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Immunology; |
Department of Histology and Medical Embriology, Istituto Pasteur-Fondazione Cenci Bolognetti, "Sapienza" University of Rome, 00161 Rome, Italy
ABSTRACT
The testis is regarded as an immunologically privileged site because it tolerates either autoantigenic germ cells or allografts. Because the blood testis barrier represents an incomplete immunological barrier, we have explored whether Sertoli cells, the somatic cells of the seminiferous epithelium, might play an active role in immune evasion. We report data indicating that B7-H1(officially known as CD274)-mediated co-inhibition, an immunomodulatory mechanism based on cell-cell interaction, can be activated in Sertoli cell-lymphocyte cocultures. We have found that, in response to interferon gamma (IFNG), mouse Sertoli cells strongly up-regulate the negative co-stimulatory ligand B7-H1 but remain devoid of positive co-stimulatory molecules. Blockade of B7-H1 on the Sertoli cell surface resulted in enhanced proliferation of CD8+ T cells cocultured with Sertoli cells. Moreover, IFNG-stimulated Sertoli cells were found to express, concurrent with B7-H1, MHC class II. Therefore, we have hypothesized that Sertoli cells could function as nonprofessional tolerogenic antigen-presenting cells by inducing enrichment in regulatory T cells (Tregs) in a mixed T lymphocyte population. Interestingly, we found that coculturing T cells with Sertoli cells can indeed induce an increase in CD4+CD25+(officially known as IL2RA)FOXP3+ Tregs and a decrease in CD4+CD25– T cells, suggesting Sertoli cell-mediated Treg conversion; this process was found to be B7-H1-independent. Altogether these data show that Sertoli cells are potentially capable of down-regulating the local immune response, on one hand by directly inhibiting CD8+ T cell proliferation through B7-H1 and, on the other hand, by inducing an increase in Tregs that might suppress other bystander T cells.
immunology, inflammation, reproductive immunology, sertoli cells, spermatogenesis, T cells, testis, tolerance/suppression/anergy
The "immunological privilege" of the testis arises from the need to prevent immune responses against meiotic and haploid germ cells expressing antigens that are considered as "non-self" because they first appear at the time of puberty, long after the establishment of self-tolerance. Yet, these new autoantigens are tolerated by the testis despite the fact that their immunogenicity is preserved; indeed, they induce strong autoimmune reactions when injected elsewhere in the body [1–3], and this orchitis can be transferred to nonimmunized recipients via activated lymphocytes [4]. The blood-testis barrier, formed by Sertoli cells in the seminiferous epithelium, does not entirely segregate differentiating germ cells from the systemic circulation [5], and it has been demonstrated to be incomplete in some areas (for review see [6]). Furthermore, Head and Billingham [7] reported that organ grafts under the testicular capsule of several species of rodents survive for considerable periods without evidence of rejection by the immune system and implicate the local environment of the testis in this immune privilege. These data suggest that immune protection is conferred by the testis itself and represents the evolutionary response to the absolute need to protect reproductive capability.
In spite of its immunologically privileged status, the testis is not isolated from the immune system, and recent reports confirmed that CD8+ memory T cells survey it effectively, though their response is hampered by immunosuppressive mechanisms present on site [8]. These observations suggest that testicular immune privilege may be a localized phenomenon affecting T cell activation and maturation events and appears to be due to a continuous process of down-regulation. Multiple immunosuppressive mechanisms reported so far include persistence of physiological self antigen [9], inadequate or inappropriate signals provided by interstitial antigen-presenting cells (APCs) [10], local production of immunosuppressive molecules [11, 12], and cytokines such as transforming growth factor beta and activin [13–15].
It has recently been demonstrated that testicular allografts are consistently rejected, though the rejection tempo is significantly slower, and that more Ag-specific CD4+CD25+ (officially known as IL2RA) regulatory T cells (Tregs) are induced than observed for grafts placed at a conventional site (under the renal capsule). Moreover, blockade of the co-stimulatory pathway CD40/CD40L (officially known as CD40LG) induced tolerance of islet allografts transplanted in the testis, but not under the kidney capsule [16]. However, the role locally played by specific cell types in contact-based interactions has not been elucidated; moreover, the involvement of negative co-stimulatory molecules in maintaining testicular immune homeostasis is still to be investigated.
Negative co-stimulation can be provided by APCs through a variety of surface molecules belonging to the B7 family that bind to receptors of the CD28 family exposed on T cells. Among negative regulatory receptors, a newly described member is referred to as programmed death-1 (PD-1, officially known as PDCD1) [17]. Two ligands for PD-1 are known: B7-H1 (also called PD-L1 and officially known as CD274) is widely expressed on nonlymphoid (endothelial and muscle) cells as well [18], whereas B7-DC (also called PD-L2 and officially known as PDCD1LG2) expression is restricted to macrophages and DCs [19]. The broad expression of B7-H1 within nonlymphoid tissues suggests that this molecule may serve to regulate self-reactive T or B cell responses in peripheral tissues and/or may serve to regulate inflammatory responses at these sites. B7-H1 binds to PD-1 and inhibits the proliferative response of PD-1+/+ but not PD-1–/– splenic activated T cells, indicating that PD-1/B7-H1 engagement results in negative signaling [20]. At the maternal-fetal interface, a site in which, similarly to the testis, tolerance to unique alloantigens is greatly required, B7-H1 has been reported to play a crucial role in controlling anti-fetal T cells because its absence in the mouse results in a dramatic loss of allogenic, but not syngenic, fetuses [21, 22].
In addition to the delivery of inhibitory cosignals that induce T cell anergy, B7-H1 has also been reported to be critical in the activation and expansion of newborn CD4+CD25+FOXP3+ Tregs in allo-immune settings [23]. CD4+CD25+ Tregs play a key role in the maintenance of immunologic tolerance to both self and foreign antigens by suppressing aggressive T cell responses, preventing autoimmunity and allograft rejection in many animal models [24–29]. Tregs contribute to restore homeostasis and self-tolerance in a number of ways, but there is still some uncertainty about whether their suppressive function is mediated by secreted cytokines or by cell-to-cell contact. Tregs can either occur as functionally mature FOXP3-expressing lymphocytes that derive from the thymus and represent a small fraction (5%–10%) of CD4+ T cells, or can possibly be generated in the periphery under certain conditions [30]. Peripherally generated suppressor cells are indistinguishable in cell-surface phenotype, lifespan, and functional aspects from intrathymically generated FOXP3+CD4+CD25+ cells and have been demonstrated to be capable of mediating tolerance in various in vivo readout systems [31].
In this work, therefore, we have investigated whether Sertoli cells can inhibit T lymphocyte response through B7-H1 directly by delivering inhibitory cosignals and/or indirectly by inducing the expansion of Tregs capable of suppressing other T cell responses. Here we report that, in response to interferon gamma (IFNG), Sertoli cells can both transmit an inhibitory cosignal to CD8+ T cells through B7-H1 and induce a B7-H1-independent increase in CD4+CD25+FOXP3+ Tregs. Our data on negative co-stimulation mediated by B7-H1 represent the first evidence of a cell-to-cell immunomodulatory interaction between Sertoli cells and T lymphocytes, which likely contributes to the down-regulation of local immune responses and to testicular immune privilege.
The animals used were 2-wk-old and 6- to 8-wk-old C57BL/6 male mice (Charles River Laboratories, Inc., Calco, Italy). Animals were kept in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were killed by CO2 asphyxia.
DNase, fetal calf serum (FCS), and IFNG (supplied by units) were purchased from Boehringer Mannheim (Mannheim, Germany). Collagenase and trypsin was obtained from Roche Molecular Biochemicals (Mannheim, Germany). ConA was obtained from Sigma-Aldrich (St. Louis, MO).
The following primary antibodies were used: anti-mouse B7-H1, B7-H3, B7-H4 (clone 9 and 188), B7-DC, MHC II, B7-1, B7-2, CD40, CD4, CD8, CD25, FOXP3, IgG2a isotype, IgG1b isotype, hamster IgG, anti-rat PE (phycoerythrin)-conjugated (all from eBioscience, San Diego, CA), and donkey anti-rat FITC-conjugated (Molecular Probes Inc., Eugene, OR). Propidium Iodide was purchased from BD Pharmingen, Heidelberg, Germany. Anti-mouse B7-H1 antibody used for Western blotting analysis was purchased from R&D systems (Minneapolis, MN).
Decapsulated testes from 2-wk-old mice were briefly digested in MEM (minimum essential medium; Invitrogen Life Technologies, Carlsbad, CA) containing 0.1% collagenase plus 10 µg/ml DNase to remove interstitial tissue. To avoid interstitial contamination, the tubules were microdissected in MEM under a stereomicroscope before further processing. Fragments of seminiferous tubules were washed with Hanks Balanced Salt Solution (HBSS; Sigma-Aldrich, St. Louis, MO) twice, then plated in plastic dishes in a veil of MEM. Segments of seminiferous tubules were cultured either in the absence or in the presence of IFNG for 24 h at 32°C in serum-free conditions in a humidified atmosphere containing 5% CO2, and then processed for Western blot analysis.
Sertoli cells were prepared from C57BL/6 mice as previously described [11]. Briefly, testes from 2-wk-old animals were sequentially digested for 20 min, first with HBSS containing 0.25% trypsin plus 10 µg/ml DNase and then with HBSS supplemented with 0.1% collagenase plus 10 µg/ml DNase to remove interstitial tissue and peritubular cells. Fragments of seminiferous epithelium, mainly composed of Sertoli cells, were cultured at 32°C in 95% air and 5% CO2 in serum-free MEM. Sertoli cell cultures were routinely checked for possible contamination by macrophages and peritubular smooth muscle cells by means of indirect immunofluorescence with anti-macrophage mAb (Mac-1 antigen CD11/b; Roche, Basel Switzerland) and by histochemical detection of alkaline phosphatase activity [32]. To remove residual germ cells present in the culture and obtain pure Sertoli cells, after 3 days the cultures were subjected to hypotonic shock by incubation at room temperature with 20 mM Tris-HCl buffer (pH 7.4, Sigma-Aldrich) for 2 min [33]. In all experiments, the monolayers used were composed of at least 95% pure Sertoli cells. On the fourth day of culture, Sertoli cell monolayers were treated with IFNG. At the times indicated, the cells were analyzed for surface-marker expression by flow cytometric analysis or lysed for Northern blotting experiments.
In the experiments of coculture with T lymphocytes, it was preferred not to expose Sertoli cells to hypotonic shock, and germ cells adhering to the monolayer were removed by three consecutive gentle washings with 1 ml of MEM per 12-well plates. Sertoli cell cultures were treated with IFNG and, after 48 h, extensivly washed; at this time no germ cells were visible in the culture. Sertoli cells were then cocultured with freshly isolated total T cells, in the presence of ConA, with or without the B7-H1 blocking antibody.
Tissues were lysed with Cell Lysis Buffer (New England Biolabs, Beverly, MA) containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3V04, 1 µg/ml leupeptin, and 1 mM PMSF (Sigma-Aldrich). Protein concentration was determined by the micro-BCA method (Pierce; Tattenhall, Cheshire, UK). Equal amounts of proteins (15 µg) were subjected to SDS-PAGE and then transferred onto a nitrocellulose membrane (Hybond C; Amersham Pharmacia Biotech, Buckinghamshire, UK). The nitrocellulose was saturated with nonfat dry milk diluted at 5% in TBST (Tris-buffered saline containing 0.1% Tween-20).
Anti-mouse B7-H1 polyclonal antibody or anti 
-tubulin mouse IgG (Sigma Chemical Co., St. Louis, MO) were diluted in TBST/BSA (Sigma-Aldrich) and incubated overnight. Respectively, donkey anti-goat IgG (Santa Cruz) and goat anti-mouse IgG (Zymed, San Francisco, CA) conjugated to horseradish peroxidase were used as secondary antibodies. Protein expression was detected by chemioluminescence measurement (ECL; Amersham Pharmacia Biotech).
Total RNA was extracted from IFNG treated or untreated murine Sertoli cells using TRIZOL Reagent (Life Technologies, Grand Island, NY). Samples were processed following the manufacturer's protocol. RNA was subjected to electrophoresis through a 1.2% agarose gel containing formaldehyde and then transferred onto a neutral nylon transfer membrane (Schleicher & Schuell, Keene, NH). Blots were preincubated in hybridization solution containing 50% formamide (Sigma), 10% dextran sulfate, 1% SDS, 1.2 M NaCl (Sigma-Aldrich), and sonicated salmon sperm (Stratagene, La Jolla, CA) at 42°C for 4 h. Full-length B7-H1 cDNA was amplified from spleen and cloned as described [34]. Hybridization was performed overnight in hybridization solution containing 1 x 106 cpm/ml of a [–32P]-labeled mouse DNA probe, and the blots were exposed to Kodak BIOMAX-MS film (Eastman Kodak, Rochester, NY). The integrity and equal loading of RNA were ascertained by ethidium bromide staining of the gel before transfer.
T Lymphocyte Preparation and CFSE Staining
Single-cell suspension of splenocytes from C57BL/6 adult mice was prepared, and erythrocytes were lysed with ACK (Sigma-Aldrich). T cells were negatively purified by using pan T cell isolation kit (Miltenyi Biotec, Inc., Auburn, CA) according to the manufacturer's instructions, resulting in >95% purity as assessed by flow cytometry. Briefly, a single-cell suspension from spleens of adult mice was prepared and resuspended in 40 µl of buffer per 107 total cells. Ten microliters of biotin-antibody cocktail per 107 total cells was added and, after mixing well, cells were incubated for 10 min at 4–8°C. After this incubation, 30 µl of buffer and 20 µl of anti-biotin microbeads per 107 total cells were added, and cells were incubated for an additional 15 min at 4–8°C. Cells were washed with buffer by adding 10–20x labeling volume, resuspended in 500 µl of buffer per 108 total cells (for fewer cells, 500 µl was used) and subjected to magnetic separation. The negative fraction collected represented the enriched T cell fraction.
To assess lymphocyte proliferation, we used carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes Inc.) labeling [35]. Before coculturing with Sertoli cells, freshly purified T cells were labeled with 1.5 µM CFSE for 10 min at 37°C and washed three times with complete medium. The intensity of CFSE was determined via flow cytometric analysis.
Freshly isolated total T cells (CD4+ and CD8+) were added to Sertoli cell cultures at the indicated effector:target (E:T) ratios. Cell were cocultured at 37°C for 5 days in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B, 10 mM HEPES, 50 µM 2-mercaptoethanol (all from Life Technologies), and 15 µg/ml gentamicin (BioWhittaker, Walkersville, MD) in the presence of ConA (1.5 µg/ml). Where indicated, anti-B7-H1 blocking mAb or respective isotype antibody (eBioscience) was added to the cocultures to delineate the functional relevance of B7-H1 for Sertoli cell-T cell interactions. Lymphocyte proliferation rate was evaluated by calculating the percentage of undivided cells at each time point. The percent inhibition of CD4+ and CD8+ lymphocyte proliferation specifically due to B7-H1 was calculated as 100 x [(% undivided cells with control IgG) – (% undivided cells with anti B7-H1 blocking Ab)] / (% undivided cells with control IgG).
For analysis of surface molecules, Sertoli cells were detached with trypsin and washed with cold PBS containing 1% BSA. Cells were incubated with either fluorochrome-conjugated primary or matched isotype control monoclonal antibodies for 30 min on ice and then washed twice. When unconjugated primary antibodies were used, cells were incubated for 30 min with anti-rat PE-conjugated secondary antibody and rinsed again.
At indicated times, T cells were harvested and stained with fluorochrome-conjugated antibodies, including anti-CD4, anti-CD8, anti-CD25, and anti-FOXP3. Intracellular FOXP3 expression was measured by using an intracellular FOXP3 detection kit (eBioscience) according to the manufacturer's instructions. Cells were gated using forward vs. side scatter and PI to exclude dead cells and debris. Cells were analysed with a Coulter Epics XL flow cytometer (Beckman Coulter, CA). Histograms of stained cells were analyzed by calculating the specific fluorescence index (geometric mean of specific antibody fluorescence divided by geometric mean of isotype control antibody fluorescence).
Frozen sections (5–6 µm) of testis were cut, air dried, briefly fixed in cold acetone, rinsed, and quenched with 2% BSA. The sections were immunostained with primary antibodies or corresponding nonimmune IgG overnight at 4°C. Antibody binding was visualized by donkey anti-rat FITC-conjugated secondary antibody. Nuclei were stained with TO-PRO 3 (Molecular Probes). Specimens were observed with a Leica laser-scanning microscope TCS SP2, and images were acquired with Leica Confocal Software (Leica, Germany).
A Student t-test for paired samples was used to assess differences among groups. P < 0.05 was considered significant.
B7-H1 Is Strongly Up-Regulated in Mouse Seminiferous Tubules Treated with IFNG
In order to study the expression of B7-H1 protein, we isolated mouse seminiferous tubules and cultured them in basal medium. By Western blot analysis, no constitutive expression of B7-H1 was detected either in total testis lysates or in control seminiferous tubules (Fig.1). Since IFNG has been shown to up-regulate B7-H1 expression in many cell types, we tested in Western blot whether treatment with increasing doses of IFNG for 24 h could affect B7-H1 expression in seminiferous tubules. As shown in Figure 1, B7-H1 was strongly induced by IFNG treatment in a dose-dependent manner.
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IFNG Induces Expression of B7-H1, but Not of Other B7 Family Inhibitory Members, in Mouse Sertoli Cells
Because Sertoli cells are the somatic cells that regulate most seminiferous tubule activities and establish particularly close contact with germ cells, they represent the primary candidate in autoantigenic testicular immune protection. Moreover, given that Sertoli cells display APC characteristics, such as phagocytic activity and interleukin production, we tested the hypothesis that they could respond to IFNG treatment and contribute to the observed increment of B7-H1 expression in seminiferous tubules. To this aim, we treated primary cultures (composed of at least 95% pure Sertoli cells in all the experiments) with increasing doses of IFNG for 24 h and analyzed B7-H1 expression by flow cytometry. As shown in Figure 2, B7-H1 protein is induced by IFNG in a dose-dependent manner. To determine the kinetics of B7-H1 induction, Sertoli cells were cultured with 500 U/ml IFNG for increasing times up to 72 h. This treatment induced B7-H1 expression and time-dependent constant increase (Fig. 3). In order to verify whether IFNG stimulation of Sertoli cells affects B7-H1 mRNA levels, we analyzed total RNA from purified Sertoli cells untreated or treated with IFNG (500 U/ml) for 2–24 h by Northern blotting. B7-H1 mRNA increase was first apparent after 4 h exposure to IFNG and remained similarly high when the treatment was continued for up to 24 h (Fig. 4).
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We have also evaluated whether Sertoli cells could express other B7 inhibitory members such as B7-DC, B7-H3 (officially known as CD276), and B7-H4 (officially known as VTCN1); however, we could not detect any constitutive or IFNG-stimulated expression of negative co-stimulatory molecules other than B7-H1 (Supplemental Fig. 1, available online at www.biolreprod.org).
IFNG Induces MHC Class II on Mouse Sertoli Cells
Although Sertoli cells are known to express MHC class I and, therefore, provide CD8+ T cell receptor stimulation [36], as yet MHC class II expression has not been reported for these cells. Because TCR (T cell receptor) engagement is a requirement for the transmission of specific inhibitory signals through B7-H1 [23, 37], after having established that Sertoli cells could express the co-inhibitory surface molecule B7-H1, we investigated their capability to present antigens to CD4+ T cells. In flow cytometry experiments, we found that MHC class II was absent on untreated Sertoli cell surfaces but, interestingly, was strongly up-regulated in the presence of IFNG concurrent with B7-H1 (Fig. 5).
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In addition, we verified Sertoli cell capability provides positive co-stimulation through CD28 family members. The expression of CD80 (B7-1), CD86 (B7-2), and CD40 was assayed by flow cytometric analysis. No positive co-stimulatory molecules were detectable on Sertoli cells either in control or in IFNG-treated cultures (Supplemental Fig. 2, available online at www.biolreprod.org).
B7-H1 Contributes to the Inhibition of CD8+ T Lymphocyte Proliferation
To evaluate the functional role of B7-H1 expressed on Sertoli cells in establishing immune tolerance, we tested the effect of B7-H1 blockade on T cell proliferation. For the co-culture experiments, Sertoli cell preparations were preincubated for 48 h with IFNG to induce B7-H1 and MHC class II expression. Subsequently, freshly isolated CFSE-labeled syngenic total T cells were added at various E:T ratios in the presence of neutralizing anti-B7-H1 mAb or isotype antibody. ConA, a strong proliferative stimulus for lymphocytes, was added to the coculture in order to evaluate the inhibitory potential of B7-H1 on T cell activation. As a positive control, total T cells were simultaneously cocultured with syngenic splenocytes. Of note, CD4+ T cells cocultured with splenocytes proliferated as expected (Supplemental Fig. 3, available online at www.biolreprod.org), but failed to undergo division when cocultured with Sertoli cells at any E:T ratio, despite the presence of ConA, with and without anti-B7-H1 blocking antibody (Fig. 6A). On the contrary, CD8+ T cells in cocultures with Sertoli cells were observed to proliferate (Fig. 6B), although at lower rates than in coculture with splenocytes (Supplemental Fig. 3). Moreover, when cocultured on Sertoli cells for 5 days, the percentage of CD8+ undivided T cells decreased as the B7-H1 pathway was blocked (t-test for paired samples, t = 12.7 and 10.1 for 1:1 and 1:2 E:T ratios respectively, P < 0.01; Fig. 6B). Figure 6C summarizes the data obtained in functional assays at two E:T ratios by showing the percentage of specific CD4+ and CD8+ T lymphocyte inhibition of proliferation due to B7-H1, calculated as described in Materials and Methods. As for the steady CD4+ T lymphocyte pool, the B7-H1-mediated contribution to the complete inhibition of proliferation observed is negligible.
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We also checked whether T cells produced interleukin 2 (IK2) during the cocoltures and verified that the cytokine amount was below the limits of detection by using mouse IL2 Simplex FlowCytomix (BioLegend, San Diego, CA; data not shown). We assessed the apoptosis rate by means of propidium iodide staining of T lymphocytes in different experimental conditions (± anti-B7-H1 at 3 and 5 days of culture) and did not observe any variations being about 15% of apoptotic cells.
These data indicate that Sertoli cells can actively transmit through B7-H1 a negative signal that contributes to inhibition of CD8+ T cell proliferation.
Induction of CD4+CD25+FOXP3+ Tregs Mediated by Sertoli Cells Is B7-H1 Independent
CD25 and FOXP3, the former being expressed on the cell surface and the latter being intracellular, are highly specific and reliable markers to identify both natural and induced Tregs from other T cells. Because it has been reported that in an allogenic setting Tregs can be induced through a B7-H1-mediated pathway [23], we examined whether an increase in CD4+CD25+ Tregs could be obtained after coculturing T cells with IFNG-stimulated Sertoli cells. As surface expression of CD25 in CD4+ T cells is characteristic of both regulatory and activated responder cells, we assessed increased expression of intracellular FOXP3 in these cells by flow cytometry. The number of CD4+CD25+FOXP3+ T cells significantly increased compared to the starting population of resting CD4+CD25+FOXP3+ T cells (Fig. 7, A and B). Because CD4+ T cells are a steady population under these conditions and all CD25+ T cells are FOXP3 positive, these data suggest that the interaction of CD4+ T cells with Sertoli cells might result in the conversion of CD4+CD25– precursors into newborn CD4+CD25+FOXP3+ Tregs within the CD4+ T cells pool (Fig. 7B). The role of B7-H1 in such induction was also investigated in blocking experiments that proved that B7-H1 is not involved; in fact, similar increases in CD4+CD25+FOXP3+ T cells were observed in the presence or absence of the blocking antibody (28.3% vs. 31.8%, respectively; P > 0.05 was considered not significant; Fig. 7, B and C).
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B7-H1 Is Constitutively Expressed by Peritubular Cells
Although in Western blot no constitutive B7-H1 expression was detected in seminiferous tubule homogenates (Fig. 1), we investigated the expression of B7-H1 by immunofluorescence examination of frozen testis sections to verify the possible presence of B7-H1 on specific subsets of cells present in low percentage. Of note, we observed that constitutive expression of B7-H1 is restricted to peritubular cells, a contractile monolayer which surrounds seminiferous tubules (Fig. 8).
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Co-stimulation has been discovered as a crucial mechanism in tuning the balance between positive and negative signaling, which ultimately directs T cells towards tolerance or rejection both to self and foreign antigens. We wondered whether negative co-stimulation was involved in establishing immune tolerance in the testis. Recently, it has been demonstrated that constitutive expression of B7-H1 on corneal endothelial and stromal cells plays a critical role in the maintenance of the immune-privileged status of corneal allografts [38]. In the work presented here, we examined the constitutive and induced expression of B7-H1 in seminiferous tubules and raised the question whether B7-H1 could attenuate T cell immune response within the testis, contributing to its privileged status. We found that in Sertoli cells, the somatic cells that enclose autoantigenic germ cells, IFNG treatment induces B7-H1 mRNA and up-regulates protein expression in a dose- and time-dependent manner. Moreover, we observed that Sertoli cells do not express, either constitutively or upon IFNG stimulation, other negative co-stimulatory molecules such as B7-DC, B7-H3, and B7-H4. Interestingly, Sertoli cells also do not express any of the B7 family positive co-stimulatory molecules. Because, following stimulation with IFNG, B7-H1 is the only B7 family member upregulated in Sertoli cell cultures, we have considered it as a receptor with an important role in Sertoli cell-mediated tolerance compared with other B7 members. In accordance with our findings regarding IFNG-mediated induction of B7-H1 on Sertoli cells, it has already been described that IFNG influences B7-H1 expression predominantly on non-lymphoid tissues and up-regulates it in many cell types such as endothelial cells, keratinocytes, and myoblasts [39–42]. Although IFNG effects can all be considered proinflammatory, there is evidence that IFNG treatment of endothelial cells down-regulates CD8+ T cell responses in vitro [43] and potentially mediates peripheral tolerance by regulating T cell function at peripheral sites. Several studies have suggested an immunosuppressive role of IFNG in transplantation [44] and in autoimmune diseases such as experimental autoimmune encephalomyelitis [45] and in muscle biopsies from patients with inflammatory myopathies, where B7-H1 increase resulted in a decreased production of cytokines by T cells [41]. Thus, the beneficial effect of IFNG reported in some autoimmune settings might partly result from the induction of B7-H1 expression at the sites of inflammation and subsequent down-regulation of immune responses by PD-1 engagement [46].
In our experiments, coculturing T cells with IFNG-stimulated Sertoli cells failed to efficiently stimulate proliferative responses to autoantigenic germ cells present in the culture. However, with the addition of ConA to the culture, CD8+, but not CD4+, T cells underwent various cycles of division, and blockade of B7-H1 with a specific mAb resulted in increased CD8+ T cell proliferation. These findings are consistent with data showing that B7-H1 engagement initiates cell cycle arrest [19] and confirm previous reports that CD8+ T cells are more susceptible to B7-H1 inhibitory pathway than CD4+ T cells [47]. Because, during coculturing with Sertoli cells, CD4+ T cells remained steady and CD8+ T cells proliferation was inhibited, our results provide new evidence for the immunosuppressive potential of Sertoli cells. In particular, we report that these cells exert inhibition on CD4+ and CD8+ subsets of lymphocytes through two different pathways, one affecting mainly CD4+ T cells by means of B7-H1-independent mechanisms and the other, B7-H1-mediated, actively contributing to CD8+ T cell proliferative hyporesponsiveness.
In addition, we found that in response to IFNG treatment concurrent with B7-H1, the surface expression of MHC class II, an essential prerequisite for the interaction with CD4+ T lymphocytes, is strongly up-regulated on Sertoli cells. On this basis, we have hypothesized that, by providing suboptimal sustained antigenic stimulation concurrently to negative co-stimulation, Sertoli cells might drive immune homeostasis towards tolerance, either directly by affecting T cell activation or indirectly supporting the expansion of Tregs (possibly by skewing CD4+ T cells towards a regulatory phenotype). Tregs are produced by the normal thymus as a functionally mature and distinct T cell subpopulation [27, 48]. However, it is also becoming clear that Tregs can be generated in the periphery, perhaps under particular conditions of antigen exposure in response to newly encountered antigens, not only antigens present throughout development [49]. Interestingly, we observed that coculturing T cells with Sertoli cells treated with IFNG, necessary to induce expression of both MHC class II and B7-H1, results in an increase in CD4+CD25+FOXP3+ Treg cells. As reported in other settings [23, 50], the generation of CD4+ Tregs occurrred upon homeostatic proliferation, which suggests the possibility of a conversion of CD25– cells into CD25+ Tregs, in agreement with the concept that proliferation has an antagonistic effect on the conversion rate of naïve T cells into suppressor cells [51]. In our syngeneic model, we did not observe involvement of B7-H1 in Sertoli cell-mediated generation of Tregs, which is regarded as essential for the induction of Treg in allo-immune settings [23, 52, 53].
In testis cryosections, constitutive expression of B7-H1 was confined to peritubular smooth muscle cells (myoid cells), a contractile monolayer which surrounds seminiferous epithelium. We suggest that peritubular cells (constitutively expressing B7-H1) and Sertoli cells (expressing IFNG-induced B7-H1), which are both involved in barrier functions [54], might provide serial lines of defense against immune attack; the former might act as a first barrier and the latter as part of an emergency negative feedback mechanism in T cell-mediated inflammatory events. Notably, cells morphologically resembling mononuclear leucocytes ("light cells") have been reported in the rat as regular peritubular components that are inserted between myoid cells and are capable of proliferating; intriguingly, these cells appear to be most frequent at stages VII-XI, which also show a maximal phagocytic activity of Sertoli cells [55]. The particular proximity of these cells to the seminiferous epithelium, together with the unusually loose arrangement of adjacent myoid cells, suggests that encounters between Sertoli cells and cells of the immune system might occur at discrete boundary sites.
In conclusion, IFNG locally produced by macrophages, Leydig cells, natural killer, and T cells during inflammation [56, 57] determines the activation of two suppressive pathways mediated by Sertoli cells: IFNG, on one hand, induces the expression of B7-H1, which might effectively contribute to inhibiting of immune responses by negatively interfering with CD8+ T cell proliferation and, on the other hand, induces the expression of MHC class II, which might mediate the increase of Tregs capable of suppressing other bystander T cells.
Based on what is known so far, we strongly believe that the involvement of several immunomodulatory mechanisms is required for the establishment and maintainance of tolerance towards germ cells within the testis in vivo, including passive, almost complete, segregation by barriers, regulation by paracrine immunosuppressive mediators, and active cell-cell interaction-mediated pathways. Among the latter, we have indicated B7-H1-mediated negative co-stimulation as a relevant mechanism provided by Sertoli cells, which function as tolerogenic APCs.
ACKNOWLEDGMENTS
We thank Fioretta Palombi for helpful discussion and critical reading.
Correspondence: 1Antonio Filippini, Departmemt of Histology and Medical Embryology, "Sapienza" University of Rome, Via A. Scarpa 14, 00161 Roma, Italy. FAX: 39 06 446 2854; e-mail: antonio.filippini{at}uniroma1.it
Received: 25 June 2007.
First decision: 19 July 2007.
Accepted: 19 October 2007.
REFERENCES
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  L. R. Guerin, J. R. Prins, and S. A. Robertson Regulatory T-cells and immune tolerance in pregnancy: a new target for infertility treatment? Hum. Reprod. Update, March 11, 2009; (2009) dmp004v1. [Abstract] [Full Text] [PDF]
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 D. Starace, R. Galli, A. Paone, P. D. Cesaris, A. Filippini, E. Ziparo, and A. Riccioli Toll-Like Receptor 3 Activation Induces Antiviral Immune Responses in Mouse Sertoli Cells Biol Reprod, October 1, 2008; 79(4): 766 - 775. [Abstract] [Full Text] [PDF]
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