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Originally published online as doi:10.1189/jlb.1003476 on February 13, 2004

Published online before print February 13, 2004
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(Journal of Leukocyte Biology. 2004;75:874-883.)
© 2004 by Society for Leukocyte Biology

Cross-regulation of CD86 by CD80 differentially regulates T helper responses from Mycobacterium tuberculosis secretory antigen-activated dendritic cell subsets

Mumtaz Yaseen Balkhi, Vinoth K. Latchumanan, Balwan Singh, Pawan Sharma and Krishnamurthy Natarajan1

Immunology Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India

1Correspondence: Immunology Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110-067, India. E-mail: natrajan{at}icgeb.res.in


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We report that stimulation of Mycobacterium tuberculosis secretory antigen- and tumor necrosis factor {alpha}-matured BALB/c mouse bone marrow dendritic cells (BMDCs) with anti-CD80 monoclonal antibody up-regulated CD86 levels on the cell surface. Coculture of these BMDCs with naïve, allogeneic T cells now down-regulated T helper cell type 1 (Th1) responses and up-regulated suppressor responses. Similar results were obtained with splenic CD11c+/CD8a– DCs but not to the same extent with CD11c+/CD8a+ DCs. Following coculture with T cells, only BMDCs and CD11c+/CD8a– DCs and not CD11c+/CD8a+ DCs displayed increased levels of surface CD86, and further, coculturing these DCs with a fresh set of T cells attenuated Th1 responses and increased suppressor responses. Not only naïve but even antigen-specific recall responses of the Th1-committed cells were modulated by DCs expressing up-regulated surface CD86. Further analyses showed that stimulation with anti-CD80 increased interleukin (IL)-10 and transforming growth factor-ß-1 levels with a concomitant reduction in IL-12p40 and interferon-{gamma} levels from BMDCs and CD11c+/CD8a– DCs and to a lesser extent, from CD11c+/CD8a+ DCs. These results suggest that cross-talk between costimulatory molecules differentially regulates their relative surface densities leading to modulation of Th responses initiated from some DC subsets, and Th1-committed DCs such as CD11c+/CD8a+ DCs may not allow for such modulation. Cognate antigen-presenting cell (APC):T cell interactions then impart a level of polarization on APCs mediated via cross-regulation of costimulatory molecules, which govern the nature of subsequent Th responses.

Key Words: MTSA • Th1 response • polarization • co-stimulation • cross-talk


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Different subsets of dendritic cells (DCs) are among the most potent antigen-presenting cells (APCs) of the innate immune system that have the ability to stimulate quiescent, naïve, or memory T lymphocytes [1 ]. DCs exist at various states of development, activation, and maturation, which are defined by distinct phenotypic and functional modalities, wherein immature DCs are proficient at antigen-capture, and mature DCs are effective T cell stimulators [2 3 ]. Another feature that also typifies DCs is their ability to exist at various states of polarization, which are reflected in the subsequent polarization of T cell responses. Various factors are known to alter and contribute toward DC polarization and are often referred to as the "third signal", which governs T cell responses [4 ]. Among these are the surface levels of costimulatory molecules and the local cytokine milieu in which DCs develop [5 ].

In addition to B7.1 (CD80) and B7.2 (CD86), a number of cell-surface molecules have now been identified that show T cell costimulatory capacity [6 7 8 ]. These molecules have structural and functional similarities to the B7 molecules. However, to date, CD80 and CD86 still remain the most characterized in terms of regulating T-dependent responses. CD80 and CD86 are members of the immunoglobulin (Ig) supergene family [9 10 11 ] and are expressed on many cell types including T cells, macrophages, and DCs [12 13 ]. We [14 ] and others [15 16 17 ] have investigated the factors regulating the expression of both of these molecules. Consequently, the roles of these molecules in shaping effector T helper (Th) responses have been well documented.

We have demonstrated in the past that the relative surface densities of CD80 and CD86 on activated B cells govern the nature of Th responses [18 ] and also that there exists a distinct "cross-talk" between the two molecules, such that stimulation of CD86 on activated B cells up-regulates surface levels of CD80 [19 ]. The up-regulated CD80 now skews the T cell response toward Th1, indicating that cross-regulation between two costimulatory molecules is yet another way of regulating Th responses. As DCs constitute the principal APCs that initiate primary T cell responses, in the present study, we extended our observations to Mycobacterium tuberculosis secretory antigen (MTSA)-activated DC subsets in an effort to investigate the existence of similar cross-talk between costimulatory molecules and its influence on subsequently generated naïve and recall T cell responses. We show that stimulation of CD80 with monoclonal antibody (mAb) or during coculture with T cells up-regulates surface CD86 levels that now generate suppressor T cell responses in certain DC subsets, and further, this cross-talk is regulated by a change in the cytokine profiles of DCs following stimulation of CD80.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Female BALB/c mice, 4–6 weeks of age, were used in the study for all experiments involving DCs. For enrichment of T cells, BALB/c or C57BL/6 mice were used. All the animals were maintained under pathogen-free, environment-controlled conditions in the small animal facility of our institute. The study was undertaken after prior approval from the Institutional Animal Ethics Committee.

Materials
Fluorescein isothiocyanate-tagged mAb against mouse cell-surface molecules CD80 (clone 1G10), CD86 (clone GL-1), biotin-conjugated antibodies to CD90 (clone 53-2.1), and purified antibodies to CD16/CD32 (Fc receptor for IgG, clone 2.4G2), CD80 (clone 1G10 NA/LE), CD86 (clone GL-1 NA/LE), mucosal addressin cell adhesion molecule-1 (MAdCAM-1; clone MECA-367 NA/LE), vascular cell adhesion molecule-1 [VCAM-1; clone 429 (MVCAM.A) NA/LE], CD49d (clone R1-2 NA/LE), CD154 (clone MR1 NA/LE), and isotype-matched control antibodies were purchased from PharMingen (San Diego, CA). Anti-CD4-, -CD8-, -CD90 (Thy 1.2)-, -B220-, -CD11b-, -CD11c-, -I-A-, and -CD19-coated magnetic beads were obtained from Miltenyi Biotec (Auburn, CA). Mouse recombinant granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor {alpha} (TNF-{alpha}), and enzyme-linked immunosorbent assay (ELISA) kits for the estimation of mouse cytokines and naïve CD4+ T cell enrichment kits were purchased from R&D Systems (Cambridge, MA). Antiphosphotyrosine antibody (clone PY20) and luminol reagents for Western blotting were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). All antibodies used in culture were F(ab')2 fragments generated by enzymatic digestion of the whole molecule followed by purification over protein G columns.

MTSA
MTSA was recombinantly expressed in Escherichia coli using the pQE31 vector (Qiagen, Valencia, CA) followed by purification over Ni-NTA columns as described earlier [20 21 ]. The recombinant protein has earlier been shown to contain negligible amounts of endotoxins.

Enrichment of DC precursors from bone marrow (BM) and generation of DCs
DCs were differentiated from leukocyte precursors by culturing them in GM-CSF as described before [21 ]. When required, these BM-derived DCs (hereafter called BMDCs) were matured with 20 µg/ml MTSA or 20 ng/ml TNF-{alpha} [21 ]. For up-regulating CD86, cytokine- or antigen-activated DCs were stimulated with 50 µg/ml purified anti-CD80 for 24 h. Cells at the end of incubation in all sets were analyzed for the levels of surface molecules by flow cytometry as described before [20 ] or cocultured with naïve, allogeneic or antigen-primed, syngeneic T cells as described below. All flow cytometric analyses were performed on CD11c+-gated cells.

Enrichment of splenic DCs
CD8a– DCs and CD8a+ DCs from the spleens were enriched as described earlier [21 ]. Enriched splenic DCs (90–95%) were stimulated with MTSA or TNF-{alpha} and processed as performed for BMDCs.

Enrichment of T lymphocytes
Naïve CD4+ T cells from the spleens of 4- to 6-week-old C57BL/6 mice were enriched using the purification kit from R&D Systems by strictly following the manufacturer’s instructions. Purification of antigen-specific T cells was done as described previously [20 21 ]. Briefly, inguinal lymph nodes from immunized BALB/c mice were first depleted of adherent cells by panning over plastic plates. From this, B lymphocytes and residual, adherent cells were then removed by two rounds of incubation with anti-CD19-, -CD45R-, -I-A-, -CD11c-, and -CD11b-coated magnetic beads, followed by separation through magnetic cell sorter columns. The purity of the resulting population of T cells obtained in this manner was 95–98%, as determined by CD90–phycoerythrin-stained cells by flow cytometry. The percentage of I-A+ cells in all the fractions was found to be less than 0.5%.

T cell stimulation: allogeneic mixed leukocyte reactions
Enriched, naïve, CD4+, allogeneic C57BL/6 T cells (3x106) from spleens were cocultured with irradiated (3000 rads) DCs at 1:5 DC:T cell ratio in 24-well plates for a period of 48 h. For some experiments, nonirradiated DCs were cocultured with naïve T cells for 48 h. From this coculture, the T cells were depleted by incubation with 1 mM EDTA for 10 min (to break the DC:T cell complexes) followed by the addition of anti-CD90 antibody for 1 h at 4°C. Rabbit complement was added to the cells and incubated for 1 h at 37°C to lyse the T cells. An aliquot of the DCs was stained for surface levels of CD80 and CD86, and the remaining DCs were then irradiated and recultured with a fresh set of naïve T cells for 48 h, and culture supernatants were then screened for the presence of cytokines as described below. The percentage of T cells following complement-mediated depletion was found to be 2–5%.

Syngeneic T cell stimulation
For measuring antigen-specific T cell responses, BALB/c mice were immunized subcutaneously at the base of tail with MTSA (50 µg/mouse) in incomplete Freund’s adjuvant for 7 days as described previously [20 ]. Inguinal lymph nodes from these mice were removed, and T cells were enriched as described above. Enriched T cells were cocultured with irradiated DCs for 48 h, and culture supernatants were analyzed for cytokines.

Estimation of cytokines
Culture supernatants of DCs or DC–T cell cocultures at the end of each incubation period were analyzed for the levels of IL-12p40, interferon-{gamma} (IFN-{gamma}), or interleukin (IL)-10 using a sandwich ELISA, as recommended by the manufacturer. The sensitivity ranges for all three cytokines were between 31.2 and 2000 pg/ml. Quantitation was made against a standard curve obtained for individual cytokine standards provided by the manufacturer. Samples were correspondingly diluted to obtain values within the linear range of the standards. Transforming growth factor-ß-1 (TGF-ß-1) levels were measured by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA extracted from cells treated in various ways. The following primers were used: forward, 5' CGG AAG CGC ATC GAA GCC ATC C-3', and reverse, 5' GGG TCA GCA GCC GGT TAC CAA-3'. Amplification of ß-actin was performed and used as loading control.

Analysis of tyrosine phosphorylation
MTSA-matured BMDCs were stimulated with 50 µg/ml anti-CD80 for various times. At the end of the incubation, cells were chilled on ice and washed once with ice-cold phosphate-buffered saline and lysed in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 M EGTA, 0.5% Nonidet P-40, and 2 µg/ml each aprotinin, leupeptin, and pepstatin, followed by centrifugation at 13,000 rpm for 2 min at 4°C. Supernatants treated as cytoplasmic extracts were then resolved on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a nitrocellulose membrane (Hybond C pure, Amersham, Arlington Heights, IL). The blots were then probed with antibody to phosphotyrosine followed by horseradish peroxidase-labeled secondary antibody. The blots were later developed by chemiluminescence using the luminol kit from Santa Cruz Biotechnologies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of BMDCs with anti-CD80 up-regulates surface CD86
We have recently shown that ligation of activated B cells with anti-CD86 antibody led to the up-regulation of CD80 with significant effects on the nature of downstream Th cell responses [19 ]. To investigate if CD80 can induce the expression of CD86, we extended the above observations on their "cross-regulation" to DC subsets—the principal cell types that are recruited at sites of infection that initiate primary immune responses [1 ]. To obtain increased levels of costimulatory molecules on DC surface for subsequent stimulation with anti-CD80, we performed the present study with antigen- and cytokine-matured DCs. For this, we used DCs matured with a 10-kDa MTSA or TNF-{alpha}. We have shown earlier that MTSA induces the maturation of DC subsets with determinant effects on immune responses to mycobacterial antigens [21 ] and hence, would be ideal to study the modulation of Th responses as a function of differential surface densities of CD80 and CD86 together with their cross-regulation-mediated effects. Further, on MTSA-matured DCs, CD80 stained at a higher fluorescence intensity when compared with CD86 levels, indicating that CD80 is perhaps expressed at a higher level than CD86 [21 ]. TNF-{alpha} is a well-known, terminal DC maturation-inducing cytokine [1 2 3 ] and hence, was used. To begin with, we stimulated MTSA- or TNF-{alpha}-matured BMDCs with anti-CD80 for various lengths of time and monitored the levels of CD86 on the cell surface. Earlier experiments in our laboratory have shown that anti-CD80 mAb remains attached to the cell surface and does not get dissociated even after 72 h in culture. As shown in Figure 1A , a and b, consistent with our earlier report, MTSA-matured BMDCs showed better staining for CD80 when compared with CD86. However, stimulation of these DCs with anti-CD80 for 24 h now increased the levels of CD86 by fivefold (see Fig. 1A , c), indicating that indeed, CD80 up-regulated the expression of CD86 on DCs and further, that there exists a cross-regulation between the two costimulatory molecules. Stimulation with an isotype-matched control antibody had no effect on CD86 levels (Fig. 1A , d). Further, stimulation with anti-CD80 for longer time points had no additive effect on the up-regulation of CD86 (data not shown), and hence, this time point was used for all subsequent experiments. Similar results were obtained with DCs matured with TNF-{alpha}, wherein like MTSA-matured DCs, these DCs also showed better staining for CD80 as compared with CD86 (see Fig. 1A , e and f), and further, stimulation with anti-CD80 now induced the expression of CD86 on these DCs (see Fig. 1A , g), indicating that up-regulation of CD86 by CD80 is not specific to MTSA-matured DCs. Although CD80-mediated CD86 up-regulation was obtained, even in unstimulated, immature DCs (data not shown), the levels were rather low, probably owing to the fact that the expression of CD80 (and other costimulatory molecules) is low in immature DCs [2 ], and therefore, all experiments have been performed on MTSA- and TNF-{alpha}-matured DCs.



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  		Figure 1. CD86 is up-regulated on BMDCs following stimulation with anti-CD80. (A) Surface levels of CD80 or CD86 on BMDCs following maturation with MTSA (a–d) or TNF-{alpha} (e–h; see Materials and Methods). (c and g) CD86 levels on MTSA- and TNF-{alpha}-matured DCs following stimulation with 50 µg/ml anti-CD80 (thick line) for 24 h and (d and h) stimulation with an isotype-matched control antibody, respectively. The dotted lines in all the histograms depict levels on unstimulated DCs, and the thin lines show staining with isotype-matched control antibody on unstimulated DCs. (B) Fold increase in relative mean fluorescence intensities (MFI) of CD86 levels over unstimulated DCs (Control) on MTSA-matured (open bars) or TNF-{alpha}-matured (solid bars) BMDCs stimulated with 50 µg/ml antibody to indicated molecules for 24 h. *, Differences in the means between indicated groups are significant at P < 0.05. Data from one of five independent experiments are shown. a, Anti.

 
Although the anti-CD80 antibody that was used had low levels of endotoxins (clone 1G10 NA/LE; see Materials and Methods), nevertheless, we estimated the endotoxin levels in the antibody vials before and following generation of F(ab')2 fragments by enzymatic digestion by using the E-Toxate endotoxin detection kit from Sigma Chemical Co. (St. Louis, MO) to rule out the possibility of the observed effects being mediated by endotoxins in the anti-CD80 vials. The endotoxin levels were found to be less than 0.03 endotoxin units/ml based on the absence of a hard gel formation in the 0.03 endotoxin units/ml standard in the kit. This indicated that the observed effects were induced by anti-CD80 stimulation alone and not by any endotoxins in the antibody vials or its F(ab')2 fragments. Further, to check if the observed effects were specific to anti-CD80, we stimulated DCs with antibodies to a number of cell-surface molecules and examined the levels of surface CD86. As shown in Figure 1B , addition of anti-CD80 alone resulted in the up-regulation of CD86, and addition with many other antibodies had no effect, indicating that the effects were specific to CD80 for MTSA- and TNF-{alpha}-matured DCs.

Up-regulated CD86 down-regulates Th1 responses
To test whether the up-regulated CD86 displays costimulatory activity and differentially modulates Th responses, we cocultured CD86-expressing DCs with naïve, allogeneic T cells and monitored the cytokine profiles of the interacting T cells. Expectedly and consistent with our earlier observations [21 ], coculture of MTSA- or TNF-{alpha}-matured DCs with allogeneic T cells resulted in the dominant secretion of IFN-{gamma} from the T cells when compared with IL-10 levels (see Fig. 2 ). However, coculture of allogeneic T cells with DCs that were stimulated with anti-CD80 (to up-regulate CD86) now resulted in a down-regulation of IFN-{gamma} by over fourfold for MTSA- and TNF-{alpha}-matured DCs (see Fig. 2 ). These results indicate that CD80 levels on DCs principally drive the secretion of IFN-{gamma} from the interacting T cells. Further, this decrease in IFN-{gamma} levels was also accompanied by a significant increase in IL-10 levels by fivefold from these cultures (see Fig. 2 ), indicating that relative distribution of CD80 and CD86 on DCs influences subsequent Th responses with CD80 governing Th1 responses and CD86 influencing the development of suppressor responses. Further, addition of anti-CD86 antibody during the coculture with T cells reduced the levels of IL-10 to near basal levels, confirming that the observed increase in IL-10 levels was indeed mediated by CD86 up-regulation on DCs. In an earlier study involving blocking of CD80 and CD86 [22 ], similar to the data obtained in Figure 1A , we have observed that coculture of T cells with DCs that have been stimulated with isotype-matched control antibody had no effect on the relative levels of IFN-{gamma} or IL-10 from the interacting T cells, thereby indicating that the effects observed were specific to CD80 and not a result of IgG stimulation.



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  		Figure 2. Up-regulation of CD86 by CD80 on DCs induces suppressor responses. MTSA- or TNF-{alpha}-matured BMDCs were stimulated with 50 µg/ml anti-CD80 (a-CD80) for 24 h and cocultured with naïve, allogeneic T cells for 48 h (see Materials and Methods). Culture supernatants were then screened for the levels of IFN-{gamma} or IL-10. In some groups, anti-CD86 (a-CD86) antibody was added at the time of coculture with T cells. Control depicts values of T cell cocultured with unstimulated DCs. *, Differences in the means between indicated groups are significant at P< 0.05. One of five experiments is shown.

 
CD80 differentially up-regulates CD86 on splenic CD11c+/CD8– and CD11c+/CD8+ DCs
Of the many DC subsets identified in the mouse spleen, the two most well studied are the CD11c+/CD8a– (hereafter, CD8a–) and CD11c+/CD8a+ (hereafter CD8a+) subsets. Although the CD8a– DCs are believed to induce Th1 or Th2 responses, the CD8a+ DCs primarily induce Th1 responses [23 ]. Therefore, we thought it would be of interest to investigate if CD80 would be able to induce the expression of CD86 on these two subsets. Enriched CD8a– DCs and CD8a+ DCs were matured with MTSA or TNF-{alpha} and subsequently stimulated with anti-CD80. Expectedly and consistent with our earlier report, MTSA- and TNF-{alpha}-matured DC CD80 stained at a higher fluorescence intensity compared with CD86 (ref. [21 ] and Fig. 3A ). CD80 stimulation of CD8a– DCs now readily induced a high-level expression (approximately threefold) of CD86 on the cell surface (see Fig. 3A ). However, CD86 levels were increased only marginally (<twofold) on CD8a+ DCs. Even a longer incubation with over fivefold excess of anti-CD80 levels failed to up-regulate CD86 to levels observed on CD8a– DCs (data not shown). This was true irrespective of whether DCs were matured with TNF-{alpha} or MTSA. These results indicate that CD8a– DCs allow CD80-induced expression of CD86, and CD8a+ DCs are more resistant to modulation of surface densities of CD86. Concomitant with the CD86 expression profiles, the subsequent, naïve, allogeneic T cell responses were also similarly modulated (see Fig. 3B ). A threefold decrease in IFN-{gamma} levels from the interacting T cells was observed when CD86-expressing CD8a– DCs were used along with a three- to fourfold increase in IL-10 levels. Although IFN-{gamma} levels of T cells cocultured with CD8a+ DCs were similarly down-regulated (owing to blocking of CD80), IL-10 levels were increased by only twofold when CD80-stimulated CD8a+ DCs were used. Again, inclusion of anti-CD86 mAb in DC:T cell cocultures brought the IL-10 levels to basal levels in anti-CD80-stimulated CD8a– DCs, confirming that the increase in the IL-10 levels was indeed mediated by the up-regulated CD86.



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  		Figure 3. CD86 is differentially up-regulated on CD8a– DCs and CD8a+ DCs following stimulation with anti-CD80. Enriched CD8a+ (solid bars) or CD8a– (hatched bars) splenic DCs were matured with MTSA or TNF-{alpha} for 24 h. This was followed by stimulation with 50 µg/ml anti-CD80 (a-CD80) or isotype-matched control antibody (a-Ig) for 24 h. An aliquot of cells was stained for levels of CD80 or CD86 (A), and remaining cells were cocultured with allogeneic, naïve T cells for 48 h, and culture supernatants were screened for the levels of cytokines (B). (A) The bars show fold increase in relative MFI of CD80 or CD86 over unstimulated controls. (A and B) Control represents MFI of unstimulated DCs and cytokine levels of unstimulated DCs cocultured with T cells, respectively. In some groups, anti-CD86 (a-CD86) was added during coculture with T cells. *, Differences in the means between indicated groups are significant at P < 0.05. Data from one of four independent experiments are shown.

 
CD86 is up-regulated during DC–T cell cognate interactions and down-regulates Th1 responses of a subsequent interaction
To give a physiological relevance to the above observations and to mimic conditions where cross-regulation of costimulatory molecules would indeed take place, we cocultured MTSA- and TNF-{alpha}-matured, various DC subsets with naïve, allogeneic T cells for 48 h. From this, T cells were removed by incubation with anti-CD90 followed by complement-mediated lysis. An aliquot of the remaining DCs was stained for the levels of CD80 and CD86, and the remaining DCs were now recultured with a fresh set of naive T cells for 48 h. Supernatants from all the groups were later screened for the relative levels of IFN-{gamma} and IL-10. The results from the above experiments are depicted in Figure 4 . Coculturing of BMDCs and CD8– DCs with T cells now resulted in an up-regulation of CD86 by twofold (Fig. 4A) , indicating that CD86 is indeed up-regulated on these DCs during a cognate interaction with T cells. Consistent with the results obtained with anti-CD80, no up-regulation of CD86 was seen on CD8+ DCs cocultured with T cells (see Fig. 4A ). It is interesting that following coculture with T cells, the CD80 levels on BMDCs and CD8a– DCs were down-regulated by over five- to sixfold. No significant decrease in CD80 levels was however noticed on CD8a+ DCs upon coculture with T cells. Further, coculture of BMDCs and CD8a– DCs with a fresh set of naïve T cells now resulted in a dramatic reduction of IFN-{gamma} production by over fourfold (see Fig. 4B ), which could possibly result from the decreased CD80 levels. This was accompanied by a significant increase (P<0.05) in the levels of IL-10 by over threefold, again indicating a shift in the net Th cell responses with a reduction in Th1 responses with the progression of the immune response. Addition of anti-CD86 during coculture with the fresh set of T cells brought the IL-10 levels to baseline, confirming that the observed increase in IL-10 levels was mediated by CD86. Further, inclusion of antibody to CD28 but not cytotoxic T-lymphocyte antigen (CTLA)-4 (the T cell counter-receptors for CD80 and CD86) during coculture of BMDCs and CD8a– DCs with T cells did not up-regulate CD86; also, coculture of CD86-expressing BMDCs and CD8a– DCs with the fresh set of T cells in the presence of anti-CD28 but not anti-CTLA-4 reduced the IL-10 levels to baseline, indicating that up-regulation of CD86 during coculture with T cells was mediated by CD28, and further, the up-regulated CD86 interacted with CD28 to modulate the subsequent Th responses (data not shown). Expectedly, no significant change in IFN-{gamma} or IL-10 levels was observed in the case of CD8a+ DCs following coculture with T cells.



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  		Figure 4. CD86 is up-regulated on DCs following coculture with T cells. BMDCs (open bars) or CD8a+ DCs (solid bars) or CD8a– DCs (hatched bars) were matured with MTSA or TNF-{alpha} and cocultured with allogeneic T cells for 48 h. T cells from the coculture were depleted by complement-mediated lysis. An aliquot of the DCs was stained for the surface levels of CD80 or CD86 (A), the remaining DCs were then cocultured with a fresh set of naïve, allogeneic T cells (FrT) for 48 h (B), and culture supernatants were screened for the levels of indicated cytokines. Antibody to CD86 (a-CD86) was added in some groups during coculture with FrT. (A) Bars show fold increase in relative MFI of CD80 or CD86 levels over unstimulated DCs. (A) Control depicts levels on unstimulated DCs; (B) Control depicts cytokine levels of unstimulated DCs cocultured with T cells or FrT cells. *, Differences in the means between indicated groups are significant at P < 0.05. One of three independent experiments is shown.

 
Ligation of CD80 modulates antigen-specific responses
We next explored whether the antigen-specific responses of MTSA would also be affected as a result of CD86 up-regulation on DCs. For this, MTSA-matured DC subsets were incubated with anti-CD80 and later cocultured with MTSA-specific T cells for 48 h. Culture supernatants were then scored for the levels of IFN-{gamma} and IL-10. As shown in Figure 5 , an increase in IL-10 levels by three- to fourfold in BMDCs and CD8a– DCs with a concomitant reduction in IFN-{gamma} levels by over threefold were observed when anti-CD80-stimulated DCs were cocultured with MTSA-specific T cells when compared in the absence of anti-CD80 stimulation. Again, IL-10 levels from MTSA-specific T cells were minimally modulated when CD8a+ DCs were used, and IFN-{gamma} levels were considerably reduced, owing to blockade of CD80. These results indicate that not only are the primary T cell responses modulated, but even the recall responses of Th1-polarized T cells are shifted, implying a significant role for CD80-mediated CD86 up-regulation in differentially regulating the progression of antigen-specific, immune responses elicited by DCs.



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  		Figure 5. CD86 up-regulation modulates antigen-specific recall T cell responses of MTSA in vitro. MTSA-matured BMDCs (open bars) or CD8a+ (solid bars) or CD8a– DCs (hatched bars) were stimulated with anti-CD80 (a-CD80) and were cocultured with MTSA-specific, enriched T cells (M-T) for 48 h. IFN-{gamma} and IL-10 levels were monitored in the culture supernatants. In some groups, anti-CD86 (a-CD86) antibody was added to DC:T cell cocultures. Control represents cytokine levels of unstimulated DCs cocultured with MTSA-specific T cells. *, Differences in the means between indicated groups are significant at P < 0.05. One of four independent experiments is shown.

 
CD80 stimulation enhances IL-10 and TGF-ß-1 levels from DCs
Polarization of DCs into type 1 or type 2 is subject to a number of parameters with differential cytokine profiles being a key factor [4 5 ]. Among the various cytokines that regulate polarization of DCs are IL-12, IFN-{gamma}, IL-10, and TGF-ß [5 ]. Therefore, to investigate if stimulation of CD80 induces a change in the cytokine-secretion profiles of DCs and also to identify possible mechanisms for CD80-mediated CD86 up-regulation in DCs, we looked at the levels of IFN-{gamma}, IL-10, and TGF-ß-1 from MTSA- and TNF-{alpha}-matured DC subsets, before and following stimulation with anti-CD80. We also looked at changes in the levels of IL-12p40 in MTSA-matured DC subsets. IL-12p40 levels were undetectable in TNF-{alpha}-matured DC subsets, and hence, the effect of anti-CD80 stimulation in these DCs could not be assessed. As shown in Figure 6A , MTSA-matured BMDCs secreted moderate levels of IFN-{gamma} and IL-10. MTSA-matured CD8a+ DCs secreted high levels of IFN-{gamma} as compared with IL-10 levels (Fig. 6B) , and the ratio of IFN-{gamma} to IL-10 was slightly lower in MTSA-matured CD8a– DCs (see Fig. 6C ). It is interesting that IFN-{gamma} levels were lower in all the three DC subsets in TNF-{alpha}-matured DCs when compared with MTSA-matured DCs. However, more significant was the fact that stimulation of DCs with anti-CD80 increased the levels of IL-10 in MTSA and TNF-{alpha} sets by over twofold in BMDCs and by two- to fivefold in CD8a– DCs, respectively. Surprisingly, the IFN-{gamma} levels from these DCs were now severely down-regulated by over fivefold. In contrast, no significant change in the IFN-{gamma} levels was observed in the CD8a+ DCs, and IL-10 levels did show a significant increase. In fact, stimulation of TNF-{alpha}-matured CD8a+ DCs with anti-CD80 increased IFN-{gamma} levels by twofold. Essentially similar results were obtained with IL-12p40 levels from MTSA-matured DC subsets. MTSA readily induced the secretion of IL-12p40 from all three DC subsets (Table 1 ). Stimulation of BMDCs and CD8a– DCs with anti-CD80 brought down IL-12p40 by three- to fourfold, and IL-12p40 levels in CD8a+ DCs were marginally reduced (just twofold; see Table 1 ) upon anti-CD80 stimulation. The down-regulation of IFN-{gamma} levels in BMDCs and CD8a– DCs following stimulation of CD80 could possibly result, owing to reduced IL-12p40 levels, as IL-12 positively regulates IFN-{gamma} levels from DCs [4 5 ]. Further, TGF-ß-1 levels that were below detectable levels in MTSA- or TNF-{alpha}-matured BMDCs and CD8a– DCs were now amplified by several-fold upon stimulation of these DCs with anti-CD80 (see Fig. 6D ). However, TGF-ß-1 levels were amplified only marginally in CD8a+ DCs. Nevertheless, these results indicate that stimulation of CD80 differentially modulates the cytokine-secretion profiles and surface densities of CD86 on DC subsets, which perhaps in turn, qualitatively regulate Th responses.



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  		Figure 6. Stimulation of CD80 induces IL-10 and TGF-ß-1 levels from DCs. MTSA- or TNF-{alpha}-matured BMDCs (A) or CD8a+ (B) or CD8a– DCs (C) were stimulated with 50 µg/ml anti-CD80 (a-CD80) for 24 h. For comparison, DCs stimulated for 24 h with MTSA or TNF-{alpha} were also used. At the end of the incubation period, supernatants from all the groups were then screened for the levels of indicated cytokines. (D) RT-PCR amplified TGF-ß-1 levels in DCs treated as in A–C. Labels on the top represent various treatments, and labels on the side represent the corresponding DC subset. ß-Actin from one of the above panels is shown as loading control. The ß-actin levels of all the groups in all the panels were the same. Control in all panels depicts cytokine levels on unstimulated DCs. *, Differences in the means between indicated groups are significant at P < 0.05. Data from one of four independent experiments are shown.

 

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  		Table 1. CD80 Stimulation Decreases IL-12p40 Levels in MTSA-Matured BMDCs and CD8a– DCsa

 
Stimulation of CD80 induces tyrosine phosphorylation of proteins
To investigate if CD80 induces signal transduction from its cytoplasmic domains, we looked for induction of tyrosine phosphorylation following stimulation of MTSA-matured BMDCs with anti-CD80 for various time intervals. As shown in Figure 7 , tyrosine phosphorylation of a number of proteins was induced within 5 min of stimulation of CD80 and was maintained for up to 2 h, implying that CD80 indeed tranduces signaling that might play a role in the up-regulation of CD86.



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  		Figure 7. Stimulation of CD80 induces tyrosine phosphorylation. MTSA-matured BMDCs were stimulated with 50 µg/ml anti-CD80 for the indicated times, and cytoplasmic extracts were prepared. Proteins (10 µg) were resolved on 8% SDS-PAGE, transferred onto nylon membranes, and Western blotted for tyrosine-phosphorylated proteins. Lanes 2–8, Stimulation with anti-CD80 for 2, 5, 10, 15, 30, 60, and 120 min, respectively. Lane 1, Profile of MTSA-matured BMDCs.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Productive T cell responses occur upon engagement of the T cell receptor (TCR) with major histocompatibility complex (MHC)/antigen along with a second signal via the CD28/B7 receptor ligand system [24 25 ]. The second signal constitutes an important step toward mature T cell responses, and any interruption of this signaling pathway with CD28 antagonists not only results in the suppression of immune responses but also induces antigen-specific tolerance. Kuchroo et al. [26 ], who examined the effects of anti-CD80 and anti-CD86 treatment on the initiation of experimental autoimmune encephalitis in mice, demonstrated the first report that CD80 (B7.1) and CD86 (B7.2) costimulatory molecules might play a role in differential signaling of precursor Th cells. Treatment of mice with anti-CD80 resulted in a reduced incidence of disease, and anti-CD86 increased disease severity. The same dichotomy in Th differentiation was observed when myelin basic protein-specific TCR-transgenic T cells were primed and restimulated in the presence of anti-CD80 and anti-CD86 mAb [17 ]. Studies by Freeman et al. [27 ] also supported an important role for CD86 in the signaling of IL-4 production, such that although transfection of CD80 induced similar levels of IL-2 and IFN-{gamma}, only CD86 transfectants were able to induce IL-4. Recent studies further suggest that B7-2 interactions are not necessary for the initiation of the type 2 immune responses but are instead required for its progression after the development of effector T cells [28 29 ]. We have also shown distinct effects—the relative surface densities of CD80 and CD86 of B cells are able to exert—on the subsequently generated T cell responses, wherein increased expression of CD80 led to an increase in the levels of IFN-{gamma} from the T cells [18 19 ]. However, CD80 and CD86 can have the reverse and often no effect in influencing Th1 and Th2 differentiation. Bluestone and colleagues [15 ] put forth a temporal explanation for the differential effects of CD80 and CD86 molecules, wherein under conditions of low antigen density (as found at early time points in the immune response) together with faster kinetics of up-regulation of CD86, the extent of initial T cell priming will function to regulate Th1 and Th2 responses. This is more predominant in cases of weak MHC/TCR interactions, for example, when using altered peptide ligands as antigens. Further, in a graft-versus-host disease (GVHD) model, it was also recently shown that blocking CD80 converted chronic GVHD to acute GVHD by inducing the secretion of Th1 cytokines from CD4+ T cells [30 ].

As DCs constitute one of the key players in regulating primary immune responses to various pathogens [31 ], we thought it would be of interest and functionally more significant to examine the existence of such a cross-talk between costimulatory molecules, CD80 and CD86, and its outcome on the ensuing immune responses. Our results show that stimulation of antigen- or cytokine-matured BMDCs with anti-CD80 did indeed increase the surface levels of CD86, which was initially expressed at low levels, indicating the existence of cross-talk between CD80 and CD86. Further, coculture of these DCs with naïve, allogeneic T cells now showed a decrease in the levels of IFN-{gamma}, together with a significant increase in the levels of IL-10, again suggesting that CD80 governs Th1 or proinflammatory responses, and CD86 controls suppressor responses from DCs. It is however also possible that along with reduced CD80, the up-regulated CD86 may contribute indirectly toward reduction of IFN-{gamma} levels by increasing the levels of Th2 cytokines, as has been shown by De Becker et al. [32 ]. It is important that this seemed to be true, irrespective of whether the DCs were matured with antigen or cytokine. Furthermore, of the two splenic DCs tested for the above activity, CD86 was up-regulated to greater levels in only the CD8a– DC subset, and the CD8a+ DCs were considerably resistant to CD80 stimulation. That such a scenario might actually take place under physiological conditions was demonstrated in our experiments, wherein coculture of T cells with DCs led to the up-regulation of CD86 on the interacting DCs. Subsequent coculture of these DCs with naïve T cells now down-regulated Th1 responses and resulted in the development of suppressor responses. It is interesting that not only were the naïve Th responses modulated, but also, even the antigen-specific recall responses showed a similar trend in the case of BMDCs and CD8a– DCs. Again, the CD8a+ DC subset did not permit such modulation, and the T cell responses were maintained as Th1.

Further, the above results demonstrate that net Th responses to antigens are likely to shift between inflammatory and suppressor responses, and the progression of the immune response and the relative surface levels of costimulatory molecules may play a determinant role in such a shift. It is important that as DCs initiate most of the primary T cell responses, the above results indicate that the nature of initial T cell responses may well depend on the relative surface densities of the B7 costimulatory molecules at the time of antigen-mediated priming of the DCs and may even induce a level of "polarization" on the DCs that may influence the quality of subsequently elicited T-dependent responses.

An associated, intriguing observation was the decrease in the levels of CD80 on BMDCs and CD8a– DCs following coculture with allogeneic T cells. This could possibly be explained based on the changes in cytokine profiles of DCs following CD80 stimulation. As IFN-{gamma} levels positively regulate CD80 levels in a number of cell types including B cells, and IL-10 levels down-regulate CD80 levels [33 34 ], a decrease in IFN-{gamma} levels together with increased IL-10 levels could possibly result in down-regulation of surface CD80 levels in these DCs. Further reduced IL-12 (IL-12p40 in the present case) might also contribute toward reduced IFN-{gamma} from these DCs. As IL-12p40 and IFN-{gamma} levels from CD8a+ DCs were not significantly affected upon stimulation with anti-CD80, hence, no decrease in CD80 levels was observed following coculture with T cells. Furthermore, higher levels of IFN-{gamma} together with the absence of enhancement in TGF-ß-1 levels upon CD80 stimulation could account for the inability of this DC subset to up-regulate CD86 levels despite increase in IL-10 levels, possibly resulting from differential kinetics of up-regulation of IL-10 and CD86 following stimulation of DCs with anti-CD80. We have observed that IL-10 is secreted from DCs within 2 h of anti-CD80 stimulation, and CD86 is not seen before 12 h (data not shown). Furthermore, incubation of anti-IL-10 before CD80 stimulation did not inhibit CD86 up-regulation, indicating that up-regulation of CD86 is not mediated by IL-10 (M. Y. Balkhi and K. Natarajan, unpublished results). This then indicates that the relative levels of IFN-{gamma} and/or TGF-ß-1 regulate the expression of CD86 on DC subsets, at least in the present system. It has been well documented that CD8a+ DCs in the mouse initiate Th1 responses, and the CD8a– DCs along with myeloid BMDCs can initiate Th1 or Th2 responses [23 ]. Our results essentially add support to the above classification (at least in the mouse system) and suggest that CD8a+ DCs show functional plasticity by possibly remaining polarized to initiate Th1 responses, and the CD8a– DCs show functional flexibility, which may perhaps be influenced by the relative distribution of the costimulatory molecules and the cytokine profiles secreted by these DCs following antigen-induced activation that subsequently governs T cell responses.

Thus, apart from their well-established role in providing key secondary signals toward T cell activation, our results identify another key functional role for these costimulatory molecules in influencing the quality of Th responses by regulating the expression of each other. With a number of costimulatory molecules being identified on APCs, identification of similar or related cross-talk between them via their T cell counter-receptors could constitute a unique mechanism of regulating immune responses. Investigations of such cross-talk between molecules such as programmed death (PD)-1 and PD ligand (PDL)-1, which induce apoptosis and suppressor responses [35 36 37 38 39 40 ], would provide valuable insights into yet-unidentified, functional attributes of these costimulatory molecules. Further, as TGF-ß also induces apoptosis in certain cell types [41 ], coupled with our results about the up-regulation of TGF-ß-1 levels upon stimulation of CD80, induction of apoptosis and/or suppressor responses following interactions between PD-1 and PDL-1 can be contemplated.

Furthermore, the above results also point toward the existence of bidirectional signaling during an APC:T cell interaction initiated from the B7 family of costimulatory molecules and their T cell counter-receptors, which could well regulate the subsequently generated T cell responses. That signal transduction from the cytoplasmic domains of CD80 indeed takes place was confirmed, wherein stimulation of CD80 induced tyrosine phosphorylation of distinct proteins. In fact, a database search identified potential protein kinase C and casein kinase II phosphorylation and binding sites on the cytoplasmic domains of CD80 [42 ]. It would be worthwhile to characterize the phosphorylation and activation status of these and other proteins, especially in the context of CD8a– and CD8a+ DC subsets, which may provide vital clues toward identifying signaling intermediates in APCs during their cognate interactions with T cells.


   ACKNOWLEDGEMENTS
 
Defense Research and Development Organization Grant No. DALS/48222/LSRB/22/ID/RD/-81 to K. N. and Department of Biotechnology Grant No. BT/PR/2423/Med/13/087/2001 to P. S. supported this work.

Received October 14, 2003; revised December 3, 2003; accepted January 6, 2004.


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 DISCUSSION
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