T cell responses are compromised in the elderly. The B7-CD28 family receptors are critical in the regulation of immune responses. We evaluated whether the B7-family and CD28-family receptors were differentially expressed in dendritic cells, macrophages, and CD4+ and CD8+ T cells from young and old mice, which could contribute to the immune dysfunction in the old. Although most of the receptors were equally expressed in all cells, >85% of the old naive CD8+ T cells expressed B7-H1 compared with 25% in the young. Considering that B7-H1 negatively regulates immune responses, we hypothesized that expression of B7-H1 would downregulate the function of old CD8+ T cells. Old CD8+ T cells showed reduced ability to proliferate, but blockade of B7-H1 restored the proliferative capacity of old CD8+ T cells to a level similar to young CD8+ T cells. In vivo blockade of B7-H1 restored antitumor responses against the B7-H1− BM-185–enhanced GFP tumor, such that old animals responded with the same efficiency as young mice. Our data also indicate that old CD8+ T cells express lower levels of TCR compared with young CD8+ T cells. However, following antigenic stimulation in the presence of B7-H1 blockade, the levels of TCR expression were restored in old CD8+ T cells, which correlated with stronger T cell activation. These studies demonstrated that expression of B7-H1 in old CD8+ T cells impairs the proper activation of these cells and that blockade of B7-H1 could be critical to optimally stimulate a CD8 T cell response in the old.
It is well established that T cell responses are compromised in old mice, rats, and humans (1–3). Many studies have demonstrated that the immune function in the old is characterized by a decline in humoral and cellular responses (4, 5). Among the alterations that diminish the immune function in aged individuals are thymic involution, resulting in the decreased number of T and B cells (6–8); modifications in the production and secretion of cytokines (9, 10); reduced cytotoxic activity of CD8+ T cells (11, 12); the qualitative deficiency of B lymphocytes with a reduced response to exogenous Ags (13); and possibly deficiencies in APCs (14, 15). The accumulation of these alterations leads to immune dysfunction, such that individuals are prone to the consequences of infectious disease, autoimmunity, and cancer (16). However, although these effects cause immune responses to be diminished in the old, the underlying mechanisms dictating the dysfunction at the cellular, molecular, or physiologic level are unclear. As such, it is imperative to identify the alterations or defects of the aged immune system to generate rational, therapeutic strategies toward restoring or optimizing immune responses in the old.
The B7 family consists of activating and inhibitory costimulatory molecules that positively and negatively regulate immune responses (17–19). The B7 family and other costimulatory molecules used by APCs directly influence and/or fine-tune T cell responses (18). The B7 family consists of seven members: B7.1 (also known as CD80), B7.2 (also known as CD86), B7-DC (also known as PD-L2 or CD273), B7-H1 (also known as PD-L1 or CD274), B7-H2 (also known as ICOSL), B7-H3 (also known as CD276), and B7-H4 (also known as B7S1 or B7x) (19). B7.1 and B7.2 provide T cell signaling through CD28 and CTLA4 receptors. The interaction of B7.1/B7.2 and CD28 provides a positive signal stimulating the immune response (20), whereas the interaction of B7.1/B7.2 with CTLA4 is inhibitory and subdues the immune response (21). B7-H1 binds to the programmed cell death 1 (PD-1; also known as CD279) receptor (22). Engagement of PD-1 with B7-H1 suppresses T cell responses (23). Furthermore, PD-1 null mice develop strain-specific autoimmune syndromes later in life (24), and B7-H1 knockout mice showed an increase susceptibility to experimental autoimmune diseases, such as hepatitis and encephalomyelitis (25). B7-H1 is highly upregulated in tumors, impairing T cell antitumor responses (26). These results indicate that the interactions of B7-H1–PD-1 have a suppressive function, inhibiting T cell effector responses (27). Expression of B7-H1 on T cells can transmit signals regulating the function of these cells (28), indicating that B7-H1 can act as a ligand and receptor to execute immunoregulatory functions. The interaction of B7-H2–ICOS is critical for the generation of Th2-type responses and regulating humoral immunity (29). B7-H3 costimulation increases the proliferation of CD4 and CD8 T cells and enhances IFN-γ production and cytotoxic activity (30). B7-H4 remains an orphan ligand; its functions are exclusively inhibitory (31).
There is no information about the expression of B7-family and CD28-family receptors in the old. Considering that the interactions of B7-family receptors with their respective ligands could fine-tune immune responses, we sought to evaluate whether differences in B7-family and CD28-family receptor expression between young and old macrophages, dendritic cells (DCs), and CD4+ and CD8+ T cells could contribute to the immune dysfunction in the aged. Although we observed that greater percentages of young macrophages, DCs, and CD4+ and CD8+ T cells express B7.1, B7.2, and ICOS, the most surprising result was that greater percentages of old CD8+ T cells express B7-H1 compared with young CD8+ T cells. Based on the regulatory function of these molecules, it can be hypothesized that the differential expression of the B7Rs could modulate or interfere with the effector function of T cells and the regulation of immune responses. Because B7.1, B7.2, and ICOS positively regulate immune responses and B7-H1 negatively regulates immune responses, we were more interested in examining the expression of B7-H1 in old CD8+ T cells for three fundamental reasons: CD8+ T cells are critical cells for tumor rejection, viral clearance, and other immune responses; numerous old CD8+ T cells expressing B7-H1 could inhibit the activation of immune responses in the aged; and for therapeutic purposes, it is more accessible to block or downregulate the expression of a receptor than to increase its expression. We hypothesized that the expression of B7-H1 could impair the proper activation of old CD8+ T cells and that blockade of B7-H1–PD1 interaction could enhance or restore the old CD8+ T cell responses. Indeed, blockade of B7-H1 in old CD8+ T cells restored the proliferation of these cells, and in vivo treatment with anti–B7-H1 mAb restored effective immune responses in aged animals. Furthermore, treatment with anti–B7-H1 also restored the expression of TCR complexes, which were otherwise constitutively downregulated in old CD8+ T cells. The implications of B7-H1 expression in old CD8+ T cells and blockade of B7-H1–PD-1 interactions to manipulate the aged immune responses are discussed.
Materials and Methods
Mice and cell lines
Young (2–3 mo) and old (18–22 mo) BALB/c mice were purchased from the National Institute of Aging (Bethesda, MD) and housed under specific pathogen-free conditions. The tumor cell lines, BM-185 wild type (WT), BM-185 expressing enhanced GFP (EGFP) (BM-185-EGFP), and BM-185-EGFP-CD80 were kindly provided by Dr. D. Kohn, University of Southern California (Los Angeles, CA). Anti–B7-H1 (clone 10B5) was obtained from Dr. Haidong Dong, Mayo Clinic Rochester. All cell lines were maintained in complete RPMI medium (RPMI 1640) supplemented with 10% FCS, 2 mM glutamine, 5 × 10−5 M 2-ME, and 50 μg/ml gentamicin.
Macrophages were detected by staining with Abs CD11b-allophycocyanin and F4/80-PE; DCs were detected by staining with anti–CD11c-PE; and CD4+ and CD8+ T cells were detected by staining with anti–CD4-PE and anti–CD8-PE, respectively. The prevalence of B7 family and CD28 family receptors in macrophages, DCs, and CD4+ and CD8+ T cells was analyzed by staining the sample with anti-B7.1, anti-B7.2, anti–B7-H1, anti–B7-H2, anti–B7-H3, anti–B7-H4, anti–B7-DC, anti-ICOS, anti–PD-1, anti-CD28, and anti-CTLA4 Abs labeled with FITC (eBioscience, San Diego, CA).
Young and old mice were implanted with 105 BM-185-EGFP cells on day 0. On days 8–10, animals were injected i.p. with 1 mg/injection of BrdU; on day 11, animals were sacrificed for analysis. Spleen cells were stained with anti–CD8-PE, anti–B7-H1-allophycocyanin. BrdU staining was performed using a commercially available kit (Invitrogen, Carlsbad, CA), following the manufacturer’s protocol.
Division and proliferation assays
+ T cell suspensions (1 × 107 cells in 1 ml). After incubation for 10 min at 37°C, cells were washed immediately and cultured with the anti-CD3/anti-CD28 beads or anti-CD3/anti-CD28/PD-1–Ig beads in the presence or absence of soluble anti–B7-H1 mAb (10 μg/ml) for 72 h. Division of cells was measured by flow cytometry. For proliferation assays, enriched young and old CD8+ T cells were plated (2 × 105/well) in a 96-well flat-bottom plate coated with 3 μg/ml anti-CD3 Ab and 1 μg/ml anti-CD28 Ab in the presence or absence of anti–B7-H1 mAb (10 μg/ml). Proliferation was measured by [3H]thymidine incorporation.
In vivo tumor studies
To evaluate the effect of blocking B7-H1 in vivo, young and old animals were injected s.c. with 105 BM-185-EGFP cells on day 0. On days 5, 12, and 19, old mice were injected with anti–B7-H1 or isotype control Ig (100 μg/injection), and animals were examined twice per week for tumor development and survival. Survival analysis used the Breslow modification of the Kaplan–Meier test.
Generation of CTL cultures and cytotoxic activity
Young and old BALB/c mice were immunized with an s.c. injection of 105 BM-185-EGFP cells. On days 4 and 8, old mice were injected with anti–B7-H1 or isotype control Ig (100 μg/injection). Two weeks after priming the animals, spleens were removed, and spleen cells were restimulated in vitro with BM-185-EGFP-CD80 cells. After 5 d, CTLs were assayed for lytic activity. The BM-185 WT and BM-185-EGFP cells were incubated with 150 μCi [51Cr]sodium chromate for 1 h at 37°C. Cells were washed three times and resuspended in RPMI 1640 medium. For the cytotoxic assay, [51Cr]-labeled target cells (104) were incubated with varying concentrations of effector cells in a final volume of 200 μl in 96-well U-bottom microtiter plates. Supernatants were recovered after 5 h of incubation at 37°C, and the percentage of lysis was determined using the following formula: percentage of specific lysis = 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release).
Immunoprecipitation detected by flow cytometry
The following Abs were used: 145-2C11 (anti-CD3ε), H57-597 (anti-TCRβ), H146 (anti-CD3ζ), A19-3 (hamster Ig), 53-2.1 (anti-Thy1.2), and 6B10 (anti-CD3ζ). The method and protocol have been described in detail (32). Briefly, immunoprecipitating Abs were covalently coupled to polystyrene latex beads (Polysciences, Warrington, PA). CD8+ T cells were lysed at a concentration of 200 × 106/ml in buffer containing 50 mM Tris, 150 mM NaCl, protease inhibitors (P2714 and A8456; Sigma-Aldrich, St. Louis, MO), and 1% digitonin (ultra-pure grade; Calbiochem, San Diego, CA). TCR protein complexes were captured by incubating the immunoprecipitating beads overnight at 4°C with postnuclear lysates. Subsequently, beads were washed, probed with PE-conjugated probe Abs, and analyzed by flow cytometry.
The statistical significance of data was determined by the Student t test to evaluate the p value. The relationship between two parameters was tested using regression analysis, and p < 0.05 was considered significant.
Analysis of B7 family and CD28 family receptor expression in young and old immune cells
The expression of B7 family and CD28 family receptors in macrophages, DCs, and CD4+ and CD8+ T cells from naive young (2–3 mo) or old (18–20 mo) BALB/c mice was analyzed. The results (Fig. 1) indicate that higher percentages of young macrophages express B7.2 and ICOS compared with old macrophages; higher percentages of young DCs express ICOS compared with old DCs; higher percentages of young CD4+ T cells are positive for B7.1 and B7.2 compared with old CD4+ T cells; higher percentages of young CD8+ T cells express B7.2 compared with old CD8+ T cells; and higher percentages of old CD8+ T cells express B7-H1 compared with young CD8+ T cells (Supplemental Fig. 1). Because B7-H1 negatively regulates immune responses, we decided to evaluate whether the expression of B7-H1 downregulated or impaired the function of old CD8+ T cells.
Expression of B7-H1 and PD-1 on activated young and old naive and memory CD8+ T cells
We evaluated whether the expression of B7-H1 and PD-1 differed between naive (CD44loCD62Lhi) and memory (CD44hiCD62Llo) CD8+ T cells from young and old mice prestimulation or following activation with anti-CD3 plus anti-CD28 mAb. As shown in Fig. 2A, significantly higher percentages of old naive CD8+ T cells express B7-H1 compared with young naive CD8+ T cells (Supplemental Fig. 2). The expression of B7-H1 remained high during and after the activation process in old naive CD8+ T cells. Following TCR activation, a higher percentage of young naive CD8+ T cells started expressing B7-H1, but it was significantly less than the percentage reached by old CD8+ T cells. Higher percentages of old memory CD8+ T cells express B7-H1 prestimulation; however, following TCR activation, young and old memory cells expressed similar levels of B7-H1. No differences in the expression of PD-1 were observed in young and old naive and memory CD8+ T cells before or after stimulation (Fig. 2B). We also observed no significant differences in the mean fluorescence intensity of B7-H1 in naive and memory young and old CD8+ T cells (Supplemental Fig. 3).
In vivo expression of B7-H1 in young and old CD8+ T cells
We evaluated the expression of B7-H1 in proliferating (activated) and nonproliferating (resting) CD8+ T cells in vivo following the stimulation of young and old BALB/c mice with the immunogenic BM-185-EGFP tumor. The treatment regimen is depicted in Fig. 3A. CD8+ proliferating and nonproliferating T cells from old animals with or without tumors showed a significantly greater expression of B7-H1 compared with young CD8+ T cells (Fig. 3B, Supplemental Fig. 4). These results indicate that at all times, under activating or resting conditions, more old CD8+ T cells express B7-H1, which might influence the function of these cells.
Blockade of B7-H1 restores the proliferation activity of old CD8+ T cells
We analyzed whether there were differences in the division and proliferation activity between young and old CD8+ T cells. We developed an in vitro model using conjugated beads. Beads were conjugated with anti-CD3+anti-CD28 or anti-CD3+anti-CD28+PD-1–Ig (Fig. 4). Young and old naive CD8+ T cells were labeled with CFSE, and cells were stimulated in the presence of the conjugated beads for 72 h. As shown in Fig. 4A, 71% of young CD8+ T cells divided, whereas only 35% of old CD8+ T cells divided following stimulation with anti-CD3+anti-CD28 beads. The presence of PD-1 (Ig–PD-1) on the beads inhibited the division of young (decrease from 71% to 40%) and old (decrease from 35% to 14%) CD8+ T cells (Fig. 4B). These data indicate that B7-H1 provides negative signals inhibiting the function of T cells. Next, we evaluated whether blockade of B7-H1 on CD8+ T cells enhanced the proliferation of old CD8+ T cells. Our results indicate that, in the presence of anti–B7-H1, old CD8+ T cells recovered their ability to proliferate; they divided almost to the same degree as young CD8+ T cells (Fig. 4C). Furthermore, in the presence of anti–B7-H1, the negative effective of PD-1 on the anti-CD3+anti-CD28+PD-1–Ig beads was neutralized (Fig. 4D). We did similar experiments to evaluate T cell proliferation by thymidine incorporation on plates coated with anti-CD3+anti-CD28 in the presence or absence of anti–B7-H1. As expected, young CD8+ T cells proliferated more than old CD8+ T cells; however, in the presence of anti–B7-H1, the proliferation of old CD8+ T cells was similar to young CD8+ T cells (Fig. 4E). These results support our hypothesis that the expression of B7-H1 in old CD8+ T cells provides negative signals inhibiting the activation of these cells, and blocking B7-H1–PD-1 interactions can restore the function of these cells.
Blockade of B7-H1 restores the immune response in old mice in vivo
Having demonstrated that blockade of B7-H1–PD-1 interactions restored the proliferation activity of old CD8+ T cells, we next evaluated whether blockade of B7-H1–PD-1 interactions enhances or restores the immune responses in old mice in vivo. We used the BM-185-EGFP tumor model, which is an immunogenic tumor that is rejected by young BALB/c mice but grows in old BALB/c mice (33). Because many preclinical and clinical studies have shown that tumors express B7-H1, which could inhibit T cell responses, and that blocking B7-H1 on tumor cells enhances the immune responses (34, 35), we confirmed that BM-185-EGFP cells do not express B7-H1 (Fig. 5A). This provides a clean system in which to test the effect of blocking B7-H1 in old CD8+ T cells in enhancing the immune responses in vivo. To evaluate whether blockade of B7-H1 restores the immune responses in old mice, old BALB/c mice were implanted with BM-185-EGFP tumor cells and then treated with weekly injections of anti–B7-H1. As shown in Fig. 5B, six of seven old BALB/c mice treated with anti–B7-H1 rejected the tumor. The animal that did not reject the tumor showed a substantial delay in tumor growth compared with the control groups. Nontreated or isotype control-treated old BALB/c mice succumbed to the tumor. As a positive control, we used young BALB/c mice implanted with BM-185-EGFP cells; as expected, 100% of the animals rejected the tumor. We also evaluated whether blocking B7-H1 restored the activation of cytotoxic responses against EGFP Ags in old mice. Injections of BM-185-EGFP cells plus isotype control did not induce an immune response in old mice, whereas immunization with BM-185-EGFP plus anti–B7-H1 restored CTL responses against BM-185-EGFP cells (Fig. 5C). Importantly, the CTL activity of old CD8+ T cells from aged BALB/c mice immunized with BM-185-EGFP cells plus anti–B7-H1 was similar to the CTL activity from young BALB/c mice immunized with BM-185-EGFP cells (Fig. 5C). No cytotoxic activity was observed against the BM-185 WT cells, indicating that the specificity of the immune response was against EGFP. These results further support our hypothesis that differential expression of B7-H1 is detrimental for the optimal in vivo activation of T cell responses in old mice. These results suggest that blockade of B7-H1 might be important to optimally activate CD8+ T cell immune responses in old mice.
Effect of blocking B7-H1 on TCR expression and subunit associations in old CD8+ T cells
It is well documented that defects in the transduction of mitogenic signals following TCR stimulation exist in old CD8+ T cells. One possibility could be decreased numbers of TCR expressed on the cell surface of old CD8+ T cells. We compared the TCR levels between freshly isolated young and old CD8+ T cells; old CD8+ T cells expressed lower surface TCR levels compared with young CD8+ T cells (Fig. 6A). We also evaluated whether the blockade of B7-H1 could correct or restore, to some degree, the TCR expression in old CD8+ T cells. Following TCR stimulation, we still observed a difference in TCR expression; in the presence of anti–B7-H1, old CD8+ T cells increased the level of surface TCR expression to one equal to that of young CD8+ T cells (Fig. 6A). It is not clear why old CD8+ T cells express less TCR than young CD8+ T cells. Because surface expression of the TCR multiprotein complex is highly regulated at the posttranslational level (36), one possibility is that old and young CD8+ T cells express equal total levels of TCR, although old cells express less at the surface. As an alternative, old T cells might express fewer TCR subunit proteins, and/or these subunits might express poor association, forming fewer complexes. To distinguish between these possibilities, we used a high-sensitivity method to detect native protein–protein interactions (immunoprecipitation of multiprotein complexes detected by flow cytometry [IP-FCM]) (32). This technique is based on the analysis of coimmunoprecipitated proteins by flow cytometry. IP-FCM allows the measurement of subunits ratios within the TCR–CD3 complex (Fig. 6B). Using this technique, we evaluated the association of several subunits of the TCR. We used beads coupled to anti-ζ immunoprecipitating mAb to capture complexes from postnuclear lysates and then probed the complexes captured on the beads with mAbs against ε, ζ (the probing Ab recognizes a different epitope than the immunoprecipitating mAb), and TCRβ. This procedure allowed us to compare the association of the TCR complex subunits between young and old naive CD8+ T cells. The TCR complex subunits were assessed from freshly isolated CD8+ T cells; after evaluating each reaction by flow cytometry, our results indicated lower subunit association in old CD8+ T cells (light blue graph indicating fluorescence level) compared with young CD8+ T cells (pink graph) (Fig. 6C). This indicates that there are fewer TCR-ζ/ε and TCR-ζ/β associations in old CD8+ T cells than in young CD8+ T cells, which correlates with and is predicted to cause lower surface TCR expression in old CD8+ T cells. Observation of a slight decrease in ζ indicates that old CD8+ T cells may also express less total subunit protein (Fig. 6C, middle panel). We evaluated other subunit associations within the complex; our results indicated that, overall, there was decreased TCR subunit association in the old CD8+ T cells compared with young CD8+ T cells (Supplemental Fig. 5). As a nonspecific staining control and to verify membrane solubilization, captured complexes were also stained with nonspecific hamster Ig-PE plus anti–Thy1.2-PE, respectively (young cells = dark blue graphs and old cells = green graphs). Very little background fluorescence and nonspecific protein association was observed. Next, we evaluated whether the association of the TCR subunits could be enhanced after blockade of B7-H1. Young and old naive CD8+ T cells were stimulated with anti-CD3+anti-CD28+isotype control or anti-CD3+anti-CD28+anti–B7-H1. As seen in complexes isolated from resting cells, less TCR subunit association was observed following TCR stimulation in old CD8+ T cells (Fig. 6D, green graph) compared with young CD8+ T cells (Fig. 6D, red graph). However, blockade of B7-H1 during stimulation resulted in restoration of subunit associations of the TCR/CD3 complex in old CD8+ T cells (Fig. 6D, pink graph), whereas no changes were observed in young CD8+ T cells (Fig. 6D, blue graph). These results correlated with the rescued surface TCR expression in old CD8+ T cells following T cell stimulation in the presence of B7-H1 blockade (Fig. 6A).
The interactions of the B7 family receptors with their respective CD28 family receptors are critical to fine-tune immune responses (17–19). The regulation of viral, tumor, and tolerance immune responses by these receptors has been studied extensively. However, no data are available about whether the expression of these receptors could influence the dysregulation of the immune responses in the old. In these studies, we sought to investigate whether B7 family and CD28 family receptors were differentially expressed in different immune subpopulations in young and old mice. Although our data indicated that the majority of the receptors were equally expressed in young and old macrophages, DCs, and CD4+ and CD8+ T cells, we observed that molecules that positively regulate immune responses, such as ICOS, CD80, and CD86, were expressed in higher percentages of young cells. In contrast, B7-H1, which negatively regulates immune responses, was significantly and highly expressed in old CD8+ T cells (p < 0.01) compared with young CD8+ T cells. One of the reasons why old mice do not respond with the same vigor as young mice could result from the differential expression of these B7 family and CD28 family receptors. We were most intrigued by the differential expression of B7-H1 in old CD8+ T cells, and we concentrated our efforts on evaluating the effect of B7-H1 expression in old CD8+ T cells, because it is well established that B7-H1 can provide negative signals that inhibit T cell immune responses (37, 38); therefore, the high expression of B7-H1 in old CD8+ T cells could downregulate the function of these cells. We hypothesized that the expression of B7-H1 on old CD8+ T cells could interact with PD- 1, restricting the proper activation of CD8+ T cell responses in the aged. Overall, in vitro and in vivo analyses indicated that higher percentages of resting and activated old CD8+ T cells express B7-H1 than young CD8+ T cells. In particular, we found that higher percentages of naive CD8+ T cells from aged mice express B7-H1 compared with young naive CD8+ T cells. This is of particular interest because the expression of B7-H1 in old naive CD8+ T cells could significantly impair or limit the activation of CD8 T cell responses to de novo Ags. Importantly, at all times under activating or resting conditions, high percentages of old CD8+ T cells express B7-H1, which might influence the function of these cells. To test our hypothesis that B7-H1–PD-1 interactions inhibit the immune response and that the blockade of B7-H1–PD-1 interactions could enhance the immune response in the old, we developed an in vitro model of T cell stimulation using conjugated beads. Our data indicate that old CD8+ T cells have less capacity to divide and proliferate than do young CD8+ T cells. We confirmed that the interactions of B7-H1–PD-1 restrict the immune response, because the presence of PD-1 (Ig–PD-1) on the beads inhibited the division of young (decreased from 71% to 40%) and old (decreased from 35% to 14%) CD8+ T cells. These results confirm that B7-H1 could provide negative signals inhibiting the function of T cells. More importantly, blockade of B7-H1 with an anti–B7-H1 mAb restored the proliferative activity of old CD8+ T cells, indicating that blockade of B7-H1–PD-1 interactions neutralized the negative signals provided by B7-H1 expressed on the surface of CD8+ T cells. Blocking with B7-H1 only rescues the proliferation of old CD8+ T cells to the same levels as untreated young CD8+ T cells. It is important to emphasize that the expression of B7-H1 is not the only defect; other defects or alterations in old CD8+ T cells influence the function of these cells (4). Therefore, by blocking B7-H1, we partially restored the function of old CD8+ T cells. To optimally activate an old CD8+ T cell, it might be necessary to block or target two or more of these defects or alterations. T cell–T cell interactions via B7-H1–PD-1 are another possible mechanism by which old CD8+ T cells are inhibited through B7-H1. In the experiments with the beads, we only added CD8+ T cells to the wells. Old CD8 T cells proliferate less than do young CD8 T cells (Fig. 4A); in the presence of anti–B7-H1, old CD8 T cells proliferate to the same degree as do young old T cells (Fig. 4C). These experiments suggest that T cell–T cell interactions via B7-H1–PD-1 inhibit T cell activation, and blockade of B7-H1–PD-1 interactions at the level of T cells might be critical to optimally stimulate an old CD8 T cell response. As previously indicated, the interaction of PD-1 and its ligand B7-H1 is critical in regulating CD8+ T cells, including the induction of apoptosis or inhibiting proliferation (26–28). As such, we cannot discard the possibility that another effect of blocking B7-H1 is that it increases the survival capacity of old CD8+ T cells. Therefore, restoring the capacity of old CD8+ T cells, by blocking B7-H1, could be important to control disease and rescue the immune responses.
There is a plethora of information indicating that tumors express B7-H1, and the expression of this molecule on tumor cells inhibits T cell immunity (34, 35). Therefore, we evaluated the in vivo effect of blocking B7-H1 on T cells using the B7-H1− BM-185-EGFP tumor cell line. Our data showed that in vivo blockade of B7-H1 restored the antitumor responses against the BM-185-EGPF tumors, allowing old animals to respond with the same efficiency as young mice. Blockade of B7-H1 also restored the cytotoxic responses against EGFP+ targets, which correlate with the antitumor responses. Immune responses against new Ags occur after activating naive T cells. Considering that the majority of old naive CD8+ T cells express B7-H1, these results suggest that the expression of B7-H1 in old naive CD8+ T cells provides negative signals that inhibit or prevent the proper activation of immune responses against nominal Ags. Importantly, our results showed that blockade of B7-H1 was able to restore the immune response in old mice, indicating that effector function of old CD8+ T cells can be rescued. Thus, blocking B7-H1 signals in old CD8+ T cells may be useful to enhance the overall immune responses in the aged.
It is known that B7-H1 interacts with PD-1; it can also interact with CD80, and the B7-H1–CD80 interactions also impair the activation of T cells (39). As such, the interactions of B7-H1 with PD-1 and/or CD80 could inhibit the function of old CD8+ T cells. Studies with PD-1 and B7-H1 KO mice demonstrated that T cells from these animals are hyperresponsive and develop autoimmune disease (40, 41), indicating that these molecules are critical in regulating immune responses by restricting autoreactive CD8 T cells (42). Considering that B7-H1 is expressed at low levels in naive cells, B7-H1 probably controls CD8+ T cell responses after T cells have been activated. However, in the old, the majority of naive CD8+ T cells express B7-H1; this early expression of B7-H1 is detrimental for the proper activation of old CD8+ T cells, because interactions via B7-H1–PD-1 or B7-H1–CD80 could significantly inhibit the function of old CD8+ T cells. Overall, our results indicate that the expression of B7-H1 in old CD8+ T cells negatively regulates immune responses, and blockade of B7-H1 could be used as a strategy to restore and optimally stimulate immune responses in old animals. Our results are in agreement with the results of Barber et al. (43), who demonstrated that blockade of B7-H1 reverses the function of exhausted CD8+ T cells after lymphocytic choriomeningitis virus infection.
Aging is accompanied by numerous functional and phenotypic changes in the immune system. We also evaluated whether there was a correlation between the lack of T cell function and TCR expression. For the first time, our results show that old CD8+ T cells express lower levels of TCR, which correlate with lower interactions of the TCR complex subunits. The lack of proper immune function in the old (e.g., proliferation and cytokine production) could be due to defects in the transduction of mitogenic signals following TCR stimulation. Indeed, evidence indicates that early signaling events during stimulation via the TCR–CD3 complex are altered in old T lymphocytes (44, 45). These defects in signal transduction in old T cells could be due to the lack of proper assembly of TCR, which results in lower TCR expression and, subsequently, defective T cell activation. Following stimulation in the presence of B7-H1, the level of surface TCR expression on old CD8+ T cells was restored to a level equal to that on young CD8+ T cells. Moreover, the interactions between the subunits of the TCR complex in old CD8+ T cells improved when stimulated in the presence of B7-H1 blockade, becoming equal to that observed in young CD8+ T cells. These results showed that blockade of B7-H1 restored TCR complex assembly and expression in old CD8+ T cells, which correlates with stronger T cell activation and immune function. We are not certain how blockade of B7-H1 enhanced or restored TCR expression in old T cells. Very little is known about the signaling events of B7-H1 on T cells. T cell inhibition by PD-1 requires close proximity to the site of the TCR engagement and activation of the TCR. Following TCR engagement, PD-1 is phosphorylated, recruiting tyrosine phosphatase SHP-2. Recruitment of tyrosine phosphatase SHP-2 dephosphorylates the CD3-ζ subunit, Syk, Zap70, and PI3K, inhibiting the activation of T cells (46). It is possible that B7-H1 has a signaling pattern similar to PD-1, negatively influencing the function of T cells; thus, by blocking B7-H1, the negative signals transmitted by this receptor are prevented.
The reason for the high expression of B7-H1 in old CD8+ T cells is not understood. During proinflammatory immune responses, such as infection, the expression of B7-H1 is elevated and persists for longer periods of time in different immune cell subpopulations (47, 48). Aging is associated with inflammatory activity reflected by increased circulating levels of Th2 proinflammatory cytokines (49). Perhaps this persistent Th2 proinflammatory status in aged mice predisposes old CD8+ T cells to express B7-H1. Additionally, it is known that much of the aging population is chronically infected with CMV (50) and other viruses (51), which might drive CD8+ T cells to a state of exhaustion, inducing the high expression of B7-H1. Further studies are needed to completely understand the reason why high percentages of old naive CD8+ T cells express B7-H1, as well as how these populations relate to other immune-regulatory effects, such as cytokines, homeostasis, and APCs. More importantly, the high expression of B7-H1 on CD8+ T cells could predispose the aged to immune dysfunction, resulting in a higher risk for immune-mediated diseases, such as cancer or infections.
The capacity of an old host to mount an effective immune response is limited by the pre-existence of counterregulatory elements. Thus, controlling these regulatory mechanisms may represent a powerful strategy for controlling chronic infection or enhancing the efficiency of vaccines in the old. In these studies, we demonstrated that high percentages of old naive CD8+ T cells expressed B7-H1, impairing the proper activation of CD8+ T cell responses in the aged. As such, the high expression of B7-H1 in old CD8+ T cells negatively regulates the immune function of these cells. However, the negative effects of B7-H1 in old CD8+ T cells could be reversed by blocking this receptor, which results in the restoration of effective immune responses in the aged. Additionally, blockade of B7-H1 prevents negative signals, helping to restore the expression of TCR in old CD8+ T cells. The higher expression of TCR levels following B7-H1 blockade correlated with the acquisition of a stronger function of old CD8+ T cells. Taken together, these results strongly suggest that blockade of B7-H1 could be critical to the optimal stimulation of a CD8+ T cell response in the old.
Disclosures The authors have no financial conflicts of interest.
This work was supported by Grants CA 114336 and AG287510 from the National Institutes of Health and the American Federation for Aging Research (to J.L.).
The online version of this article contains supplemental material.
Abbreviations used in this paper:
- dendritic cell
- enhanced GFP
- immunoprecipitation of multiprotein complexes detected by flow cytometry
- programmed cell death 1
- wild type.
- Received November 3, 2009.
- Accepted March 11, 2010.
- Copyright © 2010 by The American Association of Immunologists, Inc.