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Originally published online as doi:10.1189/jlb.0205078 on July 6, 2005

Published online before print July 6, 2005
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(Journal of Leukocyte Biology. 2005;78:595-604.)
© 2005 by Society for Leukocyte Biology

Recombinant dimeric MHC antigens protect cardiac allografts from rejection and visualize alloreactive T cells

Ari Fried*, Martina Berg*, Bhavna Sharma{dagger}, Sabrina Bonde* and Nicholas Zavazava*,{ddagger},1

* University of Iowa Hospitals and Clinics & VAMC Iowa City, Department of Internal Medicine;
{dagger} Gemini Sciences Inc., San Diego, California; and
{ddagger} Immunology Graduate Program, University of Iowa, Iowa City

1Correspondence: University of Iowa Hospitals and Clinics, C51-F, Department of Internal Medicine, 200 Hawkins Dr., Iowa City, IA 52242. E-mail: nicholas-zavazava{at}uiowa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monomeric and dimeric soluble major histocompatibility complex (MHC) molecules down-regulate activated T cells in an antigen-specific manner in vitro. This property could be exploited to modulate alloresponses in vivo but has remained difficult to demonstrate. Here, intraperitoneal infusion of a Lewis-derived rat MHC class I molecule, RT1.Al-Fc, in Dark Agouti (RT1.Aa) recipient rats prolonged cardiac graft survival, which led to permanent engraftment. This effect was mediated by T cell impairment of target cell lysis by CD8+ T cells and down-regulation of interferon-{gamma} production by CD4+ T cells. The binding of the dimeric MHC allowed ex vivo visualization of alloreactive T cells in peripheral blood, splenocytes, and allografts, revealing low frequency of alloreactive CD8+ T cells after establishment of permanent engraftment of cardiac allografts. Thus, these data show the potential of dimeric MHC molecules to promote graft survival and allow visualization of alloreactive T cells.

Key Words: transplantation • tolerance • soluble MHC • visualization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane-bound major histocompatibility complex (MHC) molecules present antigenic or endogenous peptides to T cells, leading to stimulation if costimulation is provided [1 , 2 ]. In transplantation, donor-derived, so-called "passenger leukocytes", within the graft as well as host-derived antigen-presenting cells (APCs), present alloantigen to recipient T cells leading to direct and indirect alloantigen recognition, respectively [3 ]. Little is, however, known about the immunomodulatory role of soluble MHC (sMHC) antigens physiologically shed into the circulation in vivo. We and others have characterized the biochemistry of serum-derived sMHC and shown quantitative differences between individuals of different human leukocyte antigen (HLA) haplotypes [4 5 6 7 ]. For example, HLA-A24- and HLA-B15-positive individuals constitutively express three to four times more sMHC than other individuals [6 , 7 ]. However, during inflammation, such as organ rejection episodes, sMHC class I levels are highly elevated, presumably in response to proinflammatory cytokines [8 ]. These data raise the possibility of an in vivo role of sMHC as immunoregulatory agents.

There is, however, limited data in vivo on immunomodulation of T cell-mediated immune responses by sMHC. In preliminary studies, we showed that monomeric MHC class I antigens prolong graft survival in only ~50% of the transplanted allografts [9 ]. Others specifically inhibited rejection of skin grafts using a transgenic mouse model, where the recipient mouse secreted a monomeric, truncated form of the donor-derived MHC class I/peptide molecule [10 ]. Similarly, antigen-specific inhibition of an alloreactive T cell receptor-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor [11 ], indicating that MHC molecules are powerful immunomodulatory reagents. More recently, Casares et al. [12 ] have demonstrated that a class II dimeric molecule prevented the onset of disease and restored normoglycemia to animals already diabetic [12 ]. The antidiabetogenic effects of the MHC class II dimer were shown to rely on the induction of anergy in splenic, autoreactive CD4+ T cells via alteration of early T cell receptor signaling and stimulation of interleukin 10 (IL-10)-secreting T regulatory type 1 cells in the pancreas. Therefore, there are promising data that dimeric MHC/peptide complexes can be effective tools for immunomodulation. Here, we developed a truncated MHC class I dimer of the Lewis rat strain and showed in vivo that it induces graft tolerance to donor-type cardiac allografts in Dark Agouti (DA) recipients and allowed quantification of alloreactive T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and rats
The antibodies anti-CD8{alpha}, anti-CD4, anti-CD80, anti-CD86, OX18 (anti-RT1.A, polymorphic), anti-rat immunoglobulin G2c(IgG2c), and anti-mouse IgG alkaline phosphatase-conjugated antibody were purchased from BD PharMingen (San Diego, CA). The peroxidase-conjugated rabbit anti-mouse IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The rat IgG2c control Ig was obtained from Serotec (Oxford, UK). The anti-FLAG antibody was purchased from Sigma Chemical Co. (St. Louis, MO). Eight- to 12-week-old rats were purchased from Jackson Laboratory (Bar Harbor, ME). The DA RT1.Aa, Lewis RT1.Al, and Brown Norway (BN) RT1.Au rats were purchased and maintained at the VA Medical Center Iowa City Animal Care Center (IA).

Construction and expression of dimeric RT1.Al -Fc
The full-length cDNA of RT1.Al was kindly provided by Dr. T. J. Gill III and Dr. S. K. Salgar (University of Pittsburgh, Pittsburgh, PA, GenBank-Accession No.: L26224). The first four exons of RT1.Al were amplified using polymerase chain reaction (PCR). The full-length construct served as a template. During amplification, a SmaI restriction site was attached to the 5'-end, and a XbaI restriction site was added at the 3'-end. Generated PCR fragments were ligated into the SmaI/XbaI-linearized eukaryont expression vector pRK5 (BD PharMingen). In addition, DNA encoding for the hinge-, C2-, and C3-region of a rat IgG2c molecule (GenBank-Accession No: X07189) was amplified from spleen cDNA, and the resulting PCR fragment [292–990 base pairs (bp)] was ligated into the TA-cloning site of the pCR2.1 vector (Invitrogen, Carlsbad, CA). During amplification, an XbaI restriction site was attached to the 5'-end, and a HindIII restriction site was added at the 3'-end. Subsequently, the XbaI/HindIII IgG fragment was subcloned into the RT1.Al/pRK5 vector, and DNA encoding for a FLAG tag was added by PCR at the 3'-end. The resulting SmaI/HindIII DNA fragment (see Fig. 1 , 1638 bp) was thereafter subcloned into the expression vector pcDNA3.1– (Invitrogen) and was used for generating stable transfectants expressing recombinant dimeric RT1.Al molecules (RT1.Al). Each primer set was designed with regard to the full-length sequences published in GenBank and synthesized by Life Technologies (Berlin, FRG). The generated rsRT1.Al and IgG2c sequences were confirmed by DNA sequencing using the ABI373 DNA sequencer (Perkin-Elmer, Norwalk, CT). The rat myeloma cell line Y3-Ag 1.2.3 (American Type Culture Collection, Mansassas, VA) was chosen for recombinant expression of dimeric RT1.Al-Fc, as it does not express an IgG heavy chain but ß2-microglobulin. Y3-Ag cells were transfected using effectene (Qiagen, Hilden, FRG) according to the manufacturer’s instructions.



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  		Figure 1. Schematic structure and characterization of the dimeric RT1.Al-Fc. (a) Schematic diagram of the dimer, which was made up of a FLAG tag and the Fc region of the rat IgG2c fragment fused through the hinge region to the MHC class I molecule. (b) The dimer was precipitated with the OX18 mAb (anti-RT1.A heavy chain, lane 1) and an anti-ß2 microglobulin polyclonal serum (lane 6). Lanes 2 and 5 represent the vector controls, and lanes 3 and 4 represent the precipitates with the respective IgG isotype controls. (c) To determine the molecular size of the dimer, gel filtration of the supernatant was performed. Two main peaks of 66 kD and 200 kD were observed. The dimeric MHC molecule used in these experiments was detected in the 200-kD peak, confirming that the molecule was indeed dimeric and had the expected molecular size. (d) A single band of ~67 kD representing the single RT1.Al-Fc single chain was observed in the purified sample (lane 1). Lanes 2 and 3 represent the control supernatants of the wild-type cells and that of Lewis-derived splenocytes, respectively. MW, Molecular weight.

 
Quantitative enzyme-linked immunosorbent assay (ELISA)
A monoclonal rat anti-IgG2c (BD PharMingen) antibody was used as a catching antibody for the rat Ig used as a standard protein, whereas an anti-FLAG monoclonal antibody (mAb; Sigma Chemical Co.) was used as a catching antibody for the dimer, which was quantitated using standard ELISA procedure.

Immunoprecipitation and Western blotting of recombinant RT1.Al-Fc
Culture supernatant (1 mL) of transfected cells grown in serum-free medium was incubated with shaking at 4°C with 2 µg polyclonal anti-ß2 microglobulin serum (Dako, Danemark) or 2 µg OX18, a mAb against a conformational-dependent epitope on heavy chains of rat MHC class I molecules. After 4 h, 50 µl protein-A agarose (Pharmacia, Sweden) was added to each sample, and the mixture was shaken overnight at 4°C. Samples were washed thoroughly in phosphate-buffered saline and centrifuged. The pellets were resuspended in 100 µl sodium dodecyl sulfate-loading buffer, boiled, and centrifuged. The recovered supernantant was run on a 7.5% polyacrylamide gel and blotted on a nitrocellulose membrane. The proteins were stained using an anti-FLAG mAb. Protein bands were visualized by an anti-mouse IgG alkaline phosphatase-conjugated antibody.

Transplantation of cardiac allografts
To test the efficacy of the soluble dimer to promote graft survival, DA recipient animals received 10 µg of the dimer by intraperitoneal infusion daily for 14 days. They were transplanted with a cardiac allograft on the first day according to Ono and Lindsey [13 ] and left untreated (n=6) or received a single dose of 15 mg/kg cyclosporine A (CsA; n=12). Graft function was monitored by palpation. Lewis-derived cardiac allografts were transplanted to DA recipients as we previously reported [14 ]. In this strain combination, cardiac allografts are acutely rejected on Day 7.

Immunohistochemical and flow cytometric staining
Cryostat sections recovered on Day 5 (rejecting animals) or on Day 150 (tolerant animals) were fixed in methanol, washed in Tris-HCl (pH 7.6), and incubated with the dimer and a mouse anti-FLAG alkalkaline phosphatase-conjugated mAb. Sections stained for histology were stained according to standard procedure using hemotoxylin eosin staining. For the detection of alloreactive T cells by flow cytometry, 25 µl peripheral blood was incubated with a lysis solution containing ammonium chloride to remove the erythrocytes. The peripheral blood lymphocytes or splenocytes were incubated with the dimer for 30 min on ice. The cells were washed and then incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-FLAG mAb. For CD80 and CD86 staining, peritoneal macrophages, splenocytes, or lymph node-derived cells were incubated for 30 min at 4°C with FITC-conjugated anti-CD80 or anti-CD86 mAb. The cells were then washed, and cell fluorescence was measured by a FACScan (BD PharMingen).

Proliferation and cytotoxicity assays
Here, we tested the efficacy of the soluble dimer to modulate alloresponses in vivo. Thus, DA recipient animals were immunized daily from Day 0 to Day 14 with the dimeric MHC antigen (10 µg) and Lewis-derived splenocytes (107 Lewis-derived splenocytes were infused on Day 0 and Day 7), dimer only, and splenocytes only or were left untreated (control). Animals were killed on Day 14, spleens harvested, and splenocytes used in a standard [3H]thymidine proliferation assay (Lewis and BN splenocytes were used as stimulator cells).

DA responder splenocytes from the various animal groups were also used to generate CD8+ T cells for cytotoxicity assays. DA responder splenocytes were restimulated in vitro with irradiated Lewis-derived splenocytes at a 1:1 ratio for 5 days. CD8+ T cells were isolated from bulk cultures with magnetic beads (Miltenyi Biotec, Auburn, CA) and used in a 4-h 51chromium release assay with Lewis-concanavalin A (Lew-Con A) blasts as target cells.

The inhibition assay of alloreactive T cells was performed as described previously in this lab [15 16 17 ]. Briefly, DA anti-Lewis, DA anti-BN, or Lewis anti-DA CD8+ T cells were incubated with the dimer, and their cytotoxicity against Con A target cells (Lewis, BN, and DA Con A blasts, respectively) was tested in a 4-h 51chromium release assay.

Measurement of interferon-{gamma} (IFN-{gamma}) production by enzyme-linked immunospot (ELISPOT)
To measure the secretion of IFN-{gamma} in the treated groups of animals, splenocytes were derived from animals sensitized with splenocytes only and with splenocytes and treated with the dimer or from nontreated control animals. CD4+ T cells were separated using magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. CD4+ T cells were used as responder cells to irradiated Lewis-derived splenocytes. IFN-{gamma} secretion was measured by the use of an IFN-{gamma} ELISPOT kit (BD PharMingen). Spots were counted using an automated ELISPOT reader system (Immunospot, Cellular Technology Ltd., Cleveland, OH).

Peritoneal macrophage assays
APCs were obtained by adherence separation from splenocytes, lymphn odes, or peritoneal macrophages of DA recipient animals. Separated APCs were pulsed with the indicated amount of dimer and used as stimulator cells for DA-derived CD4+ T cells in a standard [3H]thymidine proliferation assay.

To track peritoneal macrophages in vivo, DA-derived peritoneal macrophages were pulsed with the dimer for 24 h and labeled afterward with carboxyfluorescein diacetate succinimidyl ester (CFSE; Vybrant carboxyfluorescein diacetate-SE cell tracer kit, Molecular Probes, Eugene, OR), according to the manufacturer’s instructions. Cells were washed extensively and reinfused into the peritoneum of the same DA animals. After 24 h, the animals were killed, and green fluorescent cells were detected in the peritoneum, peripheral blood, spleens, and lymph nodes by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of dimeric RT1.Al-Fc
Transfected Y3-Ag cells secreted the dimer in serum-free medium as determined by a newly established conformational-dependent, quantitative ELISA. Transfectants were cloned by limiting dilution, and clones secreting >15 µg/ml of the dimer were further maintained in culture. To determine the molecular size of this molecule, the dimer was immunoprecipitated using the OX18 mAb and the anti-ß2-microglobulin serum. A major band of 67 kD was detected in samples precipitated with the OX18 or with the anti-ß2-microglobulin antibody (Fig. 1b ). In supernatants derived from some clones, an additional minor band, slightly less than the major 67-kD band, was detected. This protein was precipitated by both antibodies, suggesting that it is a spliced product of the full-length, dimeric MHC. Thus, the OX18 mAb (anti-RT1.A conformationally folded heavy chain) and the anti-ß2 microglobulin polyclonal serum were able to recover the recombinant, dimeric MHC class I molecule, indicating that the recombinant class I dimer was properly folded and heterodimeric, containing the class I heavy chain and the ß2-microglobulin. To confirm that the recombinant protein was dimeric, the dimer was purified by affinity chromatography using a protein A column and run on a G200 gel-filtration column. As the transfected cells were grown in serum-free medium, only two major protein peaks of 66 and 200 kD were observed. As tested by ELISA, most of the recombinant material was eluted in the 200-kD fraction (Fig. 1c) , which matches the predicted 170-kD protein size of the dimer. The purified dimer was run on Western blots after gel filtration and detected by the anti-FLAG antibody. As expected, a single band of 67 kD representing the single chain of the dimer was detected (Fig. 1d) .

Dimeric RT1.Al-Fc induces indefinite graft survival of allogeneic cardiac allografts
DA recipient animals were infused daily with 10 µg RT1.Al-Fc for 14 days. This protocol was a modification of our previous protocol using monomeric sMHC [9 ]. Together with the first dimer infusion, recipient animals were transplanted Lewis-derived heterotopic cardiac transplants and left untreated or received a single dose of 15 mg/kg CsA. Treatment of recipient animals with this dose of CsA alone prolonged graft survival from 7 days to 14 days (Fig. 2a ). It is surprising that the dimeric MHC antigen equally prolonged graft survival, 8.5 ± 0.1–13.2 ± 0.4 days (P value <0.0001; n=10), demonstrating the efficacy of these molecules to abrogate graft rejection. In contrast, nine out of a total of 12 animals (75%) treated with the dimer and subtherapeutic CsA showed indefinite graft survival, >150 days post-transplantation (Fig. 2a) . Third-party BN-derived cardiac allografts were rejected by Day 9. These results are highly superior to our own published findings with synthetic MHC peptides or with monomeric sMHC antigens [9 , 18 ], where only 50% prolonged graft survival was achievable.



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  		Figure 2. Dimeric sMHC class I antigens induce permanent engraftment of donor-derived cardiac allografts. (a) DA recipient rats were transplanted Lewis-derived cardiac allografts and simultaneously treated with a single dose of 10 µg dimeric RT1.Al-Fc daily for 14 days. Animals that received an additional dose of CsA permanently accepted cardiac allografts (n=12). Third-party allografts were acutely rejected. The dimer and CsA used alone moderately prolonged graft survival. (b–d) Histological sections revealed normal architecture of tolerated allografts 7 and 150 days post-transplantation, whereas rejected allografts were heavily infiltrated by mononuclear cells showing significant architectural damage to the allografts.

 
However, prolonged graft survival is not always accompanied by lack of pathology in the allograft. For example, vascular occlusion as a consequence of chronic rejection has been reported in allografts, despite prolonged graft survival. Therefore, animals were killed 7 and 150 days post-transplantation, and multiple histological sections were stained and analyzed for signs of rejection. There was no graft infiltration characteristic of rejection and it is more important that there were no signs of intimal thickening as a sign of chronic rejection after treatment with the dimer and subtherapeutic CsA (Fig. 2b and 2c) . These data show that the dimer had an impact on the homing of T cells in this model. In contrast, acutely rejected allografts showed heavy infiltration of myocardial tissue by mononuclear cells (Fig. 2d) . Tissue damage was extensive, and the muscle architecture of the allografts was obliterated, clearly indicating severe acute rejection.

Treatment with dimeric RT1.Al-Fc modulates T cell responses to alloantigen
To understand the mechanism by which donor-derived dimeric MHC protected allografts from rejection, recipient animals were treated with the dimeric MHC antigen for 14 days as described above. Together with the first dimer infusion, animals were immunized with 10 x 106 donor-derived splenocytes. This infusion was repeated on Day 7, and the animals were killed on Day 14. Spleens were harvested and their response to alloantigen analyzed. First, T cells were used as responder cells in a 4-day mixed lymphocyte assay. Control groups were animals treated with splenocytes only, the dimer only, or untreated animals. As expected, CD8+ T cells recovered from animals sensitized with splenocytes only showed strong proliferation to donor-type alloantigen (Fig. 3a ). It is surprising that proliferation of T cells recovered from animals that were treated with the dimer in addition to the splenocytes was significantly abrogated (P<0.05, n=5). The other two control groups showed limited T cell proliferation. However, alloresponse to third-party responder animals of the BN strain remained unchanged in all groups.



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  		Figure 3. Treatment of recipient rats with dimeric MHC antigens abrogates T cell proliferation and cytotoxic T cell cytotoxicity. (a) To test the effect of dimer treatment on T cell response to alloantigen presented by direct presentation, CD8+ T cells from animals treated with donor splenocytes only, donor splenocytes and the dimer, the dimer only, or control, untreated animals were used in a 4-day mixed lymphocyte reaction assay as responder cells. Irradiated Lewis-derived splenocytes were used as stimulator cells. Treatment with dimeric MHC strongly abrogated the alloproliferative response of T cells in animals that were sensitized with donor splenocytes and the dimer, compared with T cells in animals sensitized with splenocytes only (P<0.05, n=5). Third-party BN-derived stimulator cells remained unchanged in all groups. Error bars represent standard deviations from triplicate wells. (b) CD8+ T cells were used as effector cells in a 4-h 51chromium release assay after restimulation of the cells from the various animal groups (as indicated) for 5 days in vitro using Lewis-derived splenocytes as stimulator cells. T cells derived from animals sensitized with splenocytes only showed strong target cell (Lew-Con A blasts) killing, whereas the cytotoxicity of cells recovered from animals treated with splenocytes and the dimer was drastically abrogated, indicating a direct influence of the dimer. As expected, CD8+ T cells derived from control animals or from animals treated with the dimer only showed low cytotoxicity. (c) To determine whether the soluble dimer abrogates cytotoxic T cells directly, DA anti-Lewis, DA anti-BN, or Lewis anti-DA cytolytic T lymphocyte (CTL) were incubated with the dimer and their cytotoxicity against Con A target cells (Lewis, BN, and DA Con A blasts, respectively), tested in a 4-h 51chromium release assay. Anti-Lewis CTL were inhibited in a concentration-dependent manner but not the DA anti-BN or the Lewis anti-DA CTL used as controls. (d) To determine the effect of the dimer treatment on CD4+ T cells, an ELISPOT was used for measuring the number of IFN-{gamma}-secreting CD4+ T cells. Clearly, dimer treatment led to a significant reduction in IFN-{gamma} secretion by the CD4+ T cells. Error bars represent standard deviations from triplicate wells.

 
To investigate the effect of the treatment protocol on cytotoxic T cells against the donor strain, splenocytes harvested from treated animals were restimulated in vitro using irradiated Lewis-derived donor cells for 5 days. CD8+ T cells were isolated by immunomagnetic beads and tested for their cytotoxicity against donor-derived Con A blast cells. The highest cytotoxicity was observed with cells derived from the animals sensitized with donor-type splenocytes only (Fig. 3b) . However, cytotoxicity was abrogated by pretreatment with the dimer, despite sensitization with donor splenocytes. Our previous experiments had shown that alloreactive T cells were blocked by monomeric sMHC and by their synthetic peptides [15 , 19 ]. Here, we tested whether the dimeric MHC class I molecule abrogated alloreactive CTL (Fig. 3c) . Indeed, DA-derived anti-Lewis CTL were blocked by the dimer in a concentration-dependent manner as determined in a 4-h 51chromium-release assay using Lew-Con A blasts as target cells. Syngeneic (Lewis-derived) anti-DA CTL or DA-derived anti-BN CTL were not affected by the dimer in their ability to recognize alloantigen on target cells (Fig. 3c) . These data suggest that the dimer could have a direct effect on alloreactive T cells in vivo, as alloreactive T cells directly isolated from rejecting animals were blocked by the soluble dimer. To determine the effect of the dimer on CD4+ alloreactive T cells, DA responder rats were sensitized with Lewis-derived splenocytes only or with the splenocytes and dimer over 14 days. The animals were killed, and CD4+ T cells were isolated and used to determine their ability to produce IFN-{gamma} after alloantigen stimulation. Indeed, the cells derived from animals treated with the dimer and splenocytes showed significant reduction in IFN-{gamma} production after alloantigen stimulation (Fig. 3d) , as compared with the cells derived from animals sensitized with the splenocytes only. These results altogether indicated that dimer treatment effectively modulated CD4+ and CD8+ T cells.

To explain these unexpected sMHC-induced effects, we hypothesized that peritoneal macrophages are involved in the presentation of the dimer, leading to down-regulation of alloreactive T cells. CD80 and CD86 were measured on the DA-derived peritoneal macrophages after a peritoneal lavage. Expression of CD80 and CD86 was surprisingly low on peritoneal macrophages compared with splenocytes and lymph node-derived cells (Fig. 4a ). Therefore, peritoneal macrophages were likely to provide weak costimulation, leading to T cell anergy rather than stimulation. Indeed, when the peritoneal macrophages were used as stimulator cells in a proliferation assay with the dimer used as alloantigen, they failed to activate alloreactive CD4+ T cells, whereas lymph node-derived as well as splenic APCs were capable of stimulating the T cells (Fig. 4b) . To determine whether peritoneal macrophages trafficked to peripheral lymphoid organs after picking up alloantigen, we harvested peritoneal macrophages and pulsed them with the dimer over 24 h ex vivo. The cells were washed and subsequently labeled with CFSE, reinfused into the peritoneum of DA recipient animals, and killed 24 h later to determine the presence of fluorescent green cells in the peritoneum, peripheral blood, spleens, and lymph nodes. About 1% of circulating leukocytes were CFSE-labeled compared with 12.9% in the peritoneal lavage (Fig. 4c) . Less than 0.5% of splenocytes or lymph node-derived cells was CFSE-positive. Although the percentage of CFSE-labeled macrophages in splenocytes or lymph node-derived cells appears to be small, the total cell numbers could be sufficient to interfere with alloantigen presentation to T cells. These results suggest that peritoneal macrophages trafficked into the circulation and into peripheral lymphoid organs after engagement with alloantigen, where they likely interacted with T cells.



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  		Figure 4. Peritoneal macrophages present dimeric sMHC molecules and fail to induce CD4+ T cell proliferation. (a) DA-derived peritoneal macrophages, splenocytes, and lymph node-derived cells were stained with anti-CD80 or anti-CD86 mAb. Peritoneal macrophages show the lowest expression of CD80 and CD86, compared with splenocytes and lymph node-derived cells, indicating that they can only provide weak costimulation, possibly leading to T cell anergy. (b) To determine the ability of peritoneal macrophages to present the dimeric sMHC molecules, APC derived by adherence separation from splenocytes, lymph nodes, or peritoneal macrophages of DA recipient animals were used to present the soluble dimer to DA-derived CD4+ T cells. APC derived from peritoneal macrophages failed to stimulate T cells. (c) To determine whether peritoneal macrophages traffic into the circulation after picking up alloantigen in the peritoneum, DA-derived peritoneal macrophages were pulsed with the dimer for 24 h ex vivo and reinfused into the same DA animals after labeling with CFSE. After another 24 h, the animals were killed, and green fluorescent cells were detected in the peritoneum and peripheral blood (12.9% and 1%, respectively). FSC, Forward scatter; SSC, side scatter.

 
Dimeric MHC molecules visualize alloreactive T cells
We further investigated whether alloreactive T cells can be visualized by immunohistochemical staining using the dimer. Only a few cells were stained by the dimer on the tolerated allograft (Fig. 5a ). As expected, parenchymal tissue of the tolerated allografts was intact. In contrast, rejected allografts showed a diffuse to dense distribution of mononuclear cells, most of which were positive for the dimer (Fig. 5b) . Previous data by our group [9 ] indicate that CD8+ cells in rejected allografts were 50–60% of the total mononuclear cell population detected by immunohistochemistry. Control sections from animals transplanted with BN third-party allografts showed negligible dimer staining (Fig. 5c) . Thus, these data indicated that the dimer can be used for staining of alloreactive T cells in allografts and that the frequency of alloreactive CD8+ cells was high within the rejected allografts.



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  		Figure 5. Dimeric RT1.Al-Fc visualizes intragraft and circulating alloreactive T cells. Immunohistochemical sections were incubated with the dimeric MHC molecule, and binding was visualized by red alkaline phosphatase staining (anti-FLAG-AP). Low staining of peripheral blood lymphocytes of tolerated allografts (a, original x200) and strong staining in rejected allografts (b, original x200) were observed. It is interesting that in some sections from tolerated allografts, such as in Figure 2 , we observed perivascular infiltrates, which, however, were not accompanied by any pathology, as the tissue was well maintained. Control staining from third-party BN allografts was negative for dimer staining (c, original x200). In peripheral blood of animals that rejected allografts, there were more dimer-staining CD8+ T cells (d) than in tolerated allografts (e). T lymphocytes derived from nontransplanted control animals showed no significant dimer binding (f).

 
To validate these findings, peripheral blood was drawn from DA rats rejecting Lewis-derived cardiac allografts on Day 7 post-transplantation, from tolerant animals (Day 60) or from nontransplanted, control animals. Full blood was double-stained with the dimer and a fluorescein-conjugated anti-CD8 antibody. Representative stains are shown in Figure 5d 5e 5f . Approximately 1.4 ± 1.1% (n=5) of the cells were double-positive for CD8 and the dimer on tolerated allografts (Fig. 5e) , as compared with 6.2 ± 1.2% (n=6) detected in peripheral blood of rejecting animals (Fig. 5d) . T lymphocytes derived from nontransplanted, control animals showed low dimer binding (Fig. 5f) . These data indicated that three to four times more alloreactive T cells were stained by the dimer in rejecting animals than in tolerant animals, suggesting that the dimer could be a novel tool for monitoring alloresponses using whole blood as a source of alloreactive T cells.

The MHC dimer detects a high frequency of alloreactive CD8+ T cells in splenocytes of rejecting animals
To further characterize alloreactive T cells using the dimeric MHC molecules, Lewis-derived cardiac allografts were transplanted into DA recipient animals and left untreated. The organs were rejected on Day 7, and the animals were killed for the recovery of spleens. CD8+ T cells were separated from splenocytes by immunomagnetic beads and stained with the dimer. Clearly, 14.6% CD8high of the splenocytes in rejecting animals were positive for the dimer compared with only 3.3% in tolerant animals (n=6), killed on Day 40 post-transplantation (Fig. 6a ). To further confirm the specificity of this stain, splenocytes recovered from DA animals rejecting DA allografts (syngeneic control) and from third-party controls (BN transplanted in Lewis) were used as controls (Fig. 6a) . In both control groups, only 2% of the cells were stained. As the dimer has an IgG backbone and can potentially bind to the FcR on T cells, we preincubated splenocytes from animals rejecting a Lewis allograft with antibodies against CD4, CD8, FcR, or CD28. After 1 h incubation, the cells were washed to remove excess antibody and then stained with the dimer to detect any changes in the amount of dimer binding. None of these antibodies interfered with the dimer staining, clearly showing that the stains obtained were not a result of the dimer binding to the cells via the FcR or any of the tested molecules (Fig. 6b) . Further, dimer-binding cells were isolated by immunomagnetic separation and used as responder cells in a proliferation assay. Control cells were CD8+ cells obtained from a control animal. Indeed, dimer-binding cells from a rejecting animal responded to alloantigen three to four times more strongly than nondimer-binding cells (data not shown), clearly confirming that the cells binding the dimer were alloreactive. Collectively, the data presented here further confirmed that the dimeric MHC molecule indeed visualizes alloreactive T cells in an allospecific manner.



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  		Figure 6. Acutely rejecting animals show a high frequency of dimer-binding cells in the spleen. Splenocytes of acutely rejecting and those of control animals were separated for CD8+ T cells by immunomagnetic beads and stained with the dimer (a). Cell fluorescence of dimer-binding cells was highest in acutely rejecting animals (15%) and significantly lower in spleens of tolerated allografts and in the syngeneic or third-party (BN to DA) graft controls (2–3%). Preincubation of isolated CD8+ T cells from rejecting animals with anti-Fc receptor (FcR), -CD4, or -CD8 or with anti-CD28 antibodies had no significant effect on dimer binding by the CD8+ T lymphocytes of rejecting animals as shown in the overlays (b).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is increasing recognition of the potential use of sMHC antigens in biomedicine, as tracers of antigen-specific T cells in the form of tetramers [20 ] but also as immunomodulators [19 ]. Data from this laboratory demonstrated that alloreactive T cells can be abrogated by MHC/peptide complexes [16 , 21 ]. Although the nature of T cell blockage was unclear, we subsequently reported that sMHC molecules modulate immune responses by inducing CD95L-mediated apoptosis in activated T cells, indicating that the process of T cell inhibition was irreversible and involved T cell death [19 ]. However, in naive T cells, sMHC/peptide complexes induce a state of anergy [22 ]. The role of sMHC antigens in vivo has, however, remained elusive. The data presented here provide insight in the potential application of sMHC in immune-regulation of allogeneic responses.

Animals treated with sMHC dimers showed prolonged graft survival but failed to prevent graft loss by acute rejection. However, when the dimer was supplemented with a subtherapeutic single dose of CsA, which does not prevent acute graft rejection on its own, graft-specific tolerance characterized by lack of mononuclear cell infiltration and lack of signs of chronic rejection was achieved. This effect was observed in the treated animals as early as Day 7 post-transplantation and was the same 150 days post-transplantation. This unexpected result was shown to be mediated by abrogated response of CD8+ to direct presentation of alloantigen and significant reduction of IFN-{gamma} production by CD4+ T cells. Our experiments indicated that peritoneal macrophages express low levels of costimulatory molecules and also the ability to activate T cells by presentation of soluble alloantigen. Therefore, the soluble dimer likely induced T cell nonresponsiveness, which favored engraftment of the cardiac allografts, revealing an interesting route for antigen delivery in the induction of transplantation tolerance. In this model, CsA appears to prevent the initial rejection process, allowing the dimer to induce T cell nonresponsiveness.

To study whether the dimeric MHC molecules could be used to track alloreactive T cells, the dimer was used to stain histological sections. In peripheral blood, 6.2 ± 1.2% (n=6) of blood lymphocytes were donor MHC-binding and CD8+. Assuming that only ~25% of peripheral blood lymphocytes are in fact CD8+, the number of alloreactive T cells detected by the dimeric MHC is relatively high. In contrast, only 1.4 ± 1.1% (n=5) CD8+ cells were detected in tolerant animals. The presence of the alloreactive T cells after induction of permanent engraftment of cardiac allografts indicated persistence of alloreactive T cells with no apparent reactivity toward the allograft. These data were strongly supported by the immunohistochemical stains, which indicated specific binding to T cells and significant differences between the tolerant and rejected allografts. Although the majority of recombinant MHC used in the literature is generated in Escherichia coli and loaded with desired specific peptides thereafter, here, we chose to use the expression of recombinant MHC in mammalian B cells. That way, the MHC molecules are peptide-loaded by endogenous peptides. As the majority of alloreactive CD8+ T cells is against peptide-loaded allo-MHC molecules [3 , 23 ], rather than against specific peptides, we anticipated that this strategy could be successful. Indeed, these dimeric MHC class I/peptide complexes modulated and visualized alloreactive T cells. Thus, expression of allo-MHC molecules in mammalian B cells gives the secreted MHC the complete repertoire of endogenous peptides, mimicking those presented by allogeneic organs. Analysis of peptides presented by secreted, recombinant sMHC in our lab revealed little difference between their peptides and those presented by membrane-bound MHC molecules [24 ]. However, we cannot rule out the possibility that the total number of alloreactive T cells may in fact be higher than those estimated here, as a small percentage of alloreactive T cells may be specific for Lewis-derived and tissue-specific allo-peptides not expressed in the Y3-Ag 1.2.3 cell line, which was used for transfecting the sMHC alloantigen. Nonetheless, the results still allow differentiation of tolerant from acute-rejecting animals. To definitively elucidate the role of the peptide, we are working on a mouse model of these experiments that will allow us to test MHC dimers loaded with different peptides, as in the rat model, hardly any peptides have been defined.

Most recently, Casares et al. [12 ] demonstrated in a mouse model of diabetes that administration of dimeric sMHC class II molecules loaded with the hemagglutinin peptide (110–120) to prediabetic, double-transgenic mice prevented the onset of the disease or in animals that were already diabetic, restored normoglycemia. This effect was found to be mediated by induction of T cell anergy via alteration of early T cell signaling and stimulation of IL-10-secreting T regulatory cells. Clearly, our data in a transplantation model using a chimeric MHC class I molecule agree with these published data. Here, the dimeric MHC class I antigens used abrogated the development of alloreactive T cells, as revealed by their inability to kill target cells (Fig. 3b) . Altogether, these data point to a therapeutic potential of dimeric MHC/peptide complexes. We previously published data on prolonged graft survival using synthetic MHC peptides or monomeric MHC molecules [9 , 15 , 25 ]. However, none of these strategies increased graft survival in more than 50% of the cases. Additionally, graft quality was poor, as there was evidence of chronic rejection, which was not seen here, showing that the dimer is a powerful and superior regulatory molecule in this transplantation model. Indeed, compared with published data with MHC class I- or class II-derived, synthetic peptides [25 26 27 28 ], the dimer used here is more effective in modulating alloresponses. Further, the ability to stain alloreactive CD8+ by MHC-oligomers using flow cytometry and immunhistology [29 ] might allow the establishment of new, diagnostic tools, in particular, for the detection of established tolerance.


   ACKNOWLEDGEMENTS
 
This publication was made possible by Grant Number NIH/NHLBI R01 HLO73015in addition to a VA Merit Review Award and a grant from the Deutsche Forschungsgemeinschaft (Germany). A. F. and M. B. contributed equally to this manuscript.

Received February 7, 2005; revised May 11, 2005; accepted May 12, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gill, R., Coulombe, M., Lafferty, K. (1996) Pancreatic islet allograft immunity and tolerance: the two-signal hypothesis revisited Immunol. Rev. 149,75-96[CrossRef][Medline]
  2. June, C., Bluestone, J., Nadler, L., Thompson, C. (1994) The B7 and CD28 receptor families Immunol. Today 15,321-331[CrossRef][Medline]
  3. Shoskes, D., Wood, K. (1994) Indirect presentation of MHC antigens in transplantation Immunol. Today 15,32-38[CrossRef][Medline]
  4. Haga, J., She, J., Kao, K. (1991) Biochemical characterization of 39-kDa class I histocompatibility antigen in plasma J. Biol. Chem. 266,3695-3701[Abstract/Free Full Text]
  5. Kao, K., Scornik, J., Riley, W., McQueen, C. (1988) Association between phenotype and HLA concentration in plasma and platelets Hum. Immunol. 21,115-124[CrossRef][Medline]
  6. Zavazava, N., Westphal, E., Mueller-Ruchholtz, W. (1990) Characterization of soluble HLA molecules in sweat and quantitative HLA differences in serum of healthy individuals J. Immunogenetics 17,387-394[Medline]
  7. Pouletty, P., Chang, C., Kalil, J., Atwood, E., Ferrone, S., Shimizu, B., Howson, W., Mazaheri, R., Villano, B., Grumet, C. (1993) Typing of serum-soluble HLA-B27 antigen by ELISA Tissue Antigens 42,14-19[Medline]
  8. Zavazava, N., Böttcher, H., Mueller-Ruchholtz, W. (1993) Soluble MHC class I antigens (sHLA) and anti-HLA antibodies in heart and kidney allograft recipients Tissue Antigens 42,20-26[Medline]
  9. Behrens, D., Lange, K., Fried, A., Yoo-Ott, K., Richter, K., Fändrich, F., Krönke, M., Zavazava, N. (2001) Donor-derived soluble MHC antigens plus low-dose cyclosporine induce transplantation unresponsiveness independent of the thymus by down-regulating T cell-mediated alloresponses in a rat transplantation model Transplantation 72,1974-1982[CrossRef][Medline]
  10. Schneck, J., Sechler, J., O’Herrin, S., Bieler, J., Barnes, N., Rosenberg, A. (1996) Specific inhibition of graft rejection by soluble MHC superdimers Human Immunol. 47,149
  11. O’Herrin, S. M., Slansky, J., Tang, Q., Markiewicz, M., Gajewski, T., Pardoll, D., Schneck, J., Bluestone, J. (2001) Antigen-specific blockage of T cells in vivo using dimeric MHC peptide J. Immunol. 167,2555-2560[Abstract/Free Full Text]
  12. Casares, S., Hurtado, A., McEvoy, R. C., Sarukhan, A., von Boehmer, H., Brumeanu, T. D. (2002) Down-regulation of diabetogenic CD4+ T cells by a soluble dimeric peptide-MHC class II chimera Nat. Immunol. 3,383-391[CrossRef][Medline]
  13. Ono, K., Lindsey, E. (1969) Improved technique of heart transplantation in rats J. Thorac. Cardiovasc. Surg. 57,225-229[Medline]
  14. Fändrich, F., Zhu, X., Schröder, J., Dresske, B., Henne-Bruns, D., Oswald, H., Zavazava, N. (1999) Different in vivo tolerogenicity of MHC class I peptides J. Leukoc. Biol. 65,16-27[Abstract]
  15. Freese, A., Zavazava, N. (2002) HLA-B7 ß-pleated sheet-derived synthetic peptides are immunodominant T-cell epitopes regulating alloresponses Blood 99,3286-3292[Abstract/Free Full Text]
  16. Zavazava, N., Hausmann, R., Mueller-Ruchholtz, W. (1991) Inhibition of anti-HLAB7 alloreactive CTL by affinity-purified soluble HLA Transplantation 51,838-842[Medline]
  17. Charlton, R., Zmijewski, C. (1970) Soluble HL-A7 antigen: localization in the ß-lipoprotein fraction of human serum Science 170,636-637[Abstract/Free Full Text]
  18. Freese, A., Zavazava, N. (1996) Determination of HLA-B7 T-cell epitopes Transpl. Int. 9(Suppl. 1),S352-S355
  19. Zavazava, N., Krönke, M. (1996) Soluble HLA class I molecules induce apoptosis in alloreactive cytotoxic T lymphocytes Nat. Med. 2,1005-1010[CrossRef][Medline]
  20. Altman, J., Moss, P., Goulder, P., Barouch, D., McHeyzer-Williams, M., Bell, J., McMichael, A., Davis, M. (1996) Phenotypic analysis of antigen-specific T lymphocytes Science 274,94-96[Abstract/Free Full Text]
  21. . Hausmann,R.,Zavazava, N., Steinmann, J., Mueller-Ruchholtz, W. (1993) Interaction of papain-digested HLA class I molecules with human alloreactive cytotoxic T lymphocytes (CTL) Clin. Exp. Immunol. 91,183-188[Medline]
  22. Abastado, J., Lone, Y., Casrouge, A., Boulot, G., Kourilsky, P. (1995) Dimerization of soluble major histocompatibility complex-peptide complexes is sufficient for activation of T cell hybridoma and induction of unresponsiveness J. Exp. Med. 182,439-447[Abstract/Free Full Text]
  23. Obst, R., Netuschil, N., Klopfer, K., Stevanovic, S., Rammensee, H. G. (2000) The role of peptides in T cell alloreactivity is determined by self-major histocompatibility complex molecules J. Exp. Med. 191,805-812[Abstract/Free Full Text]
  24. Barnea, E., Beer, I., Patoka, R., Ziv, T., Kessler, O., Tzehoval, E., Eisenbach, L., Zavazava, N., Admon, A. (2002) Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor-specific antigens Eur. J. Immunol. 32,213-222[CrossRef][Medline]
  25. Zavazava, N., Fändrich, F., Zhu, X., Freese, A., Behrens, D., Yoo-Ott, K. (2000) Oral feeding of an immunodominant MHC-donor-derived synthetic class I peptide prolongs graft survival of heterotopic cardiac allografts in a high-responder rat strain combination J. Leukoc. Biol. 67,793-800[Abstract]
  26. Murphy, B., Mage, C., Alexander, S., Waaga, A., Snoeck, H., Vella, J., Carpenter, C., Sayegh, M. (1999) Inhibition of allorecognition by a human class II MHC-derived peptide through the induction of apoptosis J. Clin. Invest. 103,859-867[Medline]
  27. Chowdhury, N. C., Saborio, D. V., Garrovillo, M., Chandraker, A., Magee, C. C., Waaga, A. M., Sayegh, M. H., Jin, M. X., Oluwole, S. F. (1998) Comparative studies of specific acquired systemic tolerance induced by intrathymic inoculation of a single synthetic Wistar-Furth (RT1U) all-MHC class I (RT1.AU) peptide or WAG (RT1U)-derived class I peptide Transplantation 66,1059-1066[CrossRef][Medline]
  28. Oluwole, S., Jin, M., Chowdhury, N., Ohajekwe, O. (1994) Effectiveness of intrathymic inoculation of soluble antigens in the induction of specific unresponsiveness to rat islet allografts without transient recipient immunosuppresssion Transplantation 58,1077-1081[Medline]
  29. Skinner, P., Daniels, M., Schmidt, C., Jameson, S., Haase, A. (2000) Cutting edge: in situ tetramer staining of antigen-specific T cells in tissues J. Immunol. 165,613-617[Abstract/Free Full Text]




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