A.P.K. is supported by National Multiple Sclerosis Society Postdoctoral Fellowship Grant FG-1516-A-1.

² Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University, Tarry 6-718, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: s-d-miller{at}northwestern.edu

³ Abbreviations used in this paper: DC, dendritic cell; MBP, myelin basic protein; MS, multiple sclerosis; PLP, myelin proteolipid protein; R-EAE, relapsing experimental autoimmune encephalomyelitis; Tg, transgenic.

Received for publication March 7, 2006. Accepted for publication June 21, 2006.

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