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. Author manuscript; available in PMC: 2008 Nov 25.
Published in final edited form as: J Immunol. 2007 Jan 1;178(1):474–479. doi: 10.4049/jimmunol.178.1.474

The Net Effect of Costimulatory Blockers Is Dependent on the Subset and Activation Status of the Autoreactive T Cells1

Ping Zhang 1, Deming Sun 1, Yan Ke 1, Henry J Kaplan 1, Hui Shao 1,2
PMCID: PMC2587059  NIHMSID: NIHMS72086  PMID: 17182586

Abstract

In this study, we investigated whether CD4 and CD8 autoreactive T cells have different costimulatory requirements for their activation in vitro by testing the effect of a panel of Abs specific for various costimulatory molecules. Our results showed that CD8 interphotoreceptor retinoid-binding protein-specific T cells are more dependent on costimulatory molecules for activation than their CD4 counterparts. Interphotoreceptor retinoid-binding protein-specific T cells are less dependent on costimulatory molecules in the secondary response than the primary response. We also showed that blockade of costimulatory molecules can either promote or inhibit the proliferation of autoreactive T cells, depending on the degree of activation of the cells. Our results show that anti-costimulatory molecule treatment can have diverse actions on autoreactive T cell subsets, the net effect being determined by the subset of immune cells affected and the type and dose of treatment used.

Many studies have shown that costimulatory molecules play an important role in T cell activation, which requires, in addition to binding of the antigenic peptide/MHC complex to the TCR, the ligation of costimulatory molecules (1–8). Most of these studies examined the activation of CD4 T cells and it remains to be determined whether CD8 T cells use the same costimulatory molecules as the corresponding Ag-specific CD4 cells. This question is of particular importance in understanding the activation of CD8 autoreactive T cells in autoimmune diseases, as very few studies have examined the costimulatory requirements of these cells.

We have previously demonstrated that, in addition to CD4 autoreactive T cells, CD8 T subsets are major participants in both experimental autoimmune encephalomyelitis (9, 10) and experimental autoimmune uveitis (EAU)3 (11–13) and play an important role in the pathogenesis and immunoregulation of these autoimmune diseases. Given that the functions of autoreactive T cells are closely related to their activation status, we wished to determine the conditions that result in activation of CD8 autoreactive T cells and whether CD8 autoreactive T cells use similar costimulatory molecules for their activation and expansion as CD4 T cells. In the current study, we tested the effect of a panel of fusion proteins and Abs specific for the costimulatory molecules on APCs that are essential for the activation of CD4 and CD8 autoreactive T cells in an EAU model in the B6 mouse. We showed that a given costimulatory molecule was not equally important in the activation of CD8 and CD4 interphotoreceptor retinoid-binding protein (IRBP)-specific T cells. CD8 IRBP-specific T cells were more vulnerable to blocking of costimulation than their CD4 counterparts. Comparative studies of IRBP-specific T cells isolated from mice with actively induced EAU (primary response), mice with EAU induced by adoptive transfer of autoreactive cells (secondary response), and of established IRBP-specific T cell lines showed that primary IRBP-specific T cell responses were more dependent on costimulation for activation, while the secondary response was relatively resistant to costimulation blockers. Finally, costimulatory molecule blockers had an inhibitory effect on responder T cells exposed to optimal doses of Ag and APCs, but had the opposite effect on the same responder T cells exposed to a suboptimal dose of Ag and APCs. Our results demonstrated that anticostimulatory molecule treatment may generate undesired effects and that the net effect of treatment is dependent on the T cell subset involved and its activation status.

Materials and Methods

Animals and reagents

Pathogen-free female C57BL/6 mice (8–10 wk old) were purchased from The Jackson Laboratory and were housed and maintained in the animal facilities of the University of Louisville. Institutional approval was obtained and institutional guidelines regarding animal experimentation were followed. All Abs against costimulatory molecules are listed in Table I.

Table I.

mAbs against mouse costimulatory molecules

mAbs Clones Sources
B7.1 (CD80) 16-10A1 eBiosciences
B7.2 (CD86) GL-1 Biolegend
CD40 1C10 Biolegend
ICOSL HK5.36 Biolegend
OX40L (CD134L) RM134L eBiosciences
4-1BBL (CD137L) TKS-1 eBiosciences
CTLA4-Fc A gift from Dr. P. A. Morton (Pfizer, St. Louis, MO) (26)
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Preparation of IRBP1–20-specific T cells

Briefly, to prepare T cells, donor mice were immunized s.c. with 200 μl of an emulsion containing 200 μg of IRBP1–20 (aa 1–20 of human IRBP) (Sigma-Aldrich) and 500 μg of Mycobacterium tuberculosis H37Ra (Difco) in IFA (Sigma-Aldrich), distributed over six spots at the tail base and on the flank. T cells were isolated at 13 days postimmunization from lymph node cells or spleen cells by passage through a nylon wool column, then 1 × 107 cells in 2 ml of RPMI 1640 medium (Mediatech) were added to each well of a 6-well plate (Costar) and stimulated with 20 μg/ml IRBP1–20 in the presence of 1 × 107 irradiated syngeneic spleen cells as APCs. After 2 days, the activated lymphoblasts were isolated by gradient centrifugation on Lymphoprep (Robbins Scientific) and cultured in RPMI 1640 medium supplemented with 15% IL-2-containing medium (supernatant from Con A-stimulated rat spleen cells).

Adoptive transfer of EAU

Uveitis was induced in naive B6 mice by adoptive transfer of 5 × 106 IRBP1–20-specific T cells as described previously (14). The animals were examined three times a week for clinical signs of uveitis by fundoscopy, starting at week 2 posttransfer. Fundoscopic evaluation for longitudinal follow-up of disease was performed using a binocular microscope after pupil dilation using 0.5% tropicamide and 1.25% phenylephrine hydrochloride ophthalmic solutions. The incidence and severity of EAU were graded on a scale of 0–4 in half-point increments using previously described criteria (14), which are based on the type, number, and size of lesions present.

Purification of CD4 and CD8 T cells using auto-MACS columns

Purified CD4 and CD8 T cells were prepared from the draining lymph nodes and spleen using a CD4 and CD8 Isolation kit (Miltenyi Biotec) (11, 12). The lymph node and spleen cells were first incubated for 10 min at 4°C with a mixture of biotin-conjugated Abs against CD8 (CD8a, Ly-2) or CD4 (CD4, L3T4) T cells (H57-597), B cells (CD45R, B220), NK cells (CD49b,DX5), hemopoietic cells (CD11b, Mac-1), and erythroid cells (Ter119), then for 15 min at 4°C with anti-biotin Ab-conjugated microbeads. The cells were then applied to an auto-MACS separator column (Miltenyi Biotec), which was washed with 15 ml of medium and the flow-through fraction containing CD4− or CD8-enriched cells was collected. The purity of the isolated cell fraction was determined by flow cytometric analysis using FITC-conjugated anti-mouse TCR Abs and PE-conjugated Abs directed against mouse CD8 or CD4 (BD Biosciences). Data collection and analysis were performed on a FACSCalibur flow cytometer using CellQuest software.

Immunofluorescence flow cytometry

Aliquots of 2 × 105 cells were double stained with combinations of FITC-or PE-conjugated mAbs against mouse TCR, CD4, CD8, CD44, or CD62L. Data collection and analysis were performed on a FACSCalibur flow cytometer using CellQuest software.

Proliferation assay

T cells were prepared from IRBP1–20-immunized B6 mice or B6 mice which had undergone adoptive transfer with IRBP1–20 specific T cells and seeded at 4 × 105 cells/well in 96-well plates, then cultured at 37°C for 48 h in a total volume of 200 μl of medium with or without IRBP1–20 in the presence of irradiated syngeneic spleen APCs (1 × 105), and [3H]thymidine incorporation during the last 8 h was assessed using a microplate scintillation counter (Packard Instrument). The proliferative response was expressed as the mean cpm ± SD of triplicate determinations.

ELISA

Cytokines IL-2, IFN-γ, and IL-10 were measured using commercially available ELISA kits (R&D Systems).

Pathological examination

Inflammation of the eye was confirmed by histopathology. Whole eyes were collected, immersed for 1 h in 4% phosphate-buffered glutaraldehyde, then transferred to 10% phosphate-buffered formaldehyde until processed. The fixed and dehydrated tissue was embedded in methacrylate, and 5-μm sections were cut through the pupillaryoptic nerve plane and stained with H&E. Presence or absence of disease was evaluated blind by examining six sections cut at different levels for each eye. Severity of EAU was scored as described above.

Statistical analysis

The data are expressed as the mean ± SD. Statistical analysis of the results was performed using the Student t test (from Figs. 1–4). In Fig. 5, there are multiple statistical comparisons and an attempt to correct for this was made using the Dunnett’s post hoc test following the advice of a biostatistician. Values of p < 0.05 were considered significant.

FIGURE 1.

FIGURE 1

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CD8 IRBP-specific T cells are more dependent on costimulatory molecules for activation than their CD4 counterparts. Purified CD4 (top panels) or CD8 T cells (bottom panels) (4 × 105 cells/ well) from IRBP1–20-immunized B6 mice were stimulated with APCs (1 × 105) and IRBP1–20 (10 μg/ml) in the presence (2 or 10 μg/ml) or absence of costimulatory molecule blockers, as described in Materials and Methods. T cell proliferation was measured at 72 h by [3H]thymidine uptakes. Results shown are cpm from one representative experiment of five (average of triplicates). *, Statistically significant difference in values from the control group without Abs added (p < 0.05).

FIGURE 4.

FIGURE 4

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The secondary response of IRBP-specific T cells is less dependent on costimulation. Nonseparated CD4 plus CD8 T cells (4 × 105 cells/well) from IRBP1–20 immunized mice (A) or mice adoptively transferred with IRBP1–20-specific T cells (C) or IRBP1–20 CD4 T cell lines (B) (4 × 104 cells/well) were stimulated with APCs (1 × 105 cells/well) and IRBP1–20 (10 μg/ml) in the absence or presence of costimulatory molecules blockers (10 μg/ml), as described in Materials and Methods. T cell proliferation was measured at 72 h by [3H]thymidine uptake and expressed as cpm of triplicate wells for each condition (representative experiment of three). *, Statistically significant difference in values from the control group without Abs added (p < 0.05).

FIGURE 5.

FIGURE 5

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Combined use of costimulatory molecule blockers does not always have a synergistic effect. Purified CD4 cells (left panel) or CD8 cells (right panel) (4 × 105 cells/well) from IRBP1–20-immunized B6 mice were stimulated with 1 × 105 APCs and 10 μg/ml IRBP1–20 in the presence of a single costimulatory molecule blocker (10 μg/ml) or a combination of two (10 μg/ml each). The cells were harvested at 72 h after an 8-h pulse with 1 μCi/well [3H]thymidine. Shown are cpm from one representative experiment of five (average of triplicates). *, Statistically significant difference in values from the control group without Abs added (p < 0.05). Multiple statistical comparisons were made using the Dunnett’s post hoc test.

Results

CD8 IRBP-specific T cells are more dependent on costimulatory molecules for activation than their CD4 counterparts

To determine whether CD4 and CD8 IRBP-specific T cells have different requirements for costimulation, we tested the effect of a panel of costimulatory molecule blockers on the activation of CD4 and CD8 IRBP-specific T cells. Responder T cells were obtained from B6 mice immunized 13 days previously with a pathogenic dose (200 μg) of IRBP1–20 emulsified in CFA. CD4 and CD8 responder T cells were separated from the draining lymph nodes and spleen using an auto-MACS separator. The purity of the separated CD4− and CD8− T cells was ~95% (Fig. 1). Purified CD4 and CD8 were incubated for 48 h (cytokine studies) or 72 h (proliferation studies) with irradiated APCs and IRBP1–20 in the absence or presence of costimulatory molecule blockers at doses of 2 or 10 μg/ml. Fig. 1 shows the effect of the costimulatory molecule blockers on the proliferation of in vivo-primed CD4 and CD8 T cells. At the dose of 10 μg/ml, B7.1, B7.2, and anti-CD40 Abs were much more effective than the other blockers at inhibiting the CD4 response, whereas Abs against ICOSL, OX40L, and 4-1BBL were more effective in blocking the CD8 response than the CD4 response. Although both B7.1 and B7.2 mAbs inhibited CD4 and CD8 T cell proliferative response, B7.1 Ab is a stronger inhibitor for CD4 and B7.2 Ab is more effective for CD8 response. At the lower dose of 2 μg/ml, the same blockers were equally as effective at inhibiting the CD8 response, but had little or no effect on the CD4 response, showing that CD8 IRBP-specific T cells are more dependent on costimulatory molecules for activation than their CD4 counterparts. In all subsequent studies, the dose of blocker used was 10 μg/ml.

Cytokine production does not always mirror the inhibitory effect on T cell proliferation

To determine whether the costimulatory blockers also inhibited cytokine production by the responder T cells during their activation, the same experimental setup was used, but supernatants were collected from the individual test groups after 48 h of Ag stimulation in vitro and levels of three cytokines (IL-2, IFN-γ, and IL-10) measured. Fig. 2 shows cytokine levels in the different responder CD4 and CD8 T cell supernatants. As shown, inhibition of cytokine production did not always mirror the effect on T cell proliferation. Anti-CD40 Ab blocked the proliferative response of CD4 T cells, but did not affect their IL-2 production and, in fact, enhanced their IFN-γ production, while anti-B7.1 Ab, which also blocked CD4 T cell proliferation, inhibited the production of IL-2, but not of IFN-γ and IL-10, by CD4 T cells. Interestingly, none of the blockers significantly inhibited IL-10 production by CD8 T cells, even though they inhibited IL-2 and IFN production.

FIGURE 2.

FIGURE 2

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Inhibition of cytokine production does not always mirror the inhibitory effect on T cell proliferation. Purified CD4 (top panels) or CD8 (bottom panels) T cells were cultured for 2 days with APCs (1 × 105) and IRBP1–20 (10 μg/ml) in the absence of presence of anticostimulatory molecule blockers (10 μg/ml). Culture supernatants were collected at 48 h and assayed for IL-2, IFN-γ, and IL-10 by ELISA, as described in Materials and Methods. *, Statistically significant difference in values from the control group without Abs added (p < 0.05). Results of a representative experiment of three are shown.

Treatment of costimulatory molecule blockers can also promote the activation of autoreactive T cells in vitro

Because T cell activation in vitro may differ from T cell activation in vivo, because in vivo activation is often only partial, we considered it necessary to test the blocking effect on fully vs partially activated T cells. In vivo-primed CD8 IRBP-specific T cells were stimulated with either optimal doses of APCs (1 × 105) and Ag (10 μg/ml IRBP1–20) to induce full activation (Fig. 3, A and C) or suboptimal doses of APCs (0.5 × 105) and Ag (1 μg/ml IRBP1–20) to induce partial activation (Fig. 3, B and D) in the presence of costimulatory molecule blockers. As shown in Fig. 3, Abs against B7.1 B7.2, ICOSL, or OX40L inhibited the proliferative response of fully activated CD8 T cells (Fig. 3A), but stimulated the response of partially activated responder CD8 cells (Fig. 3B). In contrast, anti-CD40 Abs had little effect on fully activated cells, but blocked the response of partially activated cells. Phenotypic analysis of the responding T cells agreed with the thymidine incorporation tests by showing that, under full activation conditions (stimulated by a high dose of immunizing Ag), anti-B7 Ab-treated T cells contained a lower percentage of CD44highCD62L− cells (activated T cells) than controls (47% compared with 63% in untreated cells, Fig. 3C), whereas the percentage of these T cells increased (from 34 to 48%) when anti-B7 Abs were added to the same T cells activated by a low dose of stimulatory Ag (Fig. 3D).

FIGURE 3.

FIGURE 3

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Anti-costimulatory molecule treatment can, under certain circumstances, promote the activation of autoreactive T cells. A and B, Purified CD8 cells (4 × 105 cells/well) from IRBP1–20-immunized B6 mice were stimulated with 1 × 105 APCs and 10 μg/ml IRBP1–20 (A) or with 0.5 × 105 APCs and 1 μg/ml IRBP1–20 (B) in the absence or presence of costimulatory molecule blockers (10 μg/ml), then T cell proliferation was measured at 72 h after an 8-h pulse with 1 μCi/well [3H]thymidine. C and D, The percentage of CD44highCD62− cells in the cells in A (C) or B (D) was examined by flow cytometry. The data are represented as a dot plot for CD44 and CD62L expression after gating on CD8+ T cells (representative experiment of three).

The secondary response of CD4 IRBP-specific T cells is more resistant than the primary response to costimulatory molecule blockers

The above-described studies examined the effect of blockers on the activation of in vivo-primed autoreactive T cells. It is probable that secondary T cell responses show a different dependency on costimulatory molecules (15, 16). Because the isolation of memory T cells from diseased mice is not feasible, we decided to compare the blocking effect of different agents on in vivo-primed IRBP-specific T cells and established IRBP-specific T cell lines. As shown in Fig. 4, the primary response of nonseparated (CD4 plus CD8) in vivo-primed T cells was blocked by a number of the costimulatory molecule blockers including B7.1, CTLA-4-Fc, and CD40 (Fig. 4A), but the secondary response of IRBP1–20-specific T cell line was resistant to most blockers (Fig. 4B). Similarly, the secondary response of nonseparated IRBP-specific T cells isolated from mice 30 days after adoptive transfer of cells was also relatively resistant (Fig. 4C).

The simultaneous use of a combination of costimulatory molecule blockers does not have a synergistic effect

We then examined whether combinations of costimulatory molecule blockers had a synergistic blocking action on the activation of autoreactive CD4 and CD8 T cells. As shown in Fig. 5, the inhibitory effect of anti-B7.1 and CD40 Abs on the CD4 T cell response was unaffected by the noninhibitory Abs against costimulatory molecules ICOSL, OX40L, and 4-1BBL. However, the inhibitory effect of anti-B7.2 on the CD4 T cell response was reduced when used in combination with either OX40L or 4-1BBL. Combinations of any two of the B7.1, B7.2, ICOSL, OX40L, or 4-1BBL blockers (each 10 μg/ml) did not generate a greater inhibitory effect on the CD8 response than the single blocker alone. The noninhibitory effect of CD40 Ab is dominant for a CD8 T cell response when used alone or with other inhibitory Abs, such as B7.2, ICOSL, and 4-1 BBL, but not with Ab OX40L. Similar results were obtained using 5 μg/ml of each blocker in a combination compared with 10 μg/ml of a single blocker.

Discussion

Both the number and the activation status of autoreactive T cells are crucial for their pathogenic activity (14, 17, 18). In both experimental autoimmune encephalomyelitis and EAU, transfer of as few as 1 × 106 newly activated autoreactive T cells readily induces disease, whereas transfer of 100 times as many nonactivated T cells does not (H. Shao and D. Sun, unpublished observation). Thus, inhibition of T cell activation is one of the major strategies that can hold autoimmune diseases in check. Because the same autoimmune disease can be induced in disease-prone rodents using several different Ags and because the activation of T cells requires not only primary antigenic stimulation, but also the ligation of costimulatory molecules, it has been suggested that it would be more effective to target the costimulatory molecules rather than the T cells reacting to the different autoantigens (7, 19, 20) that may be involved in the pathogenesis of the same autoimmune disease in different individuals (20–26).

We have previously shown that treatment of EAU-prone rodents with competitive binding molecules, such as a fusion protein (26) or mAbs (27, 28) that bind to costimulatory molecules prevents the initiation of EAU, indicating that these molecules are important in the pathogenesis of this autoimmune disease. However, most previous studies, including our own, were conducted using acute and monophasic autoimmune disease models (29, 30). Our recent studies have shown that CD8 autoreactive T subsets, which become more prominently involved in the chronic disease phase, are major participants in the autoimmune response (11–13). This raises the question of whether the same costimulatory molecule blockers are equally effective in preventing disease recurrence and in the control of CD8 autoreactive T cells and whether blockade of the secondary CD8 response can impede the progression of disease initiated by CD4 autoreactive T cells.

By simultaneous testing a panel of agents blocking costimulatory molecules previously found to be essential for the activation of Ag-specific CD4 T cells, we were able to show that a given costimulatory molecule was not equally important for the activation of CD8 and CD4 IRBP-specific T cells, the CD8 cells being more vulnerable to blocking of costimulation.

Our results showed that the in vitro response of CD8 IRBP-specific T cells was more readily inhibited by anti-costimulatory molecule blockers than their CD4 T cell counterparts, especially when lower doses of blockers were used (Fig. 1). This agrees with our previous observation that CD8 autoreactive T cells are activated to a lower extent than CD4 cells under comparable activation conditions (11). By testing the effect of different doses of blockers, we found that blockade of costimulatory molecules could either promote or inhibit the activation of autoreactive T cells, depending on the amount of blocking Abs used and the activation status of the responder T cells (Fig. 3). Although the underlying mechanisms remain unclear, it is likely that some Ag-specific regulatory T cells are more dependent on costimulatory molecules for activation (31) and that these cells might be preferentially affected when only limited amounts of blocking Abs were used. Our data also demonstrated that the simultaneous use of a combination of costimulatory molecule blockers does not generate significant synergistic effect. In contrast, when two contradictory effects of Abs were used, one of the effects overrides the other. For example, B7.2 Ab, but not CD40 Ab, inhibits CD8 T cells, when used singly. However, the dominant noninhibitory role of CD40 Ab was stronger than the inhibitory effect of B7.2 Ab on CD8 proliferative response when the two were used simultaneously.

It was also interesting to note that costimulatory molecule blockers preferentially inhibited the production of the proinflammatory cytokines, IFN-γ and IL-2, but not that of the anti-inflammatory cytokine, IL-10 (Fig. 2), which suggests that, under certain circumstances, blockade of T cell activation decreases the production of proinflammatory cytokines without altering the amount of anti-inflammatory cytokines. The anti-CD40 Ab strongly blocked autoreactive CD4 T cell proliferation (Fig. 1), but had no effect on IL-2 production and enhanced IFN-γ production (Fig. 2), suggesting that anti-CD40 signaling may generate additional cellular effects, such as triggering IL-12 production by APCs, thus allowing pathogenic T cell differentiation (32).

Our previous observations that adoptive transfer of autoreactive T cells more easily induces chronic recurrent disease, whereas Ag immunization induces acute monophasic disease (14, 17, 18), and our present observation that autoreactive T cells reisolated from adoptive transferred recipients were less dependent on costimulatory molecules for their reactivation in vitro compared with the same Ag-specific T cells isolated from Ag-immunized mice (Fig. 4), agrees with our preliminary results that chronic recurrent EAU is more resistant to costimulatory molecule blocker treatment (G. M. Yike, D. Jiang, D. Sun, and H. Shao, manuscript in preparation). It is likely that treatment that prevents the monophasic disease of uveitis may be less effective in the treatment of chronic recurrent EAU and more effective treatments will be needed.

In summary, our results demonstrate that blocking of costimulation can have diverse actions on autoreactive T cell subsets and on the expansion and cytokine production of autoreactive T cells. The net effect will be determined by the subset of the immune cells affected and the type and dose of the reagent used. Under certain circumstances, costimulatory treatment enhances, rather than inhibits, autoimmune disease.

Acknowledgments

The editorial assistance of Dr. Tom Barkas is greatly appreciated.

Footnotes

1

This work was supported in part by National Institutes of Health Grants NEI EY12974 (to H.S.), EY14599 (to H.S.), and NEI-EY014366 (to D.S.), Vision Research Infrastructure Development (R24 EY015636), Grant RG3413A4 from the National Multiple Sclerosis Society, and the Commonwealth of Kentucky Research Challenge Trust Fund.

3

Abbreviations used in this paper: EAU, experimental autoimmune uveitis; IRBP, interphotoreceptor retinoid-binding protein.

Disclosures The authors have no financial conflict of interest.

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