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The Journal of Immunology, 2006, 177: 4495-4502.
Copyright © 2006 by The American Association of Immunologists, Inc.

Ligation of CD80 Is Critical for High-Level CD25 Expression on CD8+ T Lymphocytes1

Sharmila Pejawar-Gaddy and Martha A. Alexander-Miller2

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD80 and CD86 have been shown to play a critical role in the optimal activation of T cells. Although these two molecules bind the same ligand, CD28, the question of whether CD80 and CD86 provide unique signals or serve redundant roles remains controversial. Previous studies have suggested that CD80 binding to CD28 may be superior to CD86 for the activation of naive CD8+ T cells. This study provides a potential mechanism to explain these observations. Our study demonstrates a previously unappreciated role for CD80, its superiority over CD86 in promoting CD25 expression, increasing both the number of cells that express CD25 and the level expressed on a per cell basis. These findings provide new insights into the role of CD80 vs CD86 and have important implications for the design of vaccines and immunotherapeutics aimed at the generation of a robust CD8+ T cell response in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 are the most potent APCs, capable of priming both naive and memory T lymphocytes (1, 2). Recent studies have shown (3, 4) that optimal T cell activation requires at least three signals delivered by a mature DC. These include TCR binding to peptide/MHC, costimulatory molecule engagement, and the production of cytokines (i.e., IL-12 and type I IFN).

The best-characterized costimulatory molecules, CD80 and CD86, belong to the B7 family of molecules and bind a common receptor, CD28, found on T cells. This interaction promotes and sustains T cell activation and survival in concert with the TCR signal. Although the functional consequence of CD28 engagement is well known (5, 6), the significance of CD80 vs CD86 engagement in the activation of CD8+ T cells remains unclear. Although some reports (7) have suggested redundancy in these two signals, others (8, 9, 10, 11, 12) advocate that CD80 provides a superior signal. In support of the latter, in a study using transfected cells, Gajewski (11) showed that CD80 was more efficient than CD86 for CD8+ T cell activation. Furthermore, CD86 could not compensate for the lack of CD80. Fields et al. (8) extended this study, showing that CD80 was a quantitatively stronger costimulus for IL-2 production and proliferation than CD86. Additionally, Lumsden et al. (13) showed that CD80 was particularly important for T cell activation in the lung, with blocking resulting in a decreased number of virus-specific T cells. However, the mechanism(s) responsible for the increased efficacy of CD80 was not determined.

Our previous studies have shown (14) that BALB/c bone marrow-derived DC (BMDC) infected with recombinant simian virus 5 (rSV5) at a multiplicity of infection (MOI) of 10 PFU/cell significantly up-regulate the expression of CD40 and CD86, but not CD80. These studies presented us with a novel approach to address the role of CD80 and CD86 during naive CD8+ T cell activation, i.e., DC from wild-type mice that have differentially up-regulated CD80 and CD86. In this study, we present data that suggest a previously undefined role for CD80 in the up-regulation of CD25 on naive CD8+ T cells. Stimulation with rSV5-infected BMDC with minimal expression of CD80, but high expression of CD86, resulted in reduced proliferation and function compared with the CD8+ T cells cultured with DC expressing high levels of both molecules. These defects could not be overcome by increasing the CD86 signal, suggesting that a subthreshold CD28 signal was not responsible for the defects. However, provision of CD80 promoted IFN-{gamma} production and proliferation. Analysis of CD25 expression demonstrated that the absence of the CD80 signal resulted in a failure to optimally express this molecule. This result provides the first evidence that CD80 can provide a signal unique from that of CD86 in promoting CD25 expression and provides a mechanistic basis for the previously reported increased ability of CD80 to promote activation of CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

BALB/c mice were purchased from the Frederick Cancer Research and Development Center (Frederick, MD). L9.6 Listeria p60 (217–225)-specific TCR transgenic mice on a Rag-1 knockout/BALB/cJ background were a gift from Dr. E. Pamer (Memorial Sloan Kettering Cancer Center, New York, NY (15). All research performed on mice in this study complied with federal and institutional guidelines set forth by the Wake Forest University Animal Care and Use Committee.

P815 is a DBA/2-derived (H-2d) mastocytoma grown in RPMI 1640 medium (Invitrogen Life Technologies), NTCC L929 is a fibroblast cell line derived from C3H/An (H-2k) mice, and EL4 is a C57BL/6-derived (H-2b) thymoma, both grown in DMEM (Invitrogen Life Technologies). All these media were supplemented with 10% FCS (HyClone), L-glutamine, sodium pyruvate, nonessential amino acids, HEPES, penicillin, streptomycin (BioWhittaker), and 5 x 10–5 M 2-ME. EL4.CD80 and EL4.CD86 were a gift from Dr. G. J. Freeman (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA).

Generation of bone marrow-derived DC (BMDC)

The protocol used to generate BMDC was similar to the protocol used by Inaba et al. (16). Bone marrow was harvested from the femurs and tibias of mice, RBC were lysed, and then plated at 1 x 106 cells/well in a 24-well plate in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (HyClone), HEPES, gentamicin sulfate (BioWhittaker), and 5 x 10–5 M 2-ME and cultured for 7 days in the presence of 20 ng/ml rGM-CSF (BioSource International). Every 2 days, the cell culture medium of the BMDC cultures was removed followed by the addition of fresh medium with 20 ng/ml GM-CSF.

Recombinant viruses

Wild-type rSV5 were generated from cDNA clones as described previously (17, 18).

DC infections and treatments

Virus infections were performed directly in the wells where the BMDC were generated to avoid maturation of cells as a result of manipulation. rSV5 was diluted in RPMI 1640 containing 0.75% BSA (Invitrogen Life Technologies) and added to the BMDC cultures at a MOI of 10 PFU/cell. BMDC treated with 300 ng/ml LPS (Sigma-Aldrich) were used as a positive control for fully matured cells.

T cell activation assay

Day 7 BMDC from BALB/c were mock treated, LPS treated, or infected with SV5. Twenty-two hours following treatment, the cells were pulsed with Listeria p60217–225 peptide (10–10 to 10–7 M). Splenocytes from Ag-specific (L9.6 Listeria p60217–225) TCR-transgenic mice were subjected to negative selection to isolate CD8+ T cells (MiniMACS separation columns and CD8+ isolation kit; Miltenyi Biotec), labeled with 5 µM CFSE (Molecular Probes) and then cocultured with peptide-pulsed DC for 3 days at a ratio of 1:10 (DC:T cell) in 96-well plates. To test the effect of CD80 and CD86 during CD8+ T cell activation, untransfected or transfected (with CD80 or CD86) EL4 cells were added to cultures at a ratio of 1:1:10 (DC:EL4 cell:T cell). To test the effect of CD25 and CD80 during T cell activation, antagonistic mAb specific to CD25 (30 µg/ml) or CD80 (10 µg/ml) or their appropriate isotype controls were added to cultures (BD Biosciences). Proliferation was measured by the decrease in CFSE intensity. IFN-{gamma}, TNF-{alpha}, CD25, and IL-2 (BD Biosciences) were measured by intracellular cytokine staining (ICS) assay. Analysis and quantitation of data were performed using FlowJo software (TreeStar).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD8+ T cells activated by SV5-matured DC are defective in their ability to proliferate and produce IFN-{gamma}

The question of whether CD80 and CD86 provide unique or redundant signals in the activation of CD8+ T cells remains in large part unanswered. We have found previously (14) that infection of BALB/c BMDC with rSV5 (MOI = 10) resulted in the selective up-regulation of CD86, with minimal change in CD80 expression. Furthermore, we showed that stimulation with SV5-matured DC resulted in a reduced T cell proliferative response compared with T cells activated with DC expressing high levels of both CD80 and CD86 (LPS-matured DC) (14). It is important to note that SV5-matured DC may be lacking in other untested positive signals that might be present on LPS-matured DC. However, in this study, we took advantage of the model to evaluate the significance of the differential expression of CD80 vs CD86 on DC as a result of viral infection on the activation of naive CD8+ T cells.

BALB/c BMDC were treated with LPS, infected with rSV5 at an MOI of 10, or mock infected. Twenty-two hours after infection, cells were pulsed with titrated concentrations of Listeria p60217–225 peptide, washed, and cocultured with CFSE-labeled L9.6 Listeria p60 TCR-transgenic CD8+ T cells for 3 days, at which time proliferation and function were assessed. In agreement with our previous results, a smaller percentage of CD8+ T cells entered division following culture with SV5-matured compared with LPS-matured DC (e.g., 10–9 M; Fig. 1B) and T cells that divided underwent fewer rounds of division compared with those cultured with LPS-matured DC (2.6 vs 1.5; Fig. 1A). In addition, whereas 31% of cells cultured with LPS-matured DC produced IFN-{gamma}, only 10% produced IFN-{gamma} following culture with SV5-matured DC (e.g., 10–9 M; Fig. 1D). This was not simply a reflection of the decreased proliferation in the cultures stimulated with SV5-matured DC, as the percentage of cells in each division that was capable of producing IFN-{gamma} was also decreased (Fig. 1, C and D). Effector function on a per cell basis was similarly reduced as demonstrated by a diminished amount of IFN-{gamma} produced (mean fluorescence intensity (MFI) = 160 for cultures stimulated with SV5-matured DC vs 210 for cultures stimulated with LPS-matured DC). Finally, the ability to produce TNF-{alpha} concurrently with IFN-{gamma} was decreased in cells stimulated with SV5-matured vs LPS-matured DC (12 vs 29%; data not shown). These data establish the decreased ability of CD80low, partially mature, SV5-matured DC to induce early proliferation and function of naive CD8+ T cells. One trivial explanation for these findings was that the reduced proliferation was the result of reduced presentation of peptide on SV5 vs LPS-matured cells; however, this is unlikely given that H-Kd expression following infection with SV5 was higher than with LPS treatment (MFI = 271 vs MFI = 216; data not shown).


Figure 1
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  		FIGURE 1. CD8+ T cells activated by SV5-matured DC are defective in their ability to proliferate and produce IFN-ã. A, Representative histograms depicting the proliferative profiles of CD8+ T cells on day 3 poststimulation with mock-treated, LPS-treated, or SV5-infected DC pulsed with 10–9 M Listeria p60 peptide. B, Average percentage of CD8+ T cells that divided in 3 days of culture with mock-treated, LPS-treated, or SV5-infected DC pulsed with a range of peptide concentrations: 10–10 to 10–7 M (average of eight experiments). *, 10–9 M, p = 0.011, and 10–8 M, p = 0.013. C, Representative dot plots depicting IFN-{gamma} production in the ICS assay by CD8+ T cells cultured previously for 3 days with mock-infected, LPS-treated, or SV5-infected DC pulsed with 10–9 M Listeria p60 peptide. D, Average percent of CD8+ T cells that produced IFN-{gamma} in the ICS assay following culture for 3 days with mock-infected, LPS-treated, or SV5-infected DC that had been pulsed with a range of peptide concentrations: 10–7 to 10–10 M (average of nine experiments). *, 10–9 M, p = 0.036, and 10–8 M, p = 0.035. E, Total number of live CD8+ T cells present at days 3, 5, and 7 of culture with mock-treated, LPS-treated, or SV5-infected p60-pulsed DC (average of three experiments). Values of p were calculated using the Student t test.

 
It was possible that the observed decrease in T cell proliferation following culture with SV5-matured DC was due to a delay in the kinetics of proliferation. If this were true, we would predict that the number of T cells present at later times in cultures with SV5-matured DC would be similar to the number of T cells in the LPS-DC T cell cultures. Thus, we tested whether there were differences in the ability of T cells activated by SV5-matured or LPS-matured DC to proliferate with delayed kinetics past day 3. Viable CD8+ T cells on days 3, 5, and 7 after stimulation with LPS- or SV5-infected DC were enumerated by trypan blue exclusion. At each time point assessed, a greater number of CD8+ T cells was present in cultures where LPS-matured DC were used as stimulators compared with cultures where SV5-matured DC were used (day 3, 2.9-fold; day 5, 3.3-fold; and day 7, 3.5-fold; Fig. 1E). In fact, the number of cells present in SV5-matured DC stimulated cultures was not significantly different from cultures stimulated with immature (mock) DC. These results indicate that the decreased number of T cells present in the SV5-DC cultures compared with LPS-DC cultures on day 3 was not a result of a delay in the kinetics of proliferation.

Stimulation with LPS-matured vs SV5-matured BMDC resulted in differential expression of IL-2 and CD25 on responding T cells

Our previous data demonstrated that stimulation with SV5-matured DC resulted in both a reduced percentage of T cells entering division and fewer divisions in dividing cells. This result led us to hypothesize that CD80lowCD86high SV5-matured DC might be defective in either their ability to trigger production of IL-2 or to up-regulate CD25 expression or both. To test this hypothesis, CD25 expression and IL-2 secretion were analyzed on days 1, 2, and 3 of culture with mock-treated, LPS-matured, or SV5-matured DC. On day 1, the percentage of T cells secreting IL-2 was similar in all three cultures (Fig. 2A, upper panel), although the T cells in the LPS-DC cultures secreted higher amounts of IL-2 on a per cell basis (1.6-fold) compared with those in the SV5-DC or mock-DC cultures (Fig. 2A, lower panel). However, on days 2 and 3, stimulation with LPS-matured BMDC resulted in both a larger percentage of cells secreting IL-2 (2-fold for both days) (Fig. 2A, upper panel) and a higher production of IL-2 on a per cell basis (1.5-fold for both days) compared with SV5-matured BMDC (Fig. 2A, lower panel). Furthermore, a greater percentage of these cells expressed CD25 and of those positive for CD25 (Fig. 2B, upper panel), the expression was higher compared with CD8+ cells in cultures activated by SV5-matured DC (Fig. 2B, lower panel). Thus, SV5-matured DC, which expressed a high level of CD86, but a low level of CD80, were defective in their ability to trigger both maximal IL-2 production and CD25 expression.


Figure 2
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  		FIGURE 2. Stimulation with LPS-matured vs SV5-matured BMDC resulted in differential expression of IL-2 and CD25 on responding T cells. A, Percentage of CD8+ T cells that produced IL-2 (top); geometric MFI of cells that produced IL-2 following culture with 10–9 M p60-pulsed mock-treated, LPS-treated, or SV5-infected DC (bottom). B, Percentage of CD8+ T cells that expressed CD25 (top); geometric MFI of cells that expressed CD25 following culture with 10–9 M p60-pulsed mock-treated, LPS-treated, and SV5-infected DC (bottom). Data are the average of six experiments.

 
Addition of the CD80 signal during T cell activation enhanced proliferation and function

Although a difference in CD80 expression was an attractive explanation for the disparity in IL-2 production and responsiveness, we were aware that other molecules could also vary between LPS-matured and SV5-matured DC and could be responsible for the functional differences. To determine whether CD80 was responsible, we used an experimental approach in which CD80 or CD86 was added in trans. CFSE-labeled p60 CD8+ T cells were cultured with mock-, LPS-, or SV5-matured BMDC pulsed with p60 peptide. As a source of CD80 or CD86, irradiated EL4 cells expressing these molecules singly were added in trans to the DC-T cell cultures. Importantly, EL4 cells do not express the p60-restricting MHC molecule and thus cannot directly present peptide to the T cells. In addition, the amount of CD80 and CD86 on the transfected EL4 cells was higher than that on LPS-matured DC, so that these molecules should not be limiting in our system (data not shown). Following 3 days of culture, proliferation and function were determined. The addition of EL4 cells transfected with CD80 led to the partial restoration of both proliferation (2.3-fold increase) and function (3-fold increase) in the CD8+ T cells in these SV5 cultures compared with the cultures that received untransfected control EL4 cells (Fig. 3). In contrast, no further significant increase in proliferation or function was observed following addition of EL4-CD86 (Fig. 3). Consistent with the increase in proliferation, there was an increased recovery of live cells from ~5.6 x 105 cells (SV5-matured DC alone) to ~1.04 x 106 following addition of the EL4.CD80. No increased cell number over SV5-matured DC was observed with the addition of EL4.CD86. The positive effect on proliferation and function by addition of EL4.CD80 was a result of CD80 expression on the transfected cells since the addition of neutralizing CD80 Ab prevented the increased activation (Fig. 3). However, no effect was observed upon neutralizing with the isotype control for CD80. Similar results were also obtained using plate-bound CD80 and CD86 Fc chimeras (data not shown). These results suggest that CD80 provides a unique signal that cannot be compensated for by increasing the CD86 signal and, furthermore, that optimal CD8+ T cell activation requires CD80 engagement.


Figure 3
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  		FIGURE 3. The addition of the CD80 signal enhanced proliferation and function. A, The average percentage of CD8+ T cells that divided following 3 days of culture with mock-treated, LPS-treated, or SV5-infected DC pulsed with 10–9 M peptide. Where noted, EL4 cells or EL4 cells transfected with CD80 (EL4.CD80) or CD86 (EL4.CD86) were added to cultures with SV5-infected DC. Where noted, neutralizing Ab to CD80 or isotype control Ab (10 µg/ml) was added to cultures that received EL4.CD80 transfectants. Data shown are the average of eight experiments; *, p = 0.021. B, The average percentage of CD8+ T cells that were capable of producing IFN-{gamma} in the ICS assay following 3 days of culture with mock-treated, LPS-treated, or SV5-infected DC pulsed with 10–9 M peptide. Where noted, SV5 cultures received control EL4 cells, EL4.CD80, or EL4.CD86. Data shown are the average of eight experiments; *, p =0.031. Values of p were calculated using the Student paired t test.

 
CD80 is a more potent inducer of CD25 expression on CD8+ T cells than CD86

Our previous results showed that cells stimulated with SV5-matured DC were impaired in their ability to produce IL-2 and to up-regulate CD25. Because the addition of CD80 resulted in increased function and proliferation in cells activated by SV5-matured DC, it seemed likely that the provision of CD80 resulted in increased expression of IL-2 and/or CD25. Fig. 4A shows that this was the case, since addition of CD80 in trans resulted in a significantly greater percentage of cells expressing CD25 on days 2 and 3 compared with the addition of CD86 (Fig. 4B). It is important to note that addition of CD86 in trans did cause a modest increase in the percentage of cells expressing CD25 (Fig. 4B). Of note, blocking CD80 on LPS-matured DC caused a significant decrease in the expression of CD25 on CD8+ T cells, further indicating that the CD80 signal contributes to the optimal induction of CD25 (Fig. 4C). Furthermore, blocking of CD80 on LPS-matured DC caused a modest decrease in IFN-{gamma}, but not proliferation (data not shown). This modest affect on T cell activation observed following CD80 blocking of LPS-matured DC might be due to the differential up-regulation of several surface molecules on LPS- vs SV5- BMDC. We hypothesize that some other molecule up-regulated on LPS-matured DC might compensate for the lack of CD80. Interestingly, the addition of either CD80 or CD86 similarly enhanced the secretion of IL-2, although the increase was modest in both cases. However, this result suggested that CD80 provided a more robust and optimal signal for CD25 up-regulation on CD8+ T cells.


Figure 4
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  		FIGURE 4. The addition of EL4.CD80 to cultures containing SV5-infected DC cells resulted in higher CD25 expression on CD8+ T cells compared to the addition of EL4.CD86. A, Percentage of CD8+ T cells capable of producing IL-2 in the ICS assay following 3 days of culture with 10–9 M p60-pulsed SV5-infected DC. Where indicated, EL4, EL4.CD80, or EL4.CD86 was added to the cultures. B, Percentage of CD8+ T cells that expressed CD25 on day 1 (average of four experiments), day 2 (average of four experiments), or day 3 (average of seven experiments) following culture. A significant increase in CD25 in cultures receiving EL4.CD80 vs EL4.CD86 cells was detected at day 2 (p = 0.048) and at day 3 (p = 0.020). C, Percentage of cells expressing CD25 on day 3 following addition of the neutralizing Ab to CD80 or its isotype control Ab (10 µg/ml). A significant decrease in CD25 expression was observed (p = 0.031). Data are the average of three experiments. Values of p were calculated using the Student paired t test.

 
To determine whether the increase in CD25 that resulted from the addition of CD80 to the SV5-matured DC-T cell cultures was responsible for enhanced T cell activation, we blocked CD25 by the addition of an antagonist Ab (Fig. 5). Blocking CD25 significantly decreased the percentage of CD8+ T cells that produced IFN-{gamma} (Fig. 5A). However, no significant effect was observed on proliferation (Fig. 5B). The failure to see effects on early proliferation is consistent with a previous report from D’Souza and Lefrancios (19), in which early division of CD25-deficient CD8+ T cells was similar to that of wild-type cells.


Figure 5
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  		FIGURE 5. CD25 is required for the IFN-{gamma} production by CD8+ T cells cultured with SV5-matured DC. A, The average percentage of CD8+ T cells that produced IFN-{gamma} in the ICS assay following 3 days of culture with mock-treated, LPS-treated, or SV5-infected DC pulsed with 10–9 M peptide. B, The average percentage of CD8+ T cells that divided following 3 days of culture with mock-treated, LPS-treated, or SV5-infected DC pulsed with 10–9 M peptide. Where noted, EL4 cells or EL4 cells transfected with CD80 (EL4.CD80) were added to cultures with SV5-infected DC. To some of the cultures that received the CD80 transfectants, neutralizing Ab to CD25 or its isotype control Ab (30 µg/ml) was added. Data shown are the average of three experiments.

 
A limitation to the above studies was that the addition of CD80 was always measured in the context of a high CD86 signal. Thus, it was difficult to determine the independence of these signals for the functions tested. Therefore, we used a reductionist approach, stimulating CD8+ T cells in the presence of peptide-pulsed mock-treated DC (CD80lowCD86low) with the addition of either CD80 or CD86 in trans. Although the addition of CD86 in trans resulted in a modest up-regulation of CD25 expression, the addition of CD80 was superior at all days tested (Fig. 6A). Interestingly, the addition of CD80 or CD86 resulted in only a slight increase in IL-2 expression (Fig. 6B), suggesting perhaps that other signals provided by the mature DC are necessary for optimal IL-2 expression. When CD8+ T cell proliferation and function were measured, we observed a 5-fold increase in the percentage of cells that divided in cultures stimulated the presence of exogenous CD80. Although the addition of CD86 resulted in some increase in division, this effect was reduced compared with the addition of CD80 (Fig. 6C). Interestingly, the addition of either CD80 or CD86 resulted in a similar percentage of cells that were capable of producing IFN-{gamma} (Fig. 6D). Taken together, these data support the increased efficacy of CD80 compared with CD86 for triggering CD25 expression and thus proliferation.


Figure 6
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  		FIGURE 6. CD80 is a more potent inducer of CD25 expression on CD8+ T cells compared to CD86 when added to cultures stimulated with mock-infected DC. A, The percentage of CD8+T cells that expressed CD25 on day 1 (average of four experiments), day 2 (average of four experiments), or day 3 (average of seven experiments) following culture with mock-infected DC. A significant increase in cultures receiving EL4.CD80 vs EL4.CD86 cells was detected at day 3 (p = 0.009). B–D, The percentage of CD8+ T cells that produced IL-2 (B), underwent division (C), or were capable of producing IFN-{gamma} (C) following 3 days of culture with mock-treated DC at 10–8 M p60. Where noted, EL4 cells, EL4.CD80, or EL4.CD86 were added to the cultures. Values of p were calculated using the Student paired t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A number of studies have demonstrated the ability of costimulatory signals to facilitate a more robust and sustained CD8+ T cell response. Among these, CD80 and CD86 have been extensively studied (4, 5, 12). CD80 and CD86 are type I membrane proteins belonging to the B7 family and share ~25% sequence homology and bind the same receptors, CD28 and CTLA-4, on T cells (20, 21). However, the nature of the signal delivered by engagement of CD80 vs CD86 may be unique as several studies have reported that these molecules bind CD28 with different kinetics and avidities (12, 21, 22, 23). Despite intense investigation, whether these molecules have unique or redundant roles during T cell activation remains controversial.

Our previous studies (14) revealed the unexpected finding that SV5 infection of BALB/c BMDC resulted in the selective up-regulation of CD86, whereas CD80 levels remained relatively unchanged. The incomplete maturation observed is likely the result of the failure of the virus to provide a positive signal necessary for up-regulation of CD80 expression as the addition of LPS or poly(I:C) resulted in a significant increase in the expression of CD80 on SV5-infected BALB/c BMDC. Regardless, the failure of SV5-infected cells to up-regulate CD80 may be a strategy used for immune evasion (14). These partially matured SV5-infected APC were found to be reduced in their capacity to stimulate proliferation of naive CD8+ T cells. This presented us with a system that could be exploited to dissect the functional outcome of the selective up-regulation of CD86 on DC following viral infection. As shown in Fig. 1, naive CD8+ T cells activated by CD80low rSV5-infected DC poorly activated T cells at limiting concentrations of peptide, as measured by both proliferation (Fig. 1, A and B) and IFN-{gamma} production (Fig. 1, C and D). The diminished activation was associated with reduced expression of both CD25 and IL-2 (Fig. 2), both functions associated with CD28 binding (5). It is important to note that SV5-matured DC may be lacking in other untested positive signals that might be present on LPS-matured DC, our positive control. However, because CD28 signaling to T cell has been shown to be important to its activation (5), low expression of CD80 seemed a likely candidate that might contribute to the defect observed in T cell activation. Thus, we hypothesized that the selective deficiency in CD80 expression by SV5-infected DC contributed to the suboptimal activation of naive CD8+ T cells. The defects associated with low CD80 expression could be the result of the absence of a unique signal delivered by CD80 engagement, or alternatively, it was possible that it was not the absence of CD80 signal per se, but the overall weaker signal through CD28 that was responsible.

To discriminate between these two hypotheses, we added the CD80 and/or CD86 in trans to the SV5-infected DC-T cell cultures. The addition of CD80, but not CD86, resulted in a significant increase in the percentage of cells that divided and produced IFN-{gamma} (Fig. 3), as well as increased expression of CD25 (Fig. 4B). This indicated that the signal provided by CD80 was essential and could not be compensated for by the addition of additional CD86. To complement these results, we performed similar experiments using immature DC that expressed minimal levels of CD80 and CD86. These cells express low levels of both CD80 and CD86 (14). Again, the addition of CD80 was superior to CD86 for triggering CD25 up-regulation, proliferation, and function (Fig. 6).

The findings from our study provide a mechanism to explain the previously reported (8, 9, 11, 13, 24) increased ability of CD80 to promote T cell activation. For example, Gajewski et al. (25) showed, using a tumor rejection model, that only CD80 transfectants induced lytic activity and protection against tumor challenge. Similarly, Matulonis et al. (9) showed that CD80 was superior in its costimulatory capacity compared with CD86 during the induction and maintenance of T cell-mediated antileukemia immunity in vivo. Additionally, previous studies by Masten et al. (24) have shown that in vitro, T cells stimulated by murine lung DC were almost exclusively dependent on the expression of CD80, whereas CD86 only played a secondary role. In support of the role for CD80 in respiratory immunity, CD80 was reported to be superior to CD86 for promoting proliferation of and IFN-{gamma} secretion by virus-specific CD8+ T cells elicited as a result of infection with influenza virus (13). However, it should be noted that the superiority of CD80 has not been uniformly observed. In a model of myelogenous leukemia, CD86 was shown to be superior to CD80 for the induction of antitumor immunity (26). The reasons for these differences are not known; however, it may be the result of the APC used or the clearance model studied.

IL-2 has long been known as the T cell growth factor and has been shown (27) to support and expand the growth of T cells. Thus, optimal clonal expansion of Ag-specific T cells requires the expression of CD25. Relatively few studies (28, 29) have examined the signals required for optimal CD25 up-regulation. What is known is that TCR engagement of peptide/MHC can trigger the up-regulation of CD25 (28, 29). Furthermore, recent studies (30) by van Stipdonk et al. have shown sustained CD25 expression on responding cells is a function of the length of stimulation. Recently, Notch signaling has been shown (31) to increase T cell activation by enhancing CD25 expression.

Our data have shown that blocking CD25 on CD8+ T cells (as well as CD80 on the APC) caused a significant decrease in the percentage of cells that produced IFN-{gamma}, but not in the percentage of cells that proliferated (Fig. 5 and data not shown). The requirement for IL-2 for optimal function is in agreement with previous data reported by Su et al. (32), where IL-2 was required for high level IFN-{gamma} production by CD8+ T cells. The apparent lack of effect on proliferation observed at day 3 in our system may not be surprising given a report from D’Souza and Lefrancois (19). In these studies, CD25-deficient cells proliferated to similar levels as wild-type cells at 3 days postactivation, suggesting that early division is independent of IL-2.

Although unknown, it is interesting to speculate that the superiority of the signal provided by CD80 compared with CD86 for CD25 expression could be the result of differences in the affinity of CD28 for these two molecules. CD80 is present predominantly as dimers, whereas CD86 is found as monomers (12, 21). Consequently, CD80 has been shown (33) to bind to CD28 with a 5-fold higher affinity compared with CD86, potentially increasing the length of interaction of the T cell with the APC and, thereby, the duration of stimulation, thus leading to robust CD25 expression. Studies by Slavik et al. (22) using CHO-CD80 and CHO-CD86-transfected cells have shown that CD80 engagement resulted in a more robust phosphorylation of CD28 compared with CD86. Thus, the differential effect of CD80 vs CD86 on CD25 expression could be the result of quantitatively or qualitatively different signals.

Recently, signaling by CD80/CD86 has been shown (34, 35, 36) to play an important role in the development and maintenance of CD4+CD25+ T regulatory (Treg) cells. Specifically, Liang et al. (37) have shown that conversion of CD4+ CD25– T cells into functional CD4+CD25+ Treg cells in vivo requires costimulation by CD80/CD86. Although the individual role of CD80 vs CD86 was not addressed in this study, a recent report (38) has implicated CD80 as the major contributor to activation of Treg for suppressive activity. CD86 engagement, by contrast, appears to suppress Treg function. In addition to CD80 engagement, IL-2 binding to the high-affinity form of the IL-2R has been reported (39, 40, 41) to be critical for the induction, maintenance, and suppressive activity of Treg cells. Given our findings, it is possible that CD80 is more important than CD86 in the generation CD4+CD25+ suppressor T cells because of its ability to more efficiently up-regulate CD25. Further studies will be necessary to determine whether CD80 and CD86 differ in their capacity to modulate CD25 expression in these cells.

We have previously shown (14) that in the case of SV5 infection of BALB/c DC, this maturation state includes high expression of cytokines capable of providing signal 3 (IL-12 and IFN{alpha}beta) and CD86 as a source of signal 2, with minimal expression of CD80. Using this model system, we have revealed a previously unappreciated property of CD80, its increased capacity (compared with CD86) for up-regulation of CD25 expression on CD8+ T cells. This property may explain previously reported studies (8, 9, 11, 13, 24) wherein CD80 provided a superior signal for the activation of CD8+ T cells. These findings have important implications with regard to the optimal activation of naive CD8+ T cells and for the design of vaccines and immunotherapeutics, because they suggest the ability of adjuvants/pathogens to regulate CD80 may be an important parameter in shaping the CD8+ T cell response.


   Acknowledgments
 
We thank Drs. Purnima Dubey and Elizabeth Hiltbold for the critical reading of this manuscript. We are grateful to Dr. Griff Parks for provision of SV5 and to Dr. Eric Pamer for the L9.6 Listeria p60-transgenic mice.


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


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

1 This work was supported by National Institutes of Health Grant HL071985 (to M.A.A.-M.). Back

2 Address correspondence and reprint requests to Dr. Martha A. Alexander-Miller, Department of Microbiology and Immunology, Room 5053, Hanes Building, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: marthaam{at}wfubmc.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; rSV5, recombinant simian virus 5; MOI, multiplicity of infection; BMDC, bone marrow-derived DC; Treg, T regulatory; MFI, mean fluorescence intensity; ICS, intracellular cytokine staining. Back

Received for publication April 4, 2006. Accepted for publication July 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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