The activation of T lymphocytes is a hallmark of cellular rejection in allografts, and it is regulated by multiple transmembrane glycoproteins, some of which are T lymphocyte surface receptors, including T-cell receptor–CD3, CD25, and CD28, whose functions have been well defined, whereas the function of others, such as CD98, remains to be elucidated. CD98 is a 125-kDa type II membrane glycoprotein composed of an 80-kDa glycosylated heavy chain (CD98hc) and a group of 45-kDa nonglycosylated light chains. CD98 has two distinct functions: facilitating amino acid transport (1, 2) and mediating integrin signaling (3, 4), and these functions are dependent on the different domains of CD98hc (5). The extracellular domain of CD98hc binds to one of the several light chains, which function as amino acid transporters. These amino acids are important regulators of the mammalian target of rapamycin (mTOR) pathway, which governs nutrient-regulated lymphocyte function (6, 7). The intracellular and transmembrane domains of CD98hc interact with certain integrin β subunits to mediate signaling events that control cell migration, survival, and proliferation (8, 9). CD98hc, encoded by the Slc3a2 gene, was originally described as a lymphocyte activation antigen (10), but the function of CD98hc in the immune system still remains obscure, especially regarding its function in the T lymphocyte–mediated immune response to alloantigen. In the present study, we found that the CD98hc-deficient mice accepted full major histocompatibility complex–mismatched cardiac allografts, and the mechanism may involve the attenuation of lymphocyte migration and induction of regulatory T (Treg) cells. Furthermore, the blockade of CD98hc by an anti-CD98 neutralizing monoclonal antibody (mAb) was able to prolong the survival time of the cardiac allograft. These findings clarified the previously unknown functions of CD98hc in regulating the alloimmune responses and might therefore help to develop a novel approach to promoting transplantation tolerance.
T-Cell Deficiency in CD98hc Decreases the Alloantigen Response, Thus Resulting in Cardiac Allograft Acceptance
Because the germ line loss of CD98hc is embryonically lethal (11), we targeted CD98hc by flanking exon 3 of Slc3a2 with a loxP site that specified Cre recombinase–mediated deletion of the extracellular region of CD98hc, thus leading to complete loss of CD98hc expression (see Figure 1A and B, SDC 1, http://links.lww.com/TP/A659). We crossed CD98hcf/f mice with CD4-Cre+ mice bearing Cre recombinase under control of the endogenous T cell–specific CD4 locus (12), resulting in CD98hcf/f CD4-Cre+ mice with a specific deletion of CD98hc in spleen CD3+ T lymphocytes (see Figure 1C, SDC 1,http://links.lww.com/TP/A659). We did not detect any deletion in any of the CD3-negative cell populations (data not shown). We then compared the CD98hc-related cell surface molecules, including CD11a, CD11b, CD29, CD62L, CD103, and CD147 on the T cells isolated from B6.wild and B6.CD98hc−/− mouse spleens, and found that there were no changes in the expression of these molecules after deleting CD98hc (see Figure 1D, SDC 1,http://links.lww.com/TP/A659). Furthermore, we found the total cell numbers in the spleen to be not significantly different between the wild-type and deficient mice (see Figure 2, SDC 1,http://links.lww.com/TP/A659). We also characterized the macrophages and T and B cells in the bone marrow, thymus, and spleen using anti-CD3, -CD4, -CD8, -CD11b, and -B200 antibodies and found no significant differences in the expression of these markers between the wild-type and deficient mice in the bone marrow and thymus, whereas the expression of CD8 was decreased in the spleen cells of the deficient mice (see Figure 3, SDC 1,http://links.lww.com/TP/A659).
To assess whether CD98hc is important for the T-cell responses, mixed leukocyte reaction (MLR) assay was performed. Splenic T cells obtained from naive B6.wild mice showed a vigorous proliferative response to irradiated Balb/c spleen cells; however, in the T cells from naive B6.CD98hc−/− mice, the proliferative response was reduced to nearly 20% of the B6.wild level (P=0.0002; Fig. 1A). Moreover, we still stimulated the T cells obtained from B6.wild and B6.CD98hc−/− mice with immobilized anti-CD3 and anti-CD28 in the absence of antigen-presenting cells and found that the response between these two groups was not significantly different (see Figure 4, SDC 1,http://links.lww.com/TP/A659), which means that, under these circumstances, the cell proliferation is intact. All of these results indicate that the T-cell response to allogeneic antigens was significantly impaired in the B6.CD98hc−/− mice.
We next performed cardiac transplantation, wherein Balb/c mice were used as donors and B6.wild or B6.CD98hc−/− mice were used as recipients. We found that all B6.wild recipients suffered acute rejection with a median survival time (MST) of 7.4 (SD, 0.5) days. In contrast, all B6.CD98hc−/− recipients showed indefinite allograft survival, with no sign of rejection (MST>100 days, P<0.001; Fig. 1B).
We compared the histopathologic features of the allografts harvested on postoperative day (POD)7. The classic signs of acute rejection could be seen in the allografts from the B6.wild recipients, including strong interstitial infiltration of inflammatory cells, severe hemorrhage, edema, and necrosis. In contrast, the allografts from B6.CD98hc−/− recipients were free of myocardial injury and had markedly reduced inflammatory cell infiltration (Fig. 1C, top). We then analyzed the number of infiltrating CD4+, CD8+, and bromodeoxyuridine (BrdU)+ T cells in cardiac allografts on POD7 by triple immunostaining. Cardiac allografts harvested from B6.wild recipients exhibited infiltration of numerous T cells, not only BrdU+, CD4+, and CD8+ T cells but also BrdU+CD4+ and BrdU+CD8+ T cells. However, these cells, especially BrdU+CD8+ cells, in the cardiac allografts of B6.CD98hc−/− recipients significantly decreased (Fig. 1C, top). These findings were also quantitatively analyzed using the WinROOF software program; the number of CD4+, CD8+, and BrdU+ cells infiltrated into the cardiac grafts obtained from B6.CD98hc−/− recipients on POD7 was significantly lower than that from B6.wild recipients (P=0.0001, P=0.0019, and P=0.0024, respectively; Fig. 1C, bottom).
We also compared the graft-infiltrating lymphocyte (GIL) between B6.wild and B6.CD98hc−/− recipients on POD7. As shown in Figure 2(A), the number of GIL obtained from B6.CD98hc−/− recipients was significantly lower than that from the B6.wild recipients (P=0.0028). Most of the GIL from B6.wild recipients was CD8+ T cells, as determined by fluorescence-activated cell sorter (FACS). The number of these cells was much higher than that of the GIL observed in the grafts from B6.CD98hc−/− recipients (P=0.0494). However, there were no differences in the proportion of spleen CD4+ and CD8+ T cells obtained from B6.wild and B6.CD98hc−/− recipients (Fig. 2B).
CD98hc Deletion in T Cells Attenuates Lymphocyte Migration In Vitro, and the Cells Exhibit a Poor Proliferative Response and Fail to Infiltrate Into the Allograft
To examine the role of CD98hc deficiency on lymphocyte migration in vitro, lymphocytes isolated from the spleens of B6.wild or B6.CD98hc−/− recipients on POD7 were labeled with 5-carboxyfluorescein diacetate succinimidyl ester (CFSE) and loaded in Transwell filters (Mitani Corp, Tokyo, Japan), which were placed in 24-well plates containing the medium with or without CCL2 or CCL5 at 0-, 0.1-, 1-, 10-, and 100-ng/mL concentrations. After incubation, the cells in the bottom well were collected and counted by flow cytometry (FCM). We found that the number of lymphocytes that had migrated increased with increasing concentrations of CCL2 or CCL5 from both B6.wild and B6.CD98hc−/− recipients, but fewer lymphocytes from the B6.CD98hc−/− recipients had migrated compared with the B6.wild recipients, and the chemotactic index of the lymphocytes from B6.CD98−/− recipient was significantly lower than that of the B6.wild recipients in the presence of 10-ng/mL (P=0.016) and 100-ng/mL (P=0.010) concentrations of CCL5 (Fig. 3A). These results indicate that the migratory capacity of lymphocytes from B6.CD98hc−/− mice was significantly decreased in vitro compared with those from B6.wild mice.
In addition, to visualize the effect of CD98hc deficiency on the migration of lymphocytes in vivo, lymphocytes obtained from the spleens of B6.wild and B6.CD98hc−/− naive mice were labeled with CFSE, followed by immediate adoptive transfer into Balb/c B6.Rag2KO cardiac graft recipients. Compare with the untransplanted hosts (Fig. 3B, upper), on day 5 after adoptive transfer, more than 83% of the B6.wild CD3+ T cells obtained from reconstituted B6.Rag2KO recipients were found to be negative in CFSE (17% remained positive in CFSE), and nearly 10% of the CFSE-positive cells were not dividing, thus indicating that B6.wild T cells undergo alloantigen-driven proliferation (Fig. 3B, middle). However, under the same conditions, nearly 50% of the B6.CD98hc−/− CD3+ T cells remained positive in CFSE, and more than 23% of the CFSE-positive cells were not dividing (Fig. 3B, lower). Furthermore, we compared CD8+ T-cell infiltration into Balb/c cardiac allografts on day 5 after adoptive transfer of B6.wild or B6.CD98hc−/− naive mouse lymphocytes by staining the allograft tissue sections with anti-CD8, collagen IV, and BrdU. Consistent with the findings presented in Figure 1(C), very few CD8+ T cells were detected in the Balb/c cardiac grafts obtained from B6.CD98hc−/− adoptively transferred recipients (Fig. 3C). Furthermore, we examined the proportion of propidium iodide– and annexin V–positive cells between the B6.wild and B6.CD98hc−/− naive mice, MLR culture, and B6.wild and B6.CD98hc−/− recipients on POD7, and the proportion of these cells was not significantly different between these two groups (see Figure 5, SDC 1,http://links.lww.com/TP/A659). Taken together, these results suggest that CD98hc deficiency reduced the ability of T cells to proliferate in response to an alloantigen, thus resulting in a decreased accumulation of graft-infiltrating T cells.
Deletion of CD98hc in Mouse T Lymphocytes Results in Cardiac Allograft Acceptance by Increasing the Generation of Treg Cells
To examine the changes in Treg cells after cardiac allograft acceptance in the B6.CD98hc−/− mice, we analyzed the population of the Treg cells by FACS, examined the messenger RNA (mRNA) expression of Foxp3 by quantitative real-time polymerase chain reaction (qRT-PCR), and determined the number of Foxp3-positive cells by immunohistochemistry on POD3, POD7, and POD14. We found that the proportion of CD4/Foxp3-positive cells increased significantly on POD7 and POD14 by FACS analysis, but no differences were seen on POD3 between B6.wild and B6.CD98hc−/− recipients (Fig. 4A). These findings were confirmed by qRT-PCR (Fig. 4B). Furthermore, the immunohistochemical analysis and qRT-PCR assay of grafts showed the same tendency, wherein the number of cells and the mRNA expression of Foxp3 increased significantly on POD7 and POD14, but no difference was seen on POD3 between B6.wild and B6.CD98hc−/− recipients (Fig. 4C,D). Moreover, we noticed that the mRNA expression of mTOR significantly decreased in grafts and spleens of B6.CD98hc−/− mice (see Figure 6C, SDC 1,http://links.lww.com/TP/A659) and that PS6, the key kinase in the mTOR pathway, was significantly decreased in CD4-positive cells obtained from B6.CD98hc−/− recipients compared with B6.wild recipients on POD7 (see Figure 6B, SDC 1,http://links.lww.com/TP/A659). These results indicated that CD98hc deletion could induce Treg generation, and this correlated with reduced expression levels of mTOR and PS6 after cardiac transplantation.
To assess the effects of Treg cells on B6.CD98hc−/− mouse cardiac allograft acceptance, B6.CD98hc−/− mice were given two intraperitoneal injections of anti-CD25 mAb (PC61, days −7 and −3), which our previous experiments demonstrated could deplete Treg cells. The mice then received Balb/c cardiac allografts on day 0. The MST of the cardiac allografts decreased to 18.3 (SD, 4.3) days in the PC61-treated mice, which was significantly shorter than that of the B6.CD98hc−/− recipients (MST>100days, P<0.001; Fig. 4E).
Antibody Therapy Using an Anti-CD98hc Neutralizing mAb to Prolong Cardiac Allograft Survival
A high-affinity antagonistic anti-CD98hc mAb (clone 26-24) was originally generated by Tsumura et al. (13), and it was shown to suppress human immunodeficiency virus gp160-mediated cell fusion and induce the aggregation of and production of multinucleated giant cells by monocytes/macrophages. To further investigate the function of the CD98hc molecule in preventing the T-cell response, we first examined the effects of the 26-24 mAb in the MLR assay, which was performed with B6.wild spleen T cells as responders and irradiated Balb/c splenocytes as stimulators in the presence of 0-, 1-, 10-ng/μL concentrations of 26-24 mAb. We found that the T-cell proliferative response decreased in an antibody dose-dependent manner (Fig. 5A; P<0.0001 and P=0.0012 for the differences between 26-24 mAb concentrations at 0-, 1-, and 10-ng/μL, respectively).
Next, to evaluate whether the 26-24 mAb recognizes CD98hc molecule in vivo, splenocytes were obtained from B6.wild naive mice or B6 mice, which had received 26-24 mAb 3 days before, and were then stained with 26-24 mAb, followed by anti-rat IgG2a–fluorescein isothiocyanate and anti-CD4 phycoerythrin (PE)/cyanine 5. In addition, anti-CD98hc PE (clone RL388) and anti-CD4-PE/cyanine 5 were added to confirm the expression of CD98hc. We observed that the expression of CD98hc (clone 26-24) in CD4-positive cells obtained from the B6 mice that had received the 26-24 mAb significantly decreased compared with the B6.wild naive mice (Fig. 5B, top). However, no difference in the expression of CD98hc (clone RL388) was observed between these two groups (Fig. 5B, bottom). These results indicated that 26-24 mAb could specifically recognize the CD98hc molecule in vivo.
Finally, we administered the 26-24 mAb to B6.wild recipients after they received cardiac grafts from Balb/c donors. On the day of transplantation, the recipient was given one intraperitoneal injection of 500 μg per mouse of the 26-24 mAb and then 250 μg per mouse twice a week for 2 weeks. The MST of cardiac allografts was prolonged to 13.4 (SD, 2.7) days, which was significantly longer than that of B6 recipients without 26-24 mAb treatment (MST, 7.4 [SD, 0.5] days, P=0.0186; Fig. 5C).
CD98hc was one of the earliest lymphocyte activation antigens described (1, 2). However, the function of the molecule with regard to the immune response has remained unclear. Although Cantor et al. (14) showed that CD98hc is required for rapid B-cell clonal proliferation and is necessary for their subsequent differentiation into antibody-secreting plasma cells and other studies in which T cells were treated with anti-CD98hc antibody in vitro suggested that CD98hc was involved in T-cell activation (15, 16), the precise functions, particularly with regard to the response to alloantigens, were unknown. We found that T cell–specific deficiency in CD98hc can lead to cardiac allograft permanent acceptance, wherein the mechanisms might attenuate lymphocytes migration and increase in Treg cell generation.
Regarding the first possible mechanism, T lymphocyte migration and activation are important for initiating allograft rejection, and these processes involve a complex cascade of molecular interactions and cellular responses, including CD98hc-dependent migration of T cells (16, 17). The heterodimeric complexes of CD98hc and the light chains specifically promote sodium-independent exchange of neutral amino acids, including leucine (18), and these amino acids are essential for maintaining cell viability and promoting cell growth. When CD98hc was deleted from T lymphocytes, the cells failed to take up leucine, which might have affected nutrient-regulated T lymphocyte function, thereby impairing the migration of T lymphocytes. In the present Transwell assays, we found that few lymphocytes migrated in the B6.CD98hc−/− recipients compared with the B6.wild recipients in the presence of CCL2 and CCL5 (Fig. 3A), despite the fact that the CD98hc-related cell surface molecules on the T cells did not show any changes after deletion of CD98hc (see Figure 1D, SDC 1,http://links.lww.com/TP/A659). Chemokines have been shown to play a critical role in leukocyte recruitment to transplanted organs and in leukocyte localization within tissues. CCL5 is a potent chemoattractant for monocytes, T cells, eosinophils, natural killer cells, and dendritic cells and a stimulator for endothelial T-cell adhesion and proliferation. CCL2 is a potent chemoattractant for monocytes and a stimulator of monocyte adhesion to the endothelium. CCL5 and CCL2 are upregulated in cardiac allografts early after transplantation (19, 20). Using the F344-to-Lewis rat cardiac allograft model, intragraft gene transfer of CCL5 or CCL2 antagonists prolonged cardiac allograft survival significantly (21), which means that CCL5 or CCL2 plays a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. Furthermore, in the immunohistochemical study and GIL analysis, we found that CD4/CD8-positive T cells failed to infiltrate not only into grafts harvested from the B6.CD98hc−/− recipients but also in alloreactive B6.Rag2KO recipients that received adoptive transfer (Figs. 1C, 2A, and 3C). This observation was also accompanied by down-regulation of cytotoxic T cell-related (granzyme B, perforin, FasL, PD-1, and PD-L1) and other inflammatory cytokine (interferon γ, tumor necrosis factor α, heme oxygenase 1, and inducible nitric oxide synthase) mRNA expression (see Figure 7, SDC 1,http://links.lww.com/TP/A659). Second, T-cell deletion of CD98hc could impair the proliferative function of the T cells. We found that the proliferative response of naive B6.CD98hc−/− T cells to Balb/c spleen cells significantly decreased compared with B6.wild cells in the MLR assay (Fig. 1A). All of these results indicated that T-cell deletion of CD98hc impairs the migration and proliferation of T cell, thus contributing to the acceptance of the cardiac allografts in B6.CD98hc−/− mice.
The second possible mechanism was an increase in the generation of Treg cells. Treg cells play a crucial role in controlling immune responses and inducing tolerance to allografts (22–26). We found that the proportion of CD4/Foxp3-positive cells increased significantly on POD7 and POD14, and these findings were confirmed by immunohistochemistry and qRT-PCR assay (Fig. 4A–D). Furthermore, when using anti-CD25 mAb, which has been reported to delete Treg cells (27, 28), the cardiac allograft survival time significantly decreased (Fig. 4E), thus indicating that the Treg cells play a pivotal role in the allograft acceptance in CD98hc-deficient recipients. The mechanism underlying this increase in Treg cells might be the observed decrease in the expression of mTOR and PS6, which was significantly decreased in B6.CD98hc−/− recipients on POD7 (see Figure 6, SDC 1,http://links.lww.com/TP/A659). mTOR is an important signaling molecule that translates environmental cues into specific T-cell responses (29); PS6 is the key kinase in the PI3K-AKT-mTOR axis (30). Our data support a model whereby the induction of Treg cells by means of negative regulation of the PI3K-AKT-mTOR pathway.
We showed that T-cell deficiency in CD98hc could contribute to cardiac allograft permanent acceptance. However, this conclusion was based on studies performed under nonphysiological conditions. It was therefore of critical importance to investigate the role of CD98hc under more physiological conditions. We thus further investigated the effects of the anti-CD98hc mAb and demonstrated that it could specifically recognize and block the CD98hc molecule to prolong cardiac allografts survival. Consistent with the results observed using knockout mice, the proliferative response of B6.wild spleen T cells to irradiated Balb/c splenocytes was decreased in an anti-CD98hc mAb dose-dependent manner in the in vitro MLR assay (Fig. 5A). Moreover, when we applied the anti-CD98hc mAb in vivo, we found that the survival of the cardiac allografts was significantly prolonged (Fig. 5C). Although the effect of anti-CD98hc mAb was limited compared with CD98hc knockout mice, since the antibody could just block the CD98hc molecular partly, it not only confirmed our results obtained using the T cell-specific CD98hc deficient mice, but also provided a novel approach to promoting graft acceptance.
Taken together, our data demonstrated that CD98hc is involved in regulating T-cell proliferation, and T cell–specific deficiency in CD98hc can contribute to cardiac allograft permanent acceptance by attenuation of lymphocyte migration and increase in Treg cell generation. Furthermore, in vivo administration of an anti-CD98hc mAb resulted in prolongation of mouse cardiac allograft survival, thus representing a major advance toward the therapeutic use of antibody therapy for treatment of allograft rejection.
MATERIALS AND METHODS
C57BL/6 (B6, H-2Kb), Balb/c (Balb/c, H-2Kd), C57BL/6J-RAG2KO (B6.Rag2KO, H-2Kb), and C57BL/6.CD98hc-deficient mice (B6.CD98hc−/−, H-2Kb) were used in accordance with the guidelines of the animal use and care committee of our institute (see Materials and Methods, SDC 2,http://links.lww.com/TP/A660).
Splenocytes and GILs were analyzed by FCM, and the data were analyzed using the CellQuest software package (Becton Dickinson, Franklin Lakes, NJ).
Lymphocyte Proliferation Assays
The MLR was measured using cell-proliferation enzyme-linked immunosorbent assay kits.
Heterotopic Cardiac Transplantation
The heart transplantation was performed from Balb/c donor to B6.wild or B6.CD98hc−/− recipients using microsurgical techniques.
Immunohistochemical and Histologic Analysis
Triple immunostaining of cardiac allografts was performed to detect intragraft cell infiltration. The infiltration of CD4+, CD8+, BrdU+, and Foxp3+ cells were analyzed quantitatively using the WinROOF software package (31, 32).
In Vitro Migration Assays and Adoptive Transfer Study
Splenocytes were prepared from Balb/c heart–grafted B6.wild or B6.CD98hc−/− recipients on POD7 and incubated with CFSE with or without chemokines CCL2 and CCL5 at different concentrations. After incubation, cells were collected and counted by FCM.
For the adaptive transfer study, CFSE-labeled lymphocytes from the spleens of B6.wild or B6.CD98hc−/− naive mice were injected into B6.Rag2KO recipients immediately after transplantation of Balb/c heart grafts. On day 5 after the transfer, spleen and cardiac grafts of B6.Rag2KO recipients were harvested and subjected to FACS and immunohistochemical analyses.
To assess the effect of Treg cells on cardiac allograft tolerance in B6.CD98hc−/− mice, anti-CD25 mAb was injected, and then, they received a cardiac allograft. The effect of the anti-CD98hc mAb was also investigated.
Student’s t tests were used to compare the paired and unpaired analyses. A statistical evaluation of mouse survival was performed using the Kaplan-Meier test. P values less than 0.05 were considered to be statistically significant.
The authors thank Dr. H. Kimura for his critical comments and useful suggestions. The authors also thank L. Xie, K. Kato, and S. Toyama for their valuable technical assistance.
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