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Targeting LFA-1 and CD154 Suppresses the In Vivo Activation and Development of Cytolytic (CD4-Independent) CD8⁺ T Cells^1,2

Keri E. Lunsford, Mitchel A. Koester^*, Anna M. Eiring^*, Phillip H. Horne, Donghong Gao^* and Ginny L. Bumgardner^3,*,,

^* Integrated Biomedical Science Graduate Program, Department of Surgery, Division of Transplantation, The Ohio State University Medical Center, Columbus, OH 43210; and Department of Internal Medicine, Division of Digestive Diseases, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Short-term immunotherapy targeting both LFA-1 and CD40/CD154 costimulation produces synergistic effects such that long-term allograft survival is achieved in the majority of recipients. This immunotherapeutic strategy has been reported to induce the development of CD4⁺ regulatory T cells. In the current study, the mechanisms by which this immunotherapeutic strategy prevents CD8⁺ T cell-dependent hepatocyte rejection in CD4 knockout mice were examined. Combined blockade of LFA-1 and CD40/CD154 costimulation did not influence the overall number or composition of inflammatory cells infiltrating the liver where transplanted hepatocytes engraft. Expression of T cell activation markers CD43, CD69, and adhesion molecule CD103 by liver-infiltrating cells was suppressed in treated mice with long-term hepatocellular allograft survival compared to liver-infiltrating cells of untreated rejector mice. Short-term immunotherapy with anti-LFA-1 and anti-CD154 mAb also abrogated the in vivo development of alloreactive CD8⁺ cytotoxic T cell effectors. Treated mice with long-term hepatocyte allograft survival did not reject hepatocellular allografts despite adoptive transfer of naive CD8⁺ T cells. Unexpectedly, treated mice with long-term hepatocellular allograft survival demonstrated prominent donor-reactive delayed-type hypersensitivity responses, which were increased in comparison to untreated hepatocyte rejectors. Collectively, these findings support the conclusion that short-term immunotherapy with anti-LFA-1 and anti-CD154 mAbs induces long-term survival of hepatocellular allografts by interfering with CD8⁺ T cell activation and development of CTL effector function. In addition, these recipients with long-term hepatocellular allograft acceptance show evidence of immunoregulation which is not due to immune deletion or ignorance and is associated with early development of a novel CD8⁺CD25^high cell population in the liver.

	Introduction

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Hepatocellular allografts have previously been demonstrated to initiate both CD4⁺ T cell-dependent and (CD4⁺ T cell-independent) CD8⁺ T cell-dependent pathways of allograft rejection (1, 2, 3, 4). The activity of the latter CD8-dependent rejection pathway can be demonstrated following transplantation of hepatocellular allografts into CD4 knockout (KO)⁴ mice (1), CD4-depleted C57BL/6 mice (2), and CD8-reconstituted SCID mice (1, 4). The CD8-dependent pathway is of particular interest because it is resistant to immunosuppressive strategies that readily regulate CD4⁺ T cell-dependent alloimmune responses (5, 6, 7, 8, 9, 10, 11).

The CD8-dependent pathway of rejection occurs not only in response to hepatocellular allografts (1, 2, 3, 4), but also in response to intestinal allografts (10, 11, 12), and, under particular experimental conditions, to skin allografts (9, 13) and cardiac allografts (7, 8). These studies consistently demonstrate that this pathway is resistant to immunoregulation with strategies that are very effective at suppressing CD4-dependent immune activation such as Rapamycin (14), Cyclosporine (15), anti-CD4 mAb (7, 9, 16), gallium nitrate (16), anti-CD154 mAb (7, 9, 11), combined treatment with donor-specific transfusion and anti-CD154 mAb (5, 17), and CTLA4-Ig (6, 9, 10). Resistance of CD8⁺ T cells to suppression by calcineurin inhibitors has also been correlated with an increased incidence of acute allograft rejection in clinical studies (15). In experimental models, we and others have demonstrated that CD8-dependent allograft rejection operates independently of the CD28/B7 costimulatory pathway (6, 9, 10). In contrast, we have reported that CD8-dependent hepatocyte rejection is dependent upon CD40/CD154 costimulation; however, immunotherapy targeting CD40/CD154 costimulation alone only delays hepatocyte rejection without achieving long-term allograft survival (90 days) in any recipients (6). These observations prompted consideration of alternative costimulation pathways in the activation of (CD4-independent) alloreactive CD8⁺ T cells.

LFA-1 (CD11a/CD18) is a member of the ₂ family of integrins and is expressed constitutively on lymphoid cells of T and B cell lineages. Although the role for LFA-1 as an adhesion molecule is well known, a significant amount of in vitro literature has indicated a role for LFA-1 in T cell costimulation. LFA-1 lowers the threshold for naive CD4⁺ T cell activation and proliferation through TCR engagement (18), and has a requisite role in CD8⁺ T cell activation, proliferation, and cytolytic activity (19, 20, 21, 22). In addition, signaling through LFA-1/ICAM-1 interactions can provide sufficient costimulation for CD8⁺ but not CD4⁺ T cell activation in the absence of CD28/B7 costimulation (22). These data collectively suggested that LFA-1 may play a critical role in activation of CD8⁺ T cells in vivo and led us to examine the effects of targeting LFA-1 on CD8-dependent hepatocyte rejection.

Indeed, we determined that short-term blockade of LFA-1 not only suppressed CD8-dependent hepatocyte rejection but also induced indefinite acceptance of hepatocellular allografts in 29% of recipients. Furthermore, combined blockade of CD40/CD154 and LFA-1 resulted in long-term hepatocyte allograft survival in 80% of recipients (3). This is the first strategy identified which indefinitely suppresses CD8-dependent hepatocellular allograft rejection across a complete MHC mismatch. Previous studies reporting the efficacy of combined immunotherapy targeting LFA-1 and CD40/CD154 costimulation for induction of long-term islet allograft survival supports a paradigm in which this therapeutic regimen induces a regulatory CD4⁺ T cell population because this immunotherapy induced prolonged allograft survival and transferable tolerance that was abrogated by the depletion of CD4⁺ T cells (23). The observation that combined targeting of LFA-1 and CD40/CD154 costimulation controlled CD8-dependent hepatocyte rejection in the absence of CD4⁺ T cells expands upon the existing paradigm for allograft acceptance induced by this immunotherapy. Furthermore, this strategy is distinguished from other immunotherapies, which also successfully suppress CD8-dependent rejection in stringent skin (24, 25, 26, 27, 28, 29, 30, 31) and intestinal allograft (32) models by the achievement of long-term allograft survival across a complete MHC mismatch and in the absence of recipient CD4⁺ T cells.

The purpose of the current study was to determine the mechanisms by which treatment with anti-LFA-1 and anti-CD154 mAbs results in long-term suppression of (CD4-independent) CD8-dependent rejection, which occurs in response to allogeneic hepatocytes transplanted to the liver. The effect of this treatment strategy upon leukocyte trafficking, activation, and maturation of CD8⁺ T cells in vivo was examined. The persistence of circulating anti-LFA-1 mAb treatment Ab was also evaluated because this information has not previously been reported.

	Materials and Methods

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Experimental animals

Mice were purchased from The Jackson Laboratory or Taconic Farms at 4–6 wk of age and maintained in accordance with guidelines from the University Laboratory Animal Resources and Institutional Laboratory Animal Care and Use Committee of The Ohio State University Research Foundation. The recipient strains used for these experiments were C57BL/6 (H-2^b), C57BL/6 CD4 KO (disrupted for the CD4 gene, H-2^b, The Jackson Laboratory; C57BL/6-cd4^tmlMak), and transgenic donor mice. Transgenic mice expressing human -1-antitrypsin (hA1AT-FVB/N, H-2^q) were the source of donor hepatocytes. The transgenic mouse strain was created, bred, and maintained at the Biotechnology Center and Transgenic Animal Facility, The Ohio State University (33, 34).

Hepatocyte isolation and purification

Hepatocyte isolation and purification was performed using a modification of the Seglen’s perfusion technique (35, 36), as described previously (1, 34). Briefly, the liver was perfused 0.09% EGTA-containing calcium-free salt solution followed 0.05% collagenase (type IV; Sigma-Aldrich) in 1% albumin (Sigma-Aldrich). Liver tissue was minced, filtered, and washed in RPMI 1640 with 10% FBS. Hepatocytes were purified on a 50% Percoll gradient (Pharmacia Biotech). Hepatocyte viability and purity were consistently 99%, respectively.

Hepatocyte transplantation and monitoring of graft function

Purified hepatocytes were transplanted by intrasplenic injection. Graft function was determined by the presence of hA1AT in serial recipient serum samples. Serum hA1AT levels in donor and host mice were detected by sandwich ELISA and ranged from 0.5 to 40 µg/ml (1, 34). Recipient hA1AT levels varied by experiment based on hA1AT production by transgenic donors, but were consistent among recipients receiving hepatocytes from the same donor. Graft survival was determined by sustained hA1AT levels, and graft loss was considered the time point at which host serum hA1AT was <0.5 µg/ml. Serum hA1AT in hepatocyte rejectors typically decreased from baseline levels over 1 wk. Survival of hA1AT-positive donor hepatocytes can also be demonstrated by immunohistochemical staining of host liver tissue (34, 37). Conversely, there are no detectable hA1AT-positive donor hepatocytes in host liver tissue from mice that had rejected hepatocellular allografts (not shown).

In vivo host treatment

CD4 KO mice were transplanted with transgenic hA1AT-FVB/N donor hepatocytes. Experimental groups were treated with anti-LFA-1 mAb, (M17/4.4.11.9, anti-LFA-1 unit, rat IgG2a isotype, no. TIB-217; American Type Culture Collection (ATCC)) and anti-CD154 mAb (MR1, hamster anti-mouse CD154 IgG; LigoCyte Pharmaceuticals). Anti-LFA-1 mAb was expanded for in vivo use by ascite production in pristane-primed nude mice (Ncr; Taconic Farms). Ascites were purified on a GammaBind Plus Sepharose column (Pharmacia Biotech) and ELISA determined the concentration of purified Abs. Hosts received 300 µg of anti-LFA-1 mAb on days 0 through 6 and 1 mg of anti-CD154 mAb on days 0, 2, 4, and 7 relative to hepatocyte transplantation by i.p. injection. Some CD4 KO recipient mice with continued acceptance of allogeneic hepatocytes for 60 days were depleted of CD8⁺ T cells by administration of anti-CD8 mAb (2.43, 0.25 mg i.p., days 60 and 62 after transplant). This regimen achieves CD8⁺ T cell depletion, which persists for 2–3 wk (2). Depletion of CD8⁺ T cells was confirmed and monitored by serial flow cytometric analysis of PBLs.

Detection of circulating IgG by ELISA

Serum rat IgG levels in host serum were detected by a standard sandwich ELISA. High-binding 96-well assay microplates from Corning Costar were coated overnight at 4 µg/ml in 0.05 M NaHCO₃ (pH 9.6) with unconjugated rabbit anti-rat IgG (H + L), mouse absorbed (Pierce Biotechnology), as Ag-specific capture Ab. BSA (1%) in PBS was used to block for nonspecific binding. Host serum was added in 50 µl/well duplicates at serial 1/1 dilutions with PBS with an initial starting dilution of 1/20. Plates were incubated with Ag at 4°C overnight, then washed with PBS with 0.1% Tween 20. The Ag-specific indicator Ab was mouse anti-rat IgG (H + L) HRP-conjugated (Southern Biotechnology Associates). Positive binding was detected using hydrogen peroxide as the substrate and ABTS as the chromogen. The enzymatic reaction was terminated with 1% SDS.

Liver-infiltrating cell (LIC) isolation and purification

The isolation of lymphocytes was performed based on a modification of the Crispe isolation technique (38). Briefly, mouse liver perfusion was performed as described above for hepatocyte isolation. The resulting liver cell suspension was washed once with RPMI 1640 containing 10% FBS and was digested in serum-free RPMI 1640 containing 0.05% collagenase IV (Sigma-Aldrich) and 0.002% DNase I (Sigma-Aldrich) at 37°C for 40 min. The majority of hepatocytes were pelleted by centrifugation at 30 x g for 3 min. The supernatant was then centrifuged at 300 x g for 10 min to collect the intrahepatic lymphocytes along with Kupffer cells and any remaining hepatocytes. Leukocyte populations within the LICs were purified from remaining hepatocytes, endothelial cells, and cellular debris on a 36% Histodenz (Sigma-Aldrich) discontinuous gradient by centrifugation at 1500 x g for 20 min. The interface of cells was collected, washed once with media, and counted by trypan blue exclusion before being stained and analyzed. Cell viability and purity was 90% on samples used for flow cytometric analysis.

Flow cytometric analysis of LICs

For flow cytometric analysis of LICs, 2 x 10⁶ cells were resuspended in 100 of µl of washing buffer (5% goat serum and 0.01% sodium azide in PBS) containing PE- or FITC-conjugated mAbs, isotype control Abs, or were left unstained (negative control). mAbs used for cell subset analysis were anti-CD3 mAb (hamster IgG, 145-2C11), anti-CD4 mAb (rat IgG2b, GK1.1), anti-CD8 mAb (rat IgG2b, 2.43), anti- TCR mAb (mouse IgG, UC7-13D5), anti-NK1.1 (mouse IgG2a, PK136), anti-Ly6G mAb (rat IgG2b, RB6-8C5) (all BD Pharmingen), and anti-F4/80 mAb (rat IgG2b, CI:A3-1; Caltag Laboratories). For analysis of activation, and adhesion molecule expression, on CD8⁺ T cells, LICs were costained with FITC-conjugated anti-CD8 mAb and either PE-conjugated Abs to CD25 (rat IgG1, PC61), CD43-activation associate glycoform (rat IgG2a, 1B11), CD62L (rat IgG2a, MEL-14), CD69 (hamster IgG1, H1.2F3), CD103 (rat IgG2a, M290), CD154 (hamster IgG1, MR1), (all BD Pharmingen). To evaluate apoptosis in LICs, these cells were incubated with PE-conjugated Annexin V and/or the vital dye 7-aminoactinomycin D (7-AAD) and prepared using the Annexin V^PE staining protocol provided with the PE Apoptosis Detection kit I (BD Pharmingen).

Cells were subsequently analyzed by flow cytometry by incubation for 60 min at 4°C and were washed three times before resuspension in washing buffer containing 10% buffered formalin. Flow cytometry was performed using a Beckman Coulter EPICS XL flow cytometer and System II version 3.0 acquisition software. Data was saved as listmode files, and data analysis and histogram overlays were performed using Expo32 version 1.2 software (Applied Cytometry Systems). Results are expressed in histogram form with the ordinate representing the percent of the maximum cell number and the abscissa representing fluorescence intensity in arbitrary units. Cell subset numbers were calculated based on the percentage positive of the total cell population. Activation markers, integrin expression, and apoptosis were analyzed by gating on CD8⁺ lymphocytes. For each histogram, 5 x 10⁴ gated events were analyzed.

In vivo cytotoxicity assay

The in vivo cytotoxicity assay modified from the assay, which has been previously described (39, 40). Syngeneic or allogeneic target splenocytes were prepared by mechanical disruption followed by RBC lysis (0.017 M Tris and 0.75% NH₄Cl (pH 7.4)). Isolated splenocyte populations were washed twice in PBS and were counted by trypan blue exclusion. A total of 40–50 x10⁶ splenocytes were incubated at 37°C in 10 ml of warm PBS containing CFSE (Vybrant CFDA SE Cell Tracer kit; Molecular Probes) for 20 minutes. Syngeneic target splenocytes were isolated from C57BL/6 mice and were stained at 0.2 µM CFSE (CFSE^low). Allogeneic target splenocytes were isolated from FVB/N mice and were stained at 2.0 µM CFSE (CFSE^high). Stained cells were then washed and incubated in 10 ml of prewarmed DMEM containing 10% FBS for 30 min at 37°C. Hepatocyte recipient mice and control naive mice received 20 x 10⁶ CFSE-labeled syngeneic target splenocytes and 20 x 10⁶ CFSE-labeled allogeneic target splenocytes by tail vein injection mixed in a 1:1 ratio. Splenocyte target CFSE staining and ratios were verified by flow cytometric analysis prior to injection. Spleens from hepatocyte transplant recipients were harvested 18 h after CFSE-labeled target cell injection and were analyzed by flow cytometry with gating on CFSE-positive splenocytes. The percentage of cytotoxicity was calculated as:

(1)

In this formula, no. CFSE^high represents the number of allogeneic target cells and no. CFSE^low represents the number of syngeneic target cells recovered from either naive or experimental (anti-LFA-1 and anti-CD154-treated) mice.

Delayed-type hypersensitivity (DTH) assay

DTH responses were measured as footpad swelling after injection of allogeneic splenocytes or control syngeneic splenocytes into the murine footpad, as previously described (1, 33). Naive CD4 KO mice served as controls for non-Ag-specific swelling and C57BL/6 mice sensitized to FVB/N Ag by three s.c. injections of FVB/N splenocytes were used to measure the efficacy of the Ag preparations. Cellular Ag was prepared by lysis of RBCs (0.017 M Tris and 0.75% NH₄Cl (pH 7.4)) of a FVB/N or C57BL/6 splenocyte preparation followed by two washes with medium and resuspension in 1x HBSS at a concentration of 10 x 10⁶ FVB/N splenocytes in 25 µl. Splenocytes were irradiated at 2000 rad in preparation for footpad injection. Hepatocyte recipients and control mice were injected with allogeneic (FVB/N) splenocytes in the left footpad and syngeneic control (C57BL/6) splenocytes in the right footpad. Subsequent footpad swelling was measured using a micrometer (0.001 inch) 24 h after antigenic challenge.

Preparation of cell subsets for adoptive transfer

CD8⁺ T cell subsets were purified from splenocytes of C57BL/6 (all H-2^b) mice (4). C57BL/6 splenocyte subsets were enriched by negative selection using the Murine T Cell CD8⁺ Subset Column kit (R&D Systems). Purity of cell subsets was determined by flow cytometric analysis with mAbs specific for CD4 (RM4-5; BD Pharmingen) or CD8 (53-6.7; BD Pharmingen). The CD8⁺ T cell-enriched population consisted of 80–88% CD8⁺ T cells and undetectable CD4⁺ T cells. Cell counts and viability were assessed by trypan blue exclusion. A total of 2 x 10⁶ CD8⁺ T cells were adoptively transferred by tail vein injection into hepatocyte recipients with long-term allograft survival and stable serum hA1AT. Prior dose-response studies indicate that adoptive transfer of 2 x 10⁶ naive CD8⁺ T cells into serum Ig-negative SCID recipient mice results in hepatocyte rejection within 14 days (4).

Immunohistochemistry

Tissue sections from hepatocyte transplant recipients were stained for the presence of the endogenous reporter product, hA1AT, and for CD3⁺-graft infiltrating cells. Immunostaining for hA1AT was performed on frozen tissue or formalin-fixed paraffin-embedded tissue using rabbit anti-human A1AT mAb (Sigma-Aldrich) as primary Ab and the Rabbit DAKO EnVision⁺ System, HRP (diaminobenzidine (DAB)) (DAKO) for detection, as previously described (3). Immunostaining for detection of CD3⁺ graft-infiltrating cells was performed on formalin-fixed paraffin-embedded tissue sections following blocking for endogenous peroxidase with 3% H₂O₂ and Ag retrieval with Target Retrieval Solution (pH 6.0; DAKO). Tissue was blocked with 10% goat serum and stained with the DAKO Autostainer using rabbit anti-human CD3 mAb (1/150, 30 min; DAKO) as primary Ab and biotinylated goat anti-rabbit linking Ab (20 min; Vector Laboratories) as detection Ab, as previously described (4). Ab binding was detected with HRP-conjugated streptavidin (20 min; DAKO) and 3,3'-DAB as chromagen (DAKO). Immunostaining for CD3⁺ cells, rather than a specific stain for CD8⁺ cells, was used due to interference of the rat anti-LFA-1 mAb treatment Ab with detection of the rat anti-CD8 mAb primary Ab binding available for immunohistochemistry. All slides were counterstained with Richard Allen hematoxylin, dehydrated through increasing alcohol concentrations, and coverslipped. PBS was used in place of primary Ab on experimental tissue to control for nonspecific binding of secondary Ab. All positive and negative control tissues stained appropriately. Consecutive frozen and paraffin-embedded liver sections were stained using the standard H&E staining technique.

Statistical analysis

Graft survival between experimental groups was compared using Kaplan- Meier survival curves and log-rank statistics (SPSS version 11.5 for Windows). Other statistical calculations were performed using SPSS using the Student t test to analyze differences between experimental groups. A value of p < 0.05 was considered significant.

	Results

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Short-term immunotherapy targeting LFA-1 and CD40/CD154 costimulation promotes long-term hepatocyte survival beyond persistence of therapeutic agents

In previous studies using a functional hepatocellular transplantation model, it has been demonstrated that combined blockade of CD40/CD154 and LFA-1-mediated signals effectively suppresses (CD4-independent) CD8-initiated rejection responses to allogeneic hepatocytes (3). Treatment with anti-CD154 mAb (MR1, days 0, 2, 4, 7) and anti-LFA-1 mAb (days 0–6) results in long-term (90 day) survival in 85% of CD4 KO (H-2^b) mice transplanted with fully allogeneic FVB/N (H-2^q) hepatocytes (n = 13). The function of surviving hepatocytes is reflected by the detection of the transgenic reporter product, hA1AT, in recipient sera. Recipients demonstrating stable serum hA1AT levels to day 60 posttransplant generally demonstrated long-term hepatocyte allograft survival to >90 days posttransplant (Fig. 1A). For reference, all control untreated allogeneic hepatocyte rejector CD4 KO mice lose detectable serum hA1AT by 14 days posttransplant (Fig. 1B) (1).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Long-term hepatocellular allograft survival beyond clearance of rat IgG in recipients receiving short-term treatment with anti-LFA-1 mAb and anti-CD154 mAbs. Allogeneic FVB/N (H-2q) hepatocytes were transplanted into CD4 KO (H-2b) hosts and were treated with anti-LFA-1 mAb (M17/4.4.11.9, rat-anti-mouse IgG, 0.3 mg, days 0–6) and anti-CD154 mAb (MR1, hamster anti-mouse CD154 IgG, 1.0 mg, days 0, 2, 4, 7). Control hepatocellular allograft recipients received no treatment. A, CD4 KO mice treated with anti-LFA-1 and anti-CD154 mAbs accepted hepatocytes for >90 days in four of five recipients as reflected by the detection of the reporter product, hA1AT, in recipient serum. Serum rat IgG (right y-axis) was serially assayed to determine circulating levels of the anti-LFA-1 mAb (indicated by solid circles with error bars); the treatment mAb was determined to have a half-life of 18.9 days by nonlinear regression analysis (indicated by dotted line and gray shaded area). The serum hA1AT levels (left y-axis) remained stable in treated hepatocyte recipients (indicated by the thick solid lines) 30 days after clearance of anti-LFA-1 mAb. B, Untreated control CD4 KO hepatocyte recipients demonstrated rapidly decreasing serum hA1AT and rejected hepatocytes by day 10 (n = 4, indicated by the thick solid lines). Rat IgG was not detected in serum of control untreated recipients following transplantation.

Immunotherapy targeting LFA-1 and CD40/CD154 costimulation does not interfere with leukocyte accumulation in the liver after hepatocyte transplantation

To determine the effect of targeting LFA-1 and CD40/CD154 costimulation on the accumulation of immune cells in the liver following hepatocyte transplantation, CD4 KO mice were transplanted with FVB/N hepatocytes and treated with anti-LFA-1 mAb and anti-CD154 mAb. Hepatocyte allograft recipients were sacrificed 10 days following transplantation, and viable LICs were isolated and counted by trypan blue exclusion. Ten days following transplantation, anti-LFA-1 mAb and anti-CD154 mAb-treated recipients exhibited a significant increase in total number of LICs in comparison to naive CD4 KO mice (p = 0.009, Fig. 2A); however, the number of cells infiltrating the liver of treated hepatocyte allograft recipients did not differ significantly from that of untreated CD4 KO rejector mice (p = ns; Fig. 2A). LICs remained slightly, but not significantly, elevated 60 days following allogeneic hepatocyte transplantation in treated hepatocyte allograft recipients (data not shown). Thus, the total increase in cellularity at the site of hepatocyte engraftment is not appreciably influenced by targeting LFA-1 and CD154.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Accumulation of innate and adaptive immune cells in the liver of hepatocyte transplant recipients treated with anti-LFA-1 and anti-CD154 mAbs. Allogeneic FVB/N (H-2q) hepatocytes were transplanted into CD4 KO (H-2b) hosts and either received treatment with anti-LFA-1 mAb (M17/4.4.11.9, 0.3 mg, days 0–6) and anti-CD154 mAb (MR1, 1.0 mg, days 0, 2, 4, 7) or were untreated. Naive CD4 KO mice served as negative controls. LICs were isolated 10 days following transplantation of allogeneic hepatocytes to the host liver and assayed for (A) total yield and (B) composition of innate and adaptive immune cells. LICs were counted by trypan blue exclusion to determine the yield of total leukocytes. A, The absolute number of recovered LICs was significantly greater in both treated (n = 3, p < 0.001) and untreated (n = 4, p = 0.007) hepatocyte recipients than in control naive CD4 KO mice (n = 4). B, Total yield of innate cell types (macrophages, polymorphonuclear neutrophils, NK cells) and adaptive cell types (CD4+ and CD8+ T cells) was determined by immunolabeling and flow cytometry of LICs. Significantly more CD8+ T cells (p = 0.01) and neutrophils (p = 0.03) were present in the liver of anti-LFA-1 mAb and anti-CD154 mAb-treated hepatocyte recipients than in untreated recipients which rejected hepatocellular allografts. Data is presented as mean ± SD. Solid circles within each bar graph represent individual mouse values.

Given the significant accumulation of LICs (especially CD8⁺ T cells) in treated allograft recipients, immunohistochemistry was performed to assess T cell distribution in the liver after hepatocyte transplantation. Liver sections were harvested from CD4 KO hepatocyte transplant recipients 7–10 days following transplantation. Recipients were either treated with anti-LFA-1 mAb and anti-CD154 mAb or received no treatment. H&E staining demonstrated occasional foci of leukocyte clusters and periportal infiltration in both treated recipients with ongoing hepatocellular allograft function and untreated rejector mice (Fig. 3, A and B). Immunohistochemical staining for reporter product hA1AT demonstrated the presence of numerous engrafted allogeneic hepatocytes scattered throughout the liver parenchyma and adjacent to both central veins and portal tracts in treated hepatocyte allograft recipients (Fig. 3, E and F). In contrast, only a few hA1AT-positive donor hepatocytes were detected in untreated recipients with decreasing serum hA1AT levels (Fig. 3H). Due to the cross-reactivity of the rat anti-LFA-1 mAb with goat anti-rat IgG mAb typically used for immunostaining of CD8⁺ T cells, paraffin-embedded liver tissue could only be stained for CD3⁺ T cells. Immunohistochemistry for CD3⁺ cells demonstrated that CD3⁺ T cells were scattered throughout the liver parenchyma with occasional foci of leukocyte clusters in both treated (Fig. 3, I and J) and untreated (Fig. 3, K and L) hepatocyte recipients. These data suggest that T cells infiltrate the liver of both untreated hepatocyte rejectors and treated recipients with sustained hepatocyte allograft survival. Up-regulation of the ₇ integrin, _E(CD103)₇, or CD103 by CD8⁺ T cells at the graft site has been closely linked to the ability of CD8⁺ T cells to mediate allograft damage (43, 44). Furthermore, endothelial cadherin (E-cadherin), the ligand for CD103, is expressed by both hepatocytes and bile duct epithelial cells in the normal liver (45). Therefore, we next evaluated CD103 expression on CD8⁺ LICs in untreated hepatocyte allograft rejector and treated hepatocyte allograft recipient mice.

View larger version (163K): [in this window] [in a new window]   FIGURE 3. Immunohistochemistry of liver tissue from hepatocyte allograft recipients treated with anti-LFA-1 and anti-CD154 mAbs. Liver tissue from hepatocyte allograft recipients was examined by light microscopy and immunohistochemical staining. CD4 KO (H-2b) recipients were transplanted with allogeneic FVB/N (H-2q) hepatocytes and were either treated with anti-LFA-1 and anti-CD154 mAbs or were untreated. Seven days following transplantation, recipients were sacrificed and the liver was formalin fixed and embedded in paraffin for (A–D) H&E staining and for immunohistochemical staining for (E–H) hA1AT-positive cells or (I–L) CD3-positive cells. This figure (original magnification, x200 for A, C, E, and I or x400 for all others) shows that 7 days after transplantation, focal areas of mononuclear cell infiltration (arrows) were present throughout the liver parenchyma in both (A) treated hepatocyte recipients and (D) untreated hepatocyte rejectors; generally, the liver had normal histology in both groups (B and C). E and F, Immunohistochemical staining demonstrated that hA1AT-positive hepatocytes were readily detected in the liver tissue of treated recipients (arrows) both adjacent to portal tracts (PT) and central veins (CV) as well as scattered throughout liver parenchyma. G and H, Few hA1AT-positive hepatocytes were detected in untreated hepatocyte rejectors. CD3+ T cells (arrows) were present in focal areas of infiltration, in periportal infiltrate, and scattered throughout the parenchyma in both (I and J) treated hepatocyte recipients and (K and L) untreated hepatocyte rejectors.

Flow cytometric analysis of LICs from anti-LFA-1 mAb and anti-CD154 mAb treated hepatocyte allograft recipients was performed to determine whether CD8⁺ T cells infiltrating the liver expressed CD103. LICs were gated on CD8⁺ T lymphocytes (Fig. 4A). We found that CD8⁺ T cells infiltrating the liver of treated allograft acceptor mice did not up-regulate CD103 expression when compared to CD8⁺ T cells in the liver of control naive CD4 KO mice. In contrast, approximately one-third of liver-infiltrating CD8⁺ T cells expressed CD103 at 3 days (data not shown) and 10 days posttransplant in untreated CD4 KO hepatocyte allograft recipients (Fig. 4B). Collectively, these data indicate that combined blockade of LFA-1 and CD40/CD154-mediated signaling does not affect the accumulation of CD8⁺ T cells at the site of transplantation, but does influence the induction of the adhesion molecule CD103 on host CD8⁺ T cells infiltrating the liver. We next examined the expression of T cell activation markers on CD8⁺ LICs.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Flow cytometric analysis of cell surface markers on CD8+ LICs in hepatocyte transplant recipients treated with anti-LFA-1 and anti-CD154 mAbs. CD4 KO (H-2b) mice were transplanted with allogeneic FVB/N (H-2q) hepatocytes and were either treated with anti-LFA-1 and anti-CD154 mAbs (n = 3, solid line) or untreated (n = 4, dotted line). LICs were isolated 10 days following transplantation. LICs from naive CD4 KO mice served as controls (n = 4, gray shaded area). A, Cells were gated on CD8+ T cells. B, Expression of CD103 on CD8+ LICs from treated hepatocyte recipients and naive CD4 KO mice was only detected on a small percentage of cells (p = ns). In contrast, CD103 expression was detected on a significantly increased percentage of CD8+ LICs in untreated hepatocyte rejectors (p = 0.01). C, CD8+ LICs from treated hepatocyte recipients and naive CD4 KO mice did not express CD25low. In contrast, expression of CD25low was detected on CD8+ LICs in untreated hepatocyte rejectors (p = 0.009). A CD8+CD25high cell population was detected only in treated hepatocyte recipients. Expression of CD69 on CD8+ LICs from treated hepatocyte recipients and from naive CD4 KO mice was minimal. In contrast, CD69 expression was detected on a significantly increased percentage of CD8+ LICs from untreated hepatocyte rejector mice (p = 0.003). Expression of CD154 was detected on CD8+ LICs from both treated hepatocyte recipients and untreated hepatocyte rejectors (p = ns) and both were significantly higher than CD154 expression on CD8+ LICs from control naive CD4 KO mice (p = 0.03). Representative histograms from at least three separate experiments are shown. All positive and negative control cells stained appropriately. *, A value of p < 0.05 in comparison to naive mice; , p < 0.05 between untreated rejectors and treated recipients.

To determine the effect of anti-LFA-1 mAb and anti-CD154 mAb immunotherapy on CD8⁺ T cell activation, the expression of T cell activation markers on hepatocyte recipient LICs was analyzed by flow cytometry. LICs were isolated from untreated or anti-LFA-1 mAb and anti-CD154 mAb-treated hepatocyte transplant recipient mice and CD8⁺ T cells were analyzed by flow cytometry for expression of CD25, CD69, and CD154 in comparison to naive CD4 KO control mice

Transplantation of allogeneic hepatocytes resulted in the up-regulation of CD69 expression on CD8⁺ LICs 10 days after transplant (p = 0.001). In contrast, CD69 expression was not up-regulated on day 10 CD8⁺ LICs from hepatocyte transplant recipients treated with anti-LFA-1 and anti-CD154 mAbs (Fig. 4C). Transplantation of allogeneic hepatocytes also induced the expression of CD25 (the IL-2R -chain) on host CD8⁺ LICs by day 10 posttransplant (p = 0.002 compared with CD8⁺ LICs in naive mice). These CD8⁺CD25^low LICs were not observed in treated hepatocyte transplant recipients. Interestingly, anti-LFA-1 mAb and anti-CD154 mAb treatment of hepatocyte transplant recipients resulted in the accumulation of a CD8⁺CD25^high population in LICs which was not present in the CD8⁺ LIC population from recipients which rejected hepatocytes or from CD8⁺ LICs of naive mice. Expression of CD25, as reflected by mean fluorescence intensity (MFI), on this cell population was 50 times higher than CD8⁺CD25⁺ cells in untreated hepatocyte rejectors (Fig. 4C).

Expression of CD154 was up-regulated on day 10 CD8⁺ LICs from both untreated and treated hepatocyte transplant recipients mice when compared to CD8⁺ LICs from naive mice (p = 0.02 and p = 0.03, respectively; Fig. 4C). Detection of CD154 on CD8⁺ LICs in treated mice was somewhat surprising because the anti-CD154 treatment mAb would be expected to prevent detection of CD8⁺CD154⁺ cells. The anti-CD154 mAb (MR1) treatment Ab is still in circulation at the time of LIC analysis (day 10), and the detection Ab is derived from the same cell line as the anti-CD154 mAb used for flow cytometry. However, it is possible that the enzymatic methods involved with LIC isolation disrupted binding of the treatment Ab to its target. Collectively, these data suggest that targeting LFA-1 and CD154 suppresses the up-regulation of CD69 and CD25^low expression, but not CD154 on CD8⁺ LICs after hepatocyte transplant. In addition, this immunotherapy appears to induce the development of a CD8⁺CD25^high lymphocyte cell subset, which is not detected in naive mice or recipients that have rejected hepatocyte transplants.

To determine whether immunotherapy targeting LFA-1 and anti-CD154 induced CD8⁺ T cell apoptosis, day 10 LICs isolated from treated and untreated hepatocyte recipients were analyzed for apoptosis by Annexin V staining and for cell viability by binding of the vital dye 7-AAD and flow cytometry. There was no difference in apoptosis of CD8⁺ or non-CD8⁺ LICs between treated hepatocyte acceptors and untreated hepatocyte rejectors. However, the degree of apoptosis of CD8⁺ LICs in treated and untreated hepatocyte transplant recipients was significantly greater than the baseline apoptosis detected in CD8⁺ liver leukocyte populations of naive mice (p = 0.04 for each group compared to control LICs from naive mice, data not shown). There was no detectable difference in the percentage of viable cells recovered in LICs from any of the three groups. Next, we examined the influence of combined treatment with anti-LFA-1 and anti-CD154 mAbs upon the in vivo development of cytolytic CD8⁺ T cell effectors.

Targeting LFA-1 and CD154 interferes with the in vivo development of CD8⁺ cytolytic effectors after hepatocyte transplant

To determine the cytotoxic potential of CD8⁺ T cells accumulating within the liver of CD4 KO hepatocyte transplant recipients, CD8⁺ LICs were assessed for the expression of the CD43 activation-associated glycoform (CD43). CD43 is known to be up-regulated on CD8⁺ effector T cells, and its expression closely correlates with the cytotoxic potential of CD8⁺ T cells (46). CD43 expression was significantly up-regulated on a large percentage of day 10 CD8⁺ LICs from untreated hepatocyte rejector mice in comparison to CD8⁺ LICs from naive CD4 KO mice (p = 0.02). In contrast, CD43 was not up-regulated on CD8⁺ LICs from anti-LFA-1 and anti-CD154 mAb treated hepatocyte transplant recipients (p = ns in comparison to naive CD4 KO, Fig. 5A). This suggested that short-term recipient treatment with anti-LFA-1 and anti-CD154 mAbs suppresses the development of CD8⁺ CTLs.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Analysis for development of CD8+ cytolytic T cells in anti-LFA-1 and anti-CD154 mAb treated hepatocyte recipients. CD4 KO (H-2b) mice were transplanted with allogeneic FVB/N (H-2q) hepatocytes and were either treated with anti-LFA-1 and anti-CD154 mAbs (n = 3, solid line) or remained untreated (n = 4, dotted line). LICs were isolated 10 days following transplantation. Cytotoxic potential was indirectly determined by expression of CD43 activation-associated glycoform on the surface of CD8+ LICs. A, Expression of CD43 activation-associated glycoform was detected on a small percentage of CD8+ LICs from treated hepatocyte recipients as well as from naive CD4 KO mice (p = ns). In contrast, CD43 expression was detected on a significantly larger percentage of CD8+ LICs from untreated hepatocyte rejector mice (p = 0.01). All positive and negative control cells stained appropriately. B, In vivo allospecific cytotoxicity was analyzed in anti-LFA-1 and anti-CD154 mAb treated CD4 KO recipients (n = 4) (after 60 days of stable hepatocyte function), in CD4 KO hepatocyte rejector mice (n = 4), or in control naive CD4 KO mice (n = 3). In vivo cytotoxicity was also analyzed in anti-LFA-1 and anti-CD154 mAb treated CD4 KO recipients depleted of CD8+ T cells (anti-CD8 mAb, days 60 and 62) followed by adoptive transfer (AT) of 2 x 106 naive CD8+ T cells (day 108, n = 3). Mice received 20 x 106 syngeneic CFSElow C57BL/6 target cells and 20 x 106 allogeneic CFSEhigh FVB/N target cells by tail vein injection. After 18 h, splenocytes from recipient mice as well as naive control mice were isolated and analyzed by flow cytometry for the presence of CFSElow (syngeneic) and CFSEhigh (allogeneic) target cells used to calculate percent allocytotoxicity. Representative histograms from at least three separate experiments are shown. Mean cytotoxicity was determined as the average over all experiments. Zero-percent allospecific cytotoxicity was recorded when the proportion of allogeneic target cells recovered from experimental mice was equal to or greater than the proportion recovered from concurrent control naive recipients. *, A value of p < 0.05 in comparison to naive mice.

Donor-reactive DTH responses are augmented after immunotherapy targeting LFA-1 of CD40/CD154 costimulation. It has been previously demonstrated that normal, untreated CD4 KO hepatocyte allograft recipients develop detectable DTH responses following hepatocyte allograft rejection (1) (Fig. 6). Anti-CD154 mAb and anti-LFA-1 mAb-treated CD4 KO mice which had accepted FVB/N hepatocyte allografts for >60 days were assayed for DTH. DTH was measured as footpad swelling in response to allogeneic FVB/N or syngeneic C57BL/6 cellular Ag. Combined treatment with anti-CD154 mAb and anti-LFA-1 mAb resulted in donor-reactive DTH responses that were significantly increased in comparison to DTH responses of untreated hepatocyte allograft rejector mice (p = 0.01, Fig. 6). DTH response to syngeneic Ag (control for alloantigen-independent inflammation) was similar between groups (p = ns, Fig. 6). Thus, combined blockade of CD40/CD154 costimulation and LFA-1-mediated signaling did not prevent the development of in vivo donor-reactive DTH responses. Rather, an augmentation of donor-reactive DTH responses was observed which apparently did not interfere with long-term hepatocellular allograft survival.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Donor-reactive DTH responses in anti-LFA-1 and anti-CD154 mAb treated hepatocyte allograft recipients. CD4 KO (H-2b) hosts were transplanted with allogeneic hepatocytes (FVB/N, H-2q) and were either treated with anti-LFA-1 mAb (300 µg, days 0–6) and anti-CD154 mAb (1 mg, days 0, 2, 4, 7) or were untreated. DTH was measured within 2 wk of allograft rejection or at 60 days following hepatocyte transplantation in anti-LFA-1 and anti-CD154 mAb treated recipients with long-term allograft survival. DTH was assayed by footpad swelling in response to challenge with irradiated allogeneic FVB/N splenocytes (allo) or syngeneic splenocytes (syn). For reference, the DTH responses of naive CD4 KO mice (Naive) are provided. The dotted line indicates the baseline DTH response of naive CD4 KO to non-alloantigen-specific inflammation. The bar graphs represent DTH responses (mean ± SD) detected in each group. Individual values for mice in each group are represented by solid circles superimposed on the corresponding bar graphs.

Hepatocyte transplant recipients with long-term hepatocyte allograft survival (LTS) 60 days (induced by short-term therapy with anti-LFA-1 and anti-CD154 mAbs) were adoptively transferred with 2 x 10⁶ naive CD8⁺ T cells, which in prior studies are sufficient to precipitate acute hepatocyte rejection in immunocompetent mice with induced long-term allograft survival >60 days (data not shown). Naive CD8⁺ T cell adoptive transfer was performed to determine whether CD8-dependent acute rejection by naive CD8⁺ T cells would occur in these recipients with ongoing hepatocellular allograft function. The choice of 60 days posttransplant was based on previous observations that anti-LFA-1 and anti-CD154 mAb-treated recipients which develop long-term hepatocyte survival to 60 days posttransplant will continue to have ongoing hepatocyte allograft survival >90 days. Furthermore, at 60 days posttransplant both anti-LFA-1 mAb and anti-CD154 mAb are cleared from recipient serum. Following CD8⁺ T cell transfer, seven of eight mice maintained hepatocyte allograft acceptance for at least an additional 60 days following adoptive transfer, though serum hA1AT decreased from baseline levels (Fig. 7). One recipient lost graft function 56 days following CD8⁺ T cell transfer. Thus, adoptive transfer of naive CD8⁺ T cells did not trigger acute hepatocyte rejection in these recipients with long-term hepatocellular allograft survival. This suggests that short-term therapy targeting LFA-1 and CD154 may induce a state of immune regulation preventing hepatocyte rejection by naive CD8⁺ T cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Adoptive transfer of naive CD8+ T cells into CD4 KO recipients with long-term hepatocellular allograft survival induced by short-term treatment with anti-LFA-1 and anti-CD154 mAbs. CD4 KO (H-2b) hosts were transplanted with allogeneic hepatocytes (hA1AT-FVB/N, H-2q) and were treated with anti-LFA-1 mAb (300 µg, days 0–6) and anti-CD154 mAb (1 mg, days 0, 2, 4, 7). A and C, After 60 days (n = 5, black lines) or 100 days (n = 3, grey lines) of stable hepatocyte function, five recipients with long-term function received adoptive transfer of 2 x 106 naive CD8+ T cells. Two mice died of causes unrelated to transplantation 30 days following CD8+ T cell adoptive transfer with functioning hepatocellular allografts. Five of the remaining six recipients had ongoing hepatocellular allograft function 60 days despite adoptive transfer of naive CD8+ T cells. (One recipient lost graft function 56 days following adoptive transfer). B and C, Another group of anti-LFA-1 and anti-CD154 mAb treated recipients with stable hepatocyte allograft function were depleted of CD8+ T cells 60 days following transplantation (anti-CD8 mAb i.p., days 60 and 62, n = 3). Hepatocytes continued to survive for an additional 48 days following CD8 depletion. At 108 days following transplantation, these recipients received adoptive transfer of 2 x 106 naive CD8+ T cells. Hepatocytes were rejected in all recipients with MST of 11 days (n = 3).

To determine whether recipient CD8⁺ T cells were required for the maintenance of long-term hepatocyte allograft survival, CD4 KO recipients treated with anti-LFA-1 and anti-CD154 mAbs were depleted of CD8⁺ T cells by treatment with anti-CD8 mAb on days 60 and 62 (n = 3). Following CD8⁺ T cell depletion, recipients had ongoing hepatocellular allograft survival as demonstrated by detectable serum hA1AT levels (Fig. 7B). After 48 days of persistent hepatocyte acceptance, CD8-depleted recipients were adoptively transferred with 2 x 10⁶ naive CD8⁺ T cells. Hepatocytes were rejected 11–25 days following adoptive cell transfer in all recipients (Fig. 7, B and C). Following hepatocyte rejection, recipients were tested for the presence of allospecific cytolytic T cell activity. Presence of allo-CTLs was detected in vivo in all hepatocyte rejectors, although, the degree of cytotoxicity ranged from 3 to 45% (Fig. 5B, bottom panel). The results of these experiments demonstrate that ongoing hepatocellular allograft acceptance in treated recipients appears to be dependent on the presence of CD8⁺ T cells. This is indicated by the observation that after depletion of recipient CD8⁺ T cells, hepatocellular allografts became vulnerable to rejection by adoptively transferred naive CD8⁺ T cells. These hepatocyte rejectors had demonstrable allospecific cytotoxicity, which is in contrast to nondepleted recipients with long-term hepatocellular allograft function that had no detectable allospecific cytotoxicity despite adoptive transfer of naive CD8⁺ T cells (Fig. 5B).

	Discussion

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Activation of the (CD4-independent) CD8-dependent pathway of allograft rejection elicits a vigorous immune response, which is highly resistant to immunoregulation (1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13). Despite the powerful effects of anti-CD154 mAb treatment for CD4-dependent rejection in other models (47, 48, 49, 50), anti-CD154 mAb treatment only transiently suppresses CD8-dependent hepatocyte rejection (6). However, combined blockade of LFA-1 and CD40/CD154 costimulation suppresses the CD8-dependent rejection pathway (in CD4 KO mice) such that long-term hepatocyte allograft survival is achieved in the majority of recipients in the absence of CD4⁺ T cells (3). This was surprising given that the induction of long-term allograft survival in a previous study using combined blockade of LFA-1 and CD40/CD154 costimulation required the presence of CD4⁺ T cells (23). The purpose of the current study was to examine potential mechanisms by which immunotherapy targeting LFA-1 and CD154 prevents CD8-dependent hepatocyte rejection and promotes long-term hepatocellular allograft survival.

LFA-1/ICAM-1 interactions are known to play several roles in the recruitment and activation of CD8⁺ T cells in response to inflammatory stimuli. Mice deficient in LFA-1 exhibit defects in both neutrophil and T cell trafficking to peripheral inflammatory sites (42). Thus, it was expected that blockade of LFA-1 interactions in combination with CD40/CD154 blockade would prevent the accumulation of both innate and adaptive immune effectors in the liver following transplantation. In contrast to anticipated results, the number of LICs was not altered by anti-LFA-1 mAb and anti-CD154 mAb treatment in hepatocyte allograft recipients in comparison to untreated recipients (Fig. 2A). In fact, accumulation of CD8⁺ T cells and neutrophils was actually increased in comparison to CD4 KO allograft rejector mice (Fig. 2B). This increased number of neutrophils and CD8⁺ LICs in treated recipients perhaps reflected a failure of these cells to egress from the inflammatory site. Histology demonstrated that CD3⁺ T cells (presumably CD8⁺ T cells) were scattered throughout the liver parenchyma including the vicinity of transplanted hepatocytes in both treated and untreated recipients (Fig. 3). Because the liver is known to contain an abundant and unique population of resident leukocytes, this study did not distinguish between trafficking of peripheral cells to the site of hepatocyte engraftment and expansion of resident leukocytes in response to hepatocyte transplantation. However, we favor the former interpretation based on studies in which a similar profile of CD8⁺ and innate immune cell subsets traffic to the kidney after transplantation of allogeneic hepatocytes to the kidney, which has few if any resident CD8⁺ T cells (our unpublished observation). Thus, it appears that treatment with anti-LFA-1 mAb, combined with anti-CD154 mAb, does not preclude the ability of CD8⁺ T cells (or other immune mediators) from accumulating at the site of hepatocyte engraftment, but does interfere with the ability of CD8⁺ T cells to initiate hepatocyte rejection.

CD103 is an integrin that has been shown to be up-regulated on graft-infiltrating cytolytic CD8⁺ T cells and to be necessary for graft rejection in some circumstances (43, 51). Here, it was observed that CD103 is highly expressed on CD8⁺ LICs in untreated hepatocyte rejector mice but not on CD8⁺ LICs of treated mice. Given the widespread infiltration of CD8⁺ T cells in the liver of anti-CD154 mAb and anti-LFA-1 mAb-treated hepatocyte transplant recipients, CD103 expression does not appear to be necessary for access to the liver. Instead, CD103 expression on CD8⁺ T cells may facilitate interaction with E-cadherin expressed on hepatocytes (52) to play a role in CD8⁺ T cell activation or effector function. In fact, CD103 interactions with E-cadherin have previously been observed to substitute for LFA-1/ICAM-1 interactions in providing costimulation for CD8⁺ T cell proliferation and cytolytic effector function (53). Combined blockade of CD40/CD154 costimulation and LFA-1-mediated signaling prevented up-regulation of CD103 expression on CD8⁺ LICs (Fig. 4B), which may have interfered with CD8⁺ T cell costimulation and/or effector function.

These studies addressed the potential influence of targeting LFA-1 and CD154 on CD8⁺ T cell activation in vivo because both molecules have been shown to be important in T cell activation. In vitro studies demonstrate that LFA-1/ICAM-1 interactions provide costimulation signals required for the activation of CD8⁺ T cells that is efficient in the absence of CD28/B7 costimulation (22). Although the role of CD40/CD154 costimulation in CD4⁺ T cell activation is well recognized, the role of CD154 in facilitating CD8⁺ T cell responses has received less attention. Nevertheless, we and others have reported that CD40/CD154 interactions play a critical role in CD8⁺ T cell-mediated immune responses in vivo in the absence of CD4⁺ T cells (6, 54, 55, 56). Thus, it was anticipated that this treatment strategy, which targets two molecules with known in vitro costimulatory functions for CD8⁺ T cells, would also suppress CD8⁺ T cell activation in vivo. We found that expression of CD69, the very early activation Ag which is known to be rapidly induced on T cells following TCR signaling (57, 58), was readily detected on CD8⁺ LICs from rejector mice. The combined treatment strategy suppressed CD8⁺ T cell activation as reflected by the failure to up-regulate CD69 expression in response to hepatocyte transplantation (Fig. 4C). To our knowledge, this is the first evidence demonstrating the in vivo suppression of alloreactive CD8⁺ T cell activation and cytotoxic function by concurrent targeting of LFA-1- and CD154-mediated signals.

To further evaluate the effect of combined immunotherapy on CD8⁺ T cell activation, we analyzed CD25 and CD154 expression on CD8⁺ LICs from treated and untreated hepatocyte transplant recipients. Induction of CD25 expression on CD8⁺ LICs was observed after hepatocyte transplant in 10% of CD8⁺ LICs in untreated recipients. This CD8⁺CD25^low population in day 10 LICs, which was detected in hepatocyte rejector mice, was not detected in LICs from treated hepatocyte transplant recipients in day 10 LICs. Instead, analysis of CD8⁺ LICs from treated hepatocyte acceptor mice revealed a CD8⁺CD25^high population, which expressed very high levels of the IL-2R -chain (CD25) in comparison to CD8⁺CD25^low LICs from hepatocyte rejector mice (Fig. 4C). CD4⁺CD25⁺ T regulatory cells are know to constitutively express very high levels of CD25 (59) and are capable of suppressing alloreactive T cell responses in vivo (60). Recently, a CD8⁺CD25⁺ T cell subset has been isolated from the human thymus. Similar to CD4⁺CD25⁺ T cells, this CD8⁺CD25⁺ T cell population expresses FoxP3, CTLA-4, and glucocorticoid-induced TNFR and has the ability to suppress proliferation of both CD4⁺CD25^– and CD8⁺CD25^– T cell responses in vitro (61). The functional significance of the CD8⁺CD25^high T cells present in the livers of CD4 KO hepatocyte allograft acceptors and whether they express FoxP3 and other markers, which can be associated with T regulatory cells, remain to be determined. However, these findings do raise the question of whether this cell population might represent the induction of a CD8⁺ regulatory cell population.

Day 10 CD8⁺ LICs from both untreated hepatocyte transplant recipients as well as recipients treated with anti-LFA-1 and anti-CD154 mAbs expressed CD154. Approximately 15% of CD8⁺ LICs from both untreated hepatocyte rejectors and treated hepatocyte recipients with intact hepatocellular allograft function were CD154⁺ (Fig. 4C). Thus, it appears that combined therapy with anti-LFA-1 mAb and anti-CD154 mAb does not prevent the up-regulation of CD154 on CD8⁺ T cells, but rather, the anti-CD154 mAb acts to block the costimulation of CD8⁺ T cells through CD154. The combined immunotherapy targeting of LFA-1 and CD154 differentially influences the up-regulation of CD69, CD25, and CD154 activation markers on CD8⁺ T cells induced by hepatocyte transplant.

LFA-1/ICAM-1 and CD40/CD154-mediated signals are also known to play important roles in cytolytic effector T cell development or function. Anti-CD154 mAb, either alone or in combination with anti-LFA-1 mAb, prevents the in vitro development of allospecific cytolytic activity (23). Anti-LFA-1 mAb can prevent CD8-dependent cytotoxic T cell development in the absence of CD40/CD154 costimulation (CD154 KO mice) (62). LFA-1/ICAM-1 interactions can also promote interactions between lymphocytes and hepatocytes which result in the release of soluble cytotoxic factors by activated T cells (63). In the current study, we found that combined targeting of LFA-1 and CD154 abrogated the development of allo-CTLs in vivo as reflected by the absence of CD43-activation-associated glycoform expression on day 10 CD8⁺ LICs (Fig. 5A) and by the absence of cytolysis of allospecific target cells in vivo (Fig. 5B) in treated hepatocyte recipients. The latter was observed at day 60 posttransplant when circulating anti-LFA-1 mAb and anti-CD154 were no longer present. Taken together, these results suggest that the treatment regimen suppressed the development of allo-CTLs rather than allo-CTL effector function. However, it is recognized that some immunotherapies (such as the combination of CD154, CD4, and CD8 nondepleting Abs or pretransplant donor lymphocyte infusion) induce tolerance to MHC-mismatched skin allografts mediated by T regulatory cells, which inhibit the effector function of allospecific CD8⁺ T cells (27, 28, 30). In addition to the role of LFA1–1 and CD40/CD154 on cell-mediated cytotoxicity, these molecules have been demonstrated to play a role in the development of DTH. LFA-1 KO mice exhibit defective DTH responses as a result of impaired T cell trafficking and Ag responsiveness (42). In some cases, the activation of DTH effector function has also been linked to interaction of CD154 on CD4⁺ T cells with CD40 on macrophages (64). Therefore, it was expected that a treatment regimen, which interfered with LFA-1 and CD154 would result in suppression of alloreactive DTH responses. Instead, we found that long-term hepatocyte acceptor mice exhibited strong alloreactive DTH responses, which were more prominent than DTH responses of untreated hepatocyte rejector mice (Fig. 6). These findings demonstrate that long-term hepatocellular allograft acceptance is not a function of T cell ignorance or deletion, as peripheral allospecific cell-mediated immune responses are detected in these recipients.

The dichotomy between the prominent donor-reactive DTH responses and the absence of in vivo allospecific cytotoxicity was somewhat unexpected. However, perhaps the dichotomy between DTH responses and cell-mediated cytotoxicity in treated mice should not be surprising. Although DTH is a proposed mechanism of CD4-dependent graft rejection (65), there are no studies which have definitively linked DTH as an effector mechanism of CD8-dependent graft rejection. In fact, it has previously been demonstrated that perforin-mediated cytotoxicity is not necessary for the development of CD8-mediated DTH responses in vivo (66). It is possible that different CD8⁺ T cell subsets mediate in vivo alloreactive DTH responses and in vivo allocytotoxicity and that only the latter correlates with CD8-dependent graft rejection.

The observation that immune acceptance of hepatocellular allografts induced by short-term treatment with anti-LFA-1 and anti-CD154 mAbs prevented hepatocyte rejection by adoptively transferred naive CD8⁺ T cells suggests that an active regulatory mechanism may exist. In prior studies, adoptive transfer of 2 x 10⁶ naive CD8⁺ T cells into Rag-1 KO hepatocellular allograft recipients was sufficient to initiate rejection of hepatocyte allografts with a median survival time (MST) of 17 days (4). Similarly, 2 x 10⁶ naive CD8⁺ T cells adoptively transferred into immunocompetent recipients induced to accept hepatocellular allografts long-term (60 days) with a different treatment strategy triggered complete loss of hepatocellular allograft function within 3–5 wk after adoptive transfer (our unpublished observation). In the current studies, the failure of adoptively transferred naive CD8⁺ T cells (and presumably additional new CD8⁺ thymic emigrants) to initiate rejection of established hepatocellular allografts was observed at a time point after clearance of the immunotherapeutic agents administered during the first week after transplant. It is possible that induction of long-term hepatocellular allograft acceptance could be mediated by the activity of TCR⁺CD4^–CD8 ^– T regulatory cells as has been described after skin transplantation and treatment with donor specific transfusion (30). However, our studies support a role for recipient CD8⁺ T cells in the maintenance of long-term hepatocellular allograft acceptance because depletion of recipient CD8⁺ T cells resulted in the deterioration of hepatocellular allograft function and the subsequent vulnerability of hepatocellular allografts to immune damage by adoptive transfer of naive CD8⁺ T cells (Fig. 7).

Thus, the suppression of (CD4-independent) CD8-mediated hepatocyte rejection by short-term combined therapy with anti-CD154 mAb and anti-LFA-1 mAb is associated with the early suppression of CD8⁺ T cell activation and maturation of CD8⁺ T cells into allo-CTLs. It does not result from persistence of treatment Ab, T cell apoptosis, T cell ignorance, or T cell deletion. The failure to precipitate rejection following naive CD8⁺ T cell transfer into long-term hepatocyte allograft acceptor mice and the presence of a subset of CD8⁺ LICs in these mice with a regulatory phenotype (CD8⁺CD25^high) suggest that that this immunotherapeutic strategy may induce the development of a CD8⁺ regulatory cell population. However, further studies will be required to explore this possibility.

	Acknowledgments

 
We thank Brendon L. Fussnecker, Jamie K. Mack, Jordan R. Stern, and Zeeshan A. Qureshi for technical assistance as well as Tina McKeegan and Susie Jones for expert assistance with immunohistochemistry. In addition, we thank Dr. Wendy Frankel (Department of Pathology, The Ohio State University Medical Center) for review of liver tissue histology.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was presented in part at the 54th Annual Meeting of the American Association for the Study of Liver Disease, October 24–28, 2003, Boston, MA; the 2005 American Transplant Congress, May 21–25, 2005, Seattle, WA; and the 66th Annual Meeting of the Society of University Surgeon, February 9–12, 2005, Nashville, TN.

² This work was supported in part by grants from the American Digestive Health Foundation/American Gastroenterological Association, the Roche Organ Transplantation Research Foundation, the American Society of Transplant Surgeons Wyeth Mid-Level Faculty Research Award, the W. M. Keck Genetics Research Facility of the NeuroBiotechnology Center, and by National Institutes of Health Grant R01-DK52920.

³ Address correspondence and reprint requests to Dr. Ginny L. Bumgardner, Department of Surgery, Division of Transplant, The Ohio State University Medical Center, 1654 Upham Drive, 373 Means Hall, Columbus, OH 43210-1250. E-mail address: bumgardner-1{at}medctr.osu.edu

⁴ Abbreviations used in this paper: KO, knockout; hA1AT, human -1-antitrypsin; LIC, liver-infiltrating cell; 7-AAD, 7-aminoactinomycin D; DTH, delayed-type hypersensitivity; E-cadherin, endothelial cadherin; allo-CTL, allospecific CTL; MST, median survival time.

Received for publication March 14, 2005. Accepted for publication September 29, 2005.

	References

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