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The Journal of Immunology, 1999, 162: 6993-7001.
Copyright © 1999 by The American Association of Immunologists

Human Anti-Porcine T Cell Response: Blocking with Anti-Class I Antibody Leads to Hyporesponsiveness and a Switch in Cytokine Production

Harout DerSimonian1, Luying Pan, Chris Yatko, Amelie Rodrigue-Way, Eric Johnson and Albert S. B. Edge

Department of Molecular and Cellular Biology, Diacrin, Inc., Charlestown, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intervention in the molecular interactions that lead to an immune response is possible at various stages of Ag recognition and T cell activation. Perturbation of the interaction of the TCR with the MHC/peptide ligand complex is one approach that has shown promise for autoimmunity and graft rejection in blocking T cell-activated responses. In this study, we investigated the effect of altering the target MHC class I molecule by blocking with Abs. We established a system that analyzed the human T cell response against MHC class I+/class II- porcine stimulatory cell targets. The primary human response against porcine smooth muscle cells was CD8+ T cell dependent. In the presence of F(ab')2 fragments of the MHC class I-reactive Ab, PT-85, the proliferative response was inhibited and production of IL-2 and IFN-{gamma} was blocked. Moreover, in a secondary response, proliferation was reduced and type 1 cytokine levels were inhibited. In contrast, levels of IL-10 and IL-4 were sustained or slightly increased. These findings indicate that Ab against MHC class I blocked the recognition of porcine cells by the human CD8+ T cells and altered the cytokine secretion profile. Thus, a single treatment with PT-85 F(ab')2 directed against the MHC class I molecule provides an attractive approach to the induction of T cell tolerance that may provide long-term graft survival in porcine-to-human cell transplantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of the immune system to develop tolerance to peripheral Ags provides a rationale on which to base approaches to successful graft survival. Both CD4+ and CD8+ T cells need TCR-MHC interaction and costimulatory signals before clonal expansion and the development of effector activity (1, 2). The T cell may die if not stimulated or survive but become functionally inactive or anergic. Perturbation of the interaction of the TCR with MHC-Ag peptide (signal one) will lead to the development of anergy in peripheral T cells. For example, substituting altered peptide ligands that differ by a single amino acid from the native Ag in the peptide binding groove of the MHC molecule functionally inactivated the T cell against native Ag (3). Signal one can also be disrupted by changes in the conformation of MHC class I that lead to altered TCR binding (4, 5).

Alterations in TCR MHC interactions lead to altered profiles of cytokine secretion that result in tolerization. Altered peptide ligands can shift the secretion of cytokines by T cell clones from a type 1 to a type 2 profile (6, 7, 8). In allograft rejection, type 1 cytokines play an important role. In contrast, IL-10, a type 2 cytokine, has been shown to be correlated with prolonged graft survival (9, 10). Monocytes have been shown to be inactivated by IL-10 via inhibition of tyrosine kinase and Ras signaling (11), and treatment of dendritic cells with IL-10 will inhibit their APC function (12, 13). IL-10 induces long-term anergy in T cells (14, 15). In the case of xenografts, survival of hamster hearts in rats is associated with increased levels of IL-10, IL-4, and IL-13, whereas rejection has been associated with increased IL-2, IFN-{gamma}, and TNF-{alpha} transcripts at the graft site (16). Increased IL-10 and TGF-ß mRNA and low IFN-{gamma} at the intragraft site was associated with xenogeneic heart survival in another study (17). Moreover, in vitro treatment of human APCs with IL-10 inhibits the indirect pathway of presentation of porcine Ags to human CD4+ T cells (18). IL-10 may be produced in monocytes or T cells and may inhibit macrophage activation. However, one study has shown that IL-4 is increased in rejecting xenografts (19).

Recent experiments have provided evidence that the human anti-porcine lymphocyte reaction is dependent largely on the direct pathway (20, 21, 22, 23). For example, removal of human APC in a mixed leukocyte reaction in vitro does not significantly decrease the magnitude of the response. Abs against porcine MHC but not against human MHC have a marked effect on the response. In the case of allografts, depletion of passenger leukocytes (APCs) from the tissue to be transplanted can markedly prolong engraftment and in some cases leads to indefinite survival and tolerance (24). The match between the xenogeneic MHC and self-MHC must be sufficient to allow direct recognition of the target cells by the host TCR. Alternatively, foreign Ags may be recognized indirectly as peptide in association with self-MHC on host APC (23, 25). An attractive mechanism for the inhibition of xenograft rejection would be one that had an effect on both direct and indirect presentation pathways, such as an alteration in the cytokine milieu in the direction of type 2 cytokines. A change that altered cytokine secretion at the graft site would enhance graft survival specifically and allow for transplantation without generalized immunosuppression.

In cell transplantation using populations of cells that are MHC class I+/class II-, the CD8+ T cell is the effector cell that would be likely to mediate graft rejection. CD8+ T cells in the periphery may be rendered nonfunctional by a variety of mechanisms that appear to involve exposure to Ag. Cytotoxic T cells could be inactivated by clonal exhaustion in persistent viral infections in vivo (26, 27) or by exposure to their cognate Ag (28). Anergy can be induced in these cells as shown by the inability of the cells to respond to a subsequent Ag challenge. Thus, mature CD8+ T cells can be inactivated by a number of mechanisms in peripheral tissue.

In this study, we show that blocking the human anti-porcine T cell response with anti-MHC class I Ab not only inhibited proliferation and induced subsequent hyporesponsiveness but led to an increase in the type 2/type 1 cytokine ratio. Human PBMC exposed to porcine MHC class I+/class II- aortic smooth muscle cells (SMC)2 produced high levels of IL-2 and IFN-{gamma}. After treatment with PT-85 F(ab')2 Ab, proliferation was inhibited and the production of both IL-2 and IFN-{gamma} were markedly decreased, whereas the production of IL-4 and IL-10 was unchanged and, in some cases, enhanced. These results are qualitatively similar to those obtained with the altered peptide ligand or coreceptor-deficient TCR signaling that lead to a state of unresponsiveness (8). Thus, a single treatment with PT-85 F(ab')2 directed against the MHC class I molecule on donor cells provides an attractive approach to induce T cell tolerance in vitro that may provide long-term graft survival in porcine-to-human cell transplantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of porcine SMCs

SMC were derived from porcine aorta of SLAaa and SLAdd miniature pigs provided by Dr. David H. Sachs (Massachusetts General Hospital, Boston, MA). SMC were isolated according to a previously described procedure (29). Briefly, the endothelial cell layer was removed, and small pieces of tissue containing the SMC layer were cut and digested with equal volumes of collagenase P (0.8 mg/ml) and trypsin-versene (BioWhittaker, Walkersville, MD) at 37°C for 60 min. The cells were washed several times and cultured with DMEM supplemented with 10% FCS in 100-mm tissue culture dishes. Before reaching confluence, cells were harvested and replated at a 1:3 dilution. Based on morphology and staining, >98% of the cells used were SMC. Experiments were conducted using cells between passages 12–20.

Isolation of porcine embryonic brain cells (EBC)

EBC were isolated from porcine embryonic brain tissue. Embryonic porcine brain tissues from day 27 of gestation were used to isolate EBC according to the standard ventral mesencephalon cell isolation procedure as described previously (30).

Purification of human peripheral blood T cell subsets

Peripheral blood was collected from normal volunteers, and PBMC were purified by Ficoll-Paque gradient (23). B cells, macrophages, and NK cells were removed from PBMC to generate enriched T cells by incubation with mouse anti-human CD14, CD19, CD56, and CD16 followed by negative selection using goat anti-mouse IgG magnetic beads. For purification of CD4+ and CD8+ cells, T cells were incubated with anti-CD4 or anti-CD8 mAbs (5 µg/ml) at 4°C for 45 min. Goat anti-mouse IgG magnetic beads (Dynal, Oslo, Norway) were added at a 10:1 (bead:cell) ratio. After three rounds of magnetic bead separation, the negatively enriched T cells, CD4+ T cells, or CD8+ T cell subsets were >95% pure as determined by FACS analysis.

Human anti-porcine proliferation assay

SMC (2 x 104 cells/well) or EBC (4 x 104 cells/well) were plated in 96-well flat-bottom microtiter plates. Cells were incubated with medium alone or with the blocking Ab (10 µg/ml) for 1 h at 4°C. Hybridomas producing the anti-porcine MHC class I Abs PT-85 and 74-11-10 were obtained from VMRD (Pullman, WA) (31), and Dr. David Sachs (32), respectively. mAb 9.3 was raised against porcine PBL as previously described (33). These Abs react with porcine MHC class I Ags. Ab 10.14 is against porcine CD44 (33). Hybridoma producing the anti-human MHC class I mAb, W6/32, was obtained from American Type Culture Collection (ATCC; Manassas, VA) (34). Human PBMC were then added at 2 x 105 cells/well (unless otherwise specified), whereas human CD4+ or CD8+ T cells were added at 1 x 105 cells/well. All experiments were done in AIM V medium (Life Technologies, Grand Island, NY) supplemented with 5% heat-inactivated FCS (HyClone, Logan, UT). For secondary stimulation, cells were harvested and restimulated with fresh SMC, EBC, or immobilized anti-human CD3 Ab (5 µg/ml) in 96-well flat-bottom plates in the presence or absence of anti-CD28 (1 µg/ml) in the culture. Supernatants were harvested from appropriate cultures at indicated times and frozen for cytokine detection. To determine the proliferative response, cells were pulsed with [3H]thymidine (1 µCi/well) for 20 h and harvested with a cell harvester (Packard Instrument, Meriden, CT) at the indicated time points. Thymidine incorporation was determined by counting the filter plate using a microplate scintillation counter (model B9906; Packard Instrument).

Construction of chimeric MHC Class I

Total RNA was isolated from BALB/c fibroblast cell lines using RNAzol B following the manufacturer’s procedures (Tel-Test, Friendswood, TX). The first strands of cDNA from mRNA were synthesized using the Advantage cDNA PCR kit following the manufacturer’s procedures (Clontech, Palo Alto, CA). The cDNA of H-2Dd was amplified by PCR using a 5' primer containing a XhoI linker tail (CGA TCT CGA GAT GGG GGC GAT GGC TCC GCG CAC) and a 3' primer containing a HindIII linker tail (ATC GAA GCT TTC ACA CTT TAC AAT CTG GGA GAG) to facilitate cloning into pGEM-7Zf (Promega, Madison, WI). The H-2Dd clone was sequenced to assure reliable amplification. For the {alpha}3 chimera, the H-2Dd/pGEM-7Zf was linearized with BsmI and partially digested with BsrDI to excise exon 4. Exon 4 of pig PD1 (35) corresponding to the {alpha}3 domain was amplified by PCR using primers that contained the junctions of mouse exon 3/pig exon 4 with an adjacent BsmI ligation site and pig exon 4/mouse exon 5 with a BsrDI ligation site. The PCR product was digested and ligated into linearized H-2Dd/pGEM-7Zf. The presence of exon 4 of pig in this vector added a new restriction site (BglII) that was used for screening. A clone containing the pig insert was sequenced, and the new chimeric gene was transferred to the expression vector pcDNA3.TK (Invitrogen, Carlsbad, CA) at the XhoI and HindIII sites. For the {alpha}1/{alpha}2 chimeras, H-2Dd/pGEM-7Zf was linearized with NotI and partially digested with BstYI to excise exons 2–3 of H-2Dd. Exons 2–3 of PD1 corresponding to the {alpha}1 and {alpha}2 domains were amplified by PCR using the 5' primer (representing the mouse exon 1/pig exon 2 junction), containing the NotI site for insertion into H-2Dd/pGEM-7Zf, and the 3' primer (representing the pig exon 3/mouse exon 4 junction), containing a BstYI site for ligation into pGEM-7Zf.

Expression of chimeric MHC class I

Native H-2Dd and PD1 as well as chimeric porcine {alpha}3 and {alpha}1/{alpha}2 chimeric genes were transfected by electroporation into BALB/c fibroblasts and C1498 cells (ATCC) using a setting of 290 V in serum-free RPMI. Stable transfectants were selected with G418 (Life Technologies) at a concentration of 1200 µg/ml for BALB/c fibroblasts and 800 µg/ml for C1498 for 2 wk. Magnetic bead separation was performed on the PD1 and {alpha}3 chimeric transfectants using mAb 34-2-12 as the primary Ab. Dynabeads (M-450) coated with goat anti-mouse IgG (Dynal) were used to select cells expressing either protein. Magnetic bead separation was performed on the H-2Dd and {alpha}1/{alpha}2 chimeric transfectants using 34-5-8s (PharMingen, San Diego, CA) as the primary Ab. Clones were selected by limiting dilution in a 96-well plate. The selected cells were analyzed by flow cytometry (FACScan; Becton Dickinson, Mountain View, CA). A variety of primary Abs were analyzed for their domain specificity on MHC class I chimeras using FITC-conjugated goat anti-mouse IgG (H+L) secondary reagent for detection (Jackson ImmunoResearch, West Grove, PA).

FACS analysis

FITC- or PE-conjugated mAbs to CD4, CD8, CD14, and CD19 were purchased from PharMingen. Cells (2 x 105) were incubated with FITC- or PE-conjugated mAb for 60 min at 4°C. Cells were washed and analyzed using a FACScan (Becton Dickson).

ELISA

The Abs used in the IFN-{gamma}, IL-2, IL-4, and IL-10 assays were purchased from PharMingen. All assays were performed according to the manufacturer’s protocol. Briefly, plates were coated with anti-cytokine mAb first and then incubated with culture supernatants or standard samples at 4°C overnight. Cytokine captured on the plate was detected by the respective biotin-labeled anti-IFN-{gamma}, anti-IL-2, anti-IL-4, or anti-IL-10 mAbs. The plate was developed using streptavidin-HRP and substrate, and the reaction was stopped by addition of an equal volume of 1 M H2SO4. The absorbance of the assay plate was read at 450 nm using a microplate reader (model 3550, Bio-Rad, Hercules, CA). rhIFN-{gamma}, rhIL-2, rhIL-4, and rhIL-10 cytokines were used as standards, respectively. The sensitivity of the assay ranged between 1 pg/ml and 10 ng/ml for all cytokines.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation of human PBMC to class I-positive cells: inhibition with SLA class I-reactive PT-85 Ab

To assess the mechanism of PT-85 blocking in the human anti-porcine response, we established a proliferation assay that measured human T cell responses to porcine cells that expressed only MHC class I Ags. Proliferation responses of human T cells were measured using SMC from two partially inbred miniature pigs and EBC isolated from outbred pigs. Primary stimulation of human T cells with porcine SMC or EBC showed profiles that were inhibited with the anti-class I PT-85 Ab (Fig. 1GoA). The human PBMC responses to SMC from the SLAaa and SLAdd pigs and to EBC from outbred pigs were inhibited with PT-85 by >50% compared with no Ab, control Ab, or anti-human HLA class I W6/32 Ab. Moreover, restimulation as measured in a 3-day assay without addition of Ab showed hyporesponsiveness when cells previously blocked with PT-85 were compared with controls (Fig. 1GoB). The HLA class I-specific W6/32 mAb was included as control. Monomorphic anti-HLA-specific W6/32 Ab, unreactive with porcine MHC class I, recognizes class I heavy chain {alpha}2 and {alpha}3 domain sequences in association with appropriate ß2-microglobulin.



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  		FIGURE 1. A, PT-85 inhibits primary human anti-porcine response. Human PBMC were incubated with MHC class I+/II- porcine SMC or EBC for 7 days in the presence or absence of Ab, and cells were pulsed with [3H]thymidine for the last 20 h of incubation. The anti-human MHC class I-reactive W6/32 mAb is not reactive with pig MHC class I. The F(ab')2 fragment of normal mouse Ig (mIgG) was used as control. The graph shows the mean and SD of triplicate wells. B, Inhibition of secondary human anti-porcine response. Cells from day 7 primary stimulation were restimulated with porcine SMC or EBC for 3 days, and cells were pulsed with [3H]thymidine for the last 20 h of incubation. No Ab was present in the secondary stimulation.

 
Consistent inhibition was observed in proliferation studies with eight different human blood donors in 12 experiments. Primary stimulation was consistently inhibited by >50% in the human anti-porcine T cell response when blocked with the anti-class I PT-85 F(ab')2 Ab but not with the W6/32 or mouse IgG F(ab')2 control Abs (Fig. 2GoA). Inhibition of proliferation was >60% by day 3 of a secondary stimulation for the initially masked group (Fig. 2GoB). These results suggested that blocking the MHC class I molecule with the PT-85 Ab may have led to T cell unresponsiveness. Blocking with the HLA-specific W6/32 Ab had no detectable effect on the human anti-porcine T cell response.



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  		FIGURE 2. A, PT-85 inhibits primary human anti-porcine response. Tissue cultures were set up as described in the legend to Fig. 1GoA. Thymidine incorporation was converted to percentage of no Ab control. Responder cell alone was <10% of the full response. Stimulator cell alone was <1% of the full response. The graph represents a summary of 12 experiments with PBMC from eight individuals. B, Inhibition of secondary human anti-porcine response. Tissue cultures were set up as described in the legend to Fig. 1GoB. Thymidine incorporation was converted to percentage of control (no-Ab group for SMC or mouse IgG group for EBC). Data represent a summary of eight experiments with PBMC from six individuals for SMC as stimulators and two experiments with PBMC from two individuals for EBC as stimulators.

 
Domain specificity of the anti-MHC class I Abs

To delineate differences in domain specificity between several anti-MHC class I mAbs and to determine whether these differences correlated to function, we established porcine and murine "exon-shuffled" MHC class I-transfected mouse cell lines. The porcine and mouse MHC class I chimeric molecules were generated using {alpha}1, {alpha}2, and {alpha}3 domains from pig PD1 (35) and mouse H-2 D genes. The recombinant genes were transfected and expressed in C1498 cells and BALB/c fibroblasts. Stably transfected lines were tested for reactivity with anti-porcine MHC class I mAbs (Table IGo). Porcine and murine MHC class I {alpha}1, {alpha}2, or {alpha}3 domain constructs are represented in Table IGo as P and M, respectively. Murine MHC class I-reactive mAbs 34-5-8s ({alpha}1/{alpha}2) and 34-2-12 ({alpha}3) of known specificities were used to confirm construct domains. Only mAb 9-3 required the PD1 {alpha}3 domain for reactivity, whereas mAb PT-85, 74-11-10, and 2-27-3 did not require the {alpha}3 domain for reactivity. These three mAbs required the PD1 {alpha}1 and/or {alpha}2 domains for reactivity. In contrast, mAb 7-34-1 required the pig ß2-microglobulin for proper recognition of pig MHC class I molecules, since it only reacted with PD1 transfected mouse cells in the presence of pig ß2-microglobulin found in pig serum (data not shown).


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  		Table I. Specificity determination of anti-porcine MHC-class I Abs1

 
The direct human anti-porcine response is initiated by CD8+ T cells and inhibited by anti-MHC class I and anti-CD8 Abs

Experiments were designed to evaluate the role of anti-class I PT-85 F(ab')2 Ab blocking in the human anti-porcine T cell response using purified populations of T cells. Proliferation of PBMC and purified CD4+ or CD8+ T cells was tested against MHC class I-positive porcine SMC. Human PBMC response to porcine SMC was blocked by two other anti-MHC class I Abs, although to a lesser degree than PT-85 (Fig. 3GoA). This response was also inhibited partially using OKT4 or OKT8 mAbs (data not shown), suggesting that both CD4+ and CD8+ T cells contributed to the proliferation. Purified CD8+ T cells proliferated in response to the SMC and as shown in Fig. 3GoB, the response was completely inhibited with PT-85, 9.3, and 74-11-10, but not with the human class I-specific W6/32 nor with the porcine CD44-specific 10.14 mAb F(ab')2 (33). Moreover, this proliferation was completely inhibited with the anti-CD8 but not with the anti-CD4 mAb (data not shown). Whereas the purified CD8+ T cells (1 x 105) responded to porcine MHC class I+ SMC in most experiments, the proliferation was relatively lower than that seen with PBMC (3 x 105) from the same donor. In contrast, purified CD4+ T cells showed no direct proliferation against the porcine SMC (data not shown). Similar data for the purified T cell responses were obtained from four different PBMC donors. These findings indicate that CD8+ T cells are the initial responders to MHC class I+ SMC stimulation and that the anti-porcine MHC class I Ab prevents this activation.



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  		FIGURE 3. Inhibition of CD8+ T cells in primary human anti-porcine response. Human PBMC, CD8+ T cells, or CD4+ T cells were incubated with porcine SMC for 7 days in the presence or absence of Ab, and cells were pulsed with [3H]thymidine for the last 8 h of incubation. 9-3 and 74-11-10 Ab are reactive with porcine MHC class I (see Table IGo). Monoclonal Ab 10.14 is reactive with porcine CD44.

 
Blocking with PT-85 F(ab')2 Ab inhibits IL-2 and IFN-{gamma} and induces IL-4 and IL-10 production

To assess whether inhibition of proliferation in the primary and secondary human anti-porcine T cell responses was associated with changes in cytokine production, we measured the levels of IL-2, IFN-{gamma}, IL-4, and IL-10 in the supernatants. IL-2 production was evident in the supernatants of the unmasked human anti-porcine cocultures between days 2 and 5 (Fig. 4Go). The level of IL-2 was decreased by days 6 and 7 during culture primarily because of consumption by T cell proliferation (data not shown). However, supernatants from PT-85 masked cell cultures showed a striking decrease in levels of IL-2 from days 2 to 5. The level of IFN-{gamma} production was also inhibited in cultures that were PT-85 masked (Fig. 5Go). The actual levels detected in the absence of the PT-85 blocking varied between 20 and 400 pg/ml for IL-2 and between 800 and 3200 pg/ml for IFN-{gamma}. The inhibition of both type 1 cytokines was consistent in all of the donors tested (n = 8). The production of IL-2 and IFN-{gamma} was not affected by the addition of control mouse IgG F(ab')2 (Figs. 4Go and 5Go) or the anti-HLA class I W6/32 F(ab')2 Abs (data not shown).



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  		FIGURE 4. PT-85 inhibits IL-2 production in primary PBMC response against porcine SMC. Tissue cultures were set up as in Fig. 1GoA. Supernatants were harvested on days as indicated and used for ELISA. The results show IL-2 levels in the culture supernatants using PBMC from three individuals.

 


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  		FIGURE 5. PT-85 inhibits IFN-{gamma} production in primary PBMC response against porcine SMC. Tissue cultures were set up as in Fig. 1GoA. Supernatants were harvested on days as indicated and used for ELISA. The results show IFN-{gamma} levels in the culture supernatants using PBMC from three individuals.

 
In contrast to type 1 cytokine inhibition, the production of IL-10 in the presence of blocking Ab did not decrease relative to control conditions (Fig. 6Go). While the IL-10 levels in the day 7 supernatant ranged between 15 and 100 pg/ml depending on donor PBMC, none decreased with PT-85 treatment. The IL-4 levels in supernatants were below the limit of detection (<2 pg/ml) in these primary cultures (data not shown).



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  		FIGURE 6. Sustained IL-10 production in primary PBMC response against porcine SMC. Tissue cultures were set up as in Fig. 1GoA. Supernatants were harvested on days as indicated and used for ELISA. The results show IL-10 levels in the culture supernatants using PBMC from three individuals.

 
Since the persistent production of the type 2 cytokine, IL-10, in masked conditions suggested a shift in the type 1/type 2 cytokine ratio, we examined IFN-{gamma}, IL-4, and IL-10 levels after 2 days of a secondary stimulation using 1) fresh porcine SMC, 2) anti-CD3 Ab, or 3) anti-CD3/CD28 Abs. These conditions were selected to examine both Ag-specific and Ag-nonspecific secondary responses using anti-CD3 or anti-CD3 and anti-CD28 mAbs. Production of IFN-{gamma} after 48 h by human cells previously exposed to PT-85 masked SMC was markedly inhibited during the secondary stimulation with SMC as compared with the control groups (Fig. 7Go). Cells incubated with control mouse IgG F(ab')2 during primary stimulation had similar levels of IFN-{gamma} compared with cells from the no-Ab group. In contrast, levels of IL-10 in the same culture supernatants increased (Fig. 8Go). Moreover, the production of IL-10 was increased during the secondary stimulation with anti-CD3 or the combination of anti-CD3/CD28 Abs (Fig. 9Go). Likewise, the levels of IL-4 were increased during secondary stimulation with anti-CD3 or with anti-CD3/CD28 mAbs (Fig. 10Go). Overall, when IFN-{gamma} and IL-10 levels in the primary (and secondary) cultures were compared, PT-85 blocking consistently showed a decrease in the ratio of type 1 to type 2 cytokines (Table IIGo). Together, the results suggest a shift in the type 1/type 2 cytokine ratio, since IL-2 and IFN-{gamma} decreased dramatically, whereas levels of IL-10 and IL-4 were maintained or slightly increased.



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  		FIGURE 7. Inhibition of IFN-{gamma} production during secondary response against porcine SMC. Tissue cultures were set up as in Fig. 1GoB. Supernatants were harvested at 48 h and used for ELISA. The results show IFN-{gamma} levels in culture supernatants using PBMC from three individuals.

 


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  		FIGURE 8. Enhanced IL-10 production during secondary anti-porcine SMC response. Tissue cultures were set up as in Fig. 1GoB. Supernatants were harvested at 48 h and used for ELISA. The results show IL-10 levels in culture supernatants using PBMC from three individuals.

 


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  		FIGURE 9. Enhanced IL-10 production during secondary stimulation with anti-CD3 or anti-CD3/CD28 mAbs. Cells from day 7 primary stimulation were restimulated with immobilized anti-CD3 in the presence or absence of anti-CD28. Culture supernatants were harvested at 48 h and used for ELISA. The results show IL-10 levels in culture supernatants using PBMC from three individuals.

 


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  		FIGURE 10. Enhanced IL-4 production during secondary stimulation with anti-CD28. Cells from day 7 primary stimulation were restimulated with SMC or immobilized anti-CD3 in the presence or absence of anti-CD28. Culture supernatants were harvested at 48 h and used for ELISA. The results show IL-4 levels in culture supernatants using PBMC from three individuals.

 

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  		Table II. Masking with PT-85 leads to decreased type 1 to type 2 cytokine ratio

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In these studies we have shown that mAbs against porcine MHC class I block proliferation of human T cells in an in vitro mixed culture of porcine SMCs with human PBMC and CD8+ T cells. Abs directed to the {alpha}1/{alpha}2 domains as well as to the {alpha}3 domain of class I on the class I+/class II- SMC inhibited proliferation of the human cells in a primary response, and human cells that were cultured with the Ab-treated porcine cells in the primary incubation retained their functional phenotype in a secondary stimulation with porcine SMC. Human cells harvested from the PT-85-treated cultures showed a marked decrease in proliferation as compared with human cells obtained from untreated primary cultures.

Purified human CD8+ T cells proliferated in response to the porcine SMC in the absence of the blocking Ab. Our in vitro experiments showed that blocking the porcine MHC class I Ags on the class I+/class II- SMC inhibited CD8+ T cell proliferation. Although CD8+ cells are normally thought of as effector cells while CD4+ cells play a regulatory role, CD8+ cells have been implicated as immunoregulatory in a number of systems (36, 37). CD8+ cells can secrete low levels of IL-2 in addition to high levels of IFN-{gamma} (38). It has also been demonstrated that CD8+ cells are capable of producing other regulatory cytokines, including IL-4 and IL-10. Thus, if the early CD8+ T cell response is blocked by Ab directed to MHC class I on the targeted cells, it may influence the overall immune response and graft survival in cellular xenotransplantation.

Since most activated CD8+ T cells secrete a type 1 pattern of cytokines (38), it is reasonable that they can skew the cytokine profile of simultaneously activated CD4+ T cells toward a type 1 phenotype (39). Further support for this conclusion from in vivo data shows that removal of CD8+ T cells inhibits IFN-{gamma} production by CD4+ T cells (40, 41). Moreover, preventing or blocking CD8 activation may have a profound effect on the subsequent CD4+ T cell activation toward a type 2 phenotype (42). The alterations in cytokine profile seen in our study comprised a decrease in IFN-{gamma} and IL-2 production and a sustained or enhanced IL-10 secretion in the human anti-porcine response when PT-85, an anti-porcine MHC class I Ab, was used to block the response. This altered cytokine pattern could provide a tolerogenic signal that permits increased graft survival.

The mechanism for the recognition of a foreign-species Ag can involve direct recognition by the T cell of the foreign MHC-peptide complexes on the graft or indirect recognition of the foreign Ags as processed peptides in association with self-APC (25, 43). In the absence of porcine MHC class II up-regulation, it is reasonable to suggest that CD4+ T cells are capable of responding to foreign Ag on self-APC through the indirect pathway. Our findings indicate that the human response to MHC class I+/class II- porcine cells is dominated by MHC class I-reactive CD8+ T cells that are capable of producing cytokines that could subsequently activate additional cell types, including CD4+ lymphocytes, monocytes, and dendritic cells. Addition of anti-MHC class I Ab blocks activation of CD8+ T cells and potent type I cytokine production, which may subsequently diminish or alter stimulation via the indirect pathway.

These findings help explain some of the protective elements of MHC class I masking for cell transplantation reported by a number of groups. Stock et al. showed that treatment of islets from DBA/2J or B10.BR mice with anti-MHC class I Abs before mixed lymphocyte-islet culture blocked the formation of cytotoxic T cells from C57/BL mice (44). A second group found that saturating anti-MHC class I Abs in a bulk mixed lymphocyte culture inhibited the generation of CTLs (45). Masking of other Ags on APCs has been shown to have a similar effect. It has been observed in a number of transplant models that masking the donor cells with anti-class I mAb leads to long-term survival. For example, porcine fetal neuronal cells pretreated with PT-85 have been shown to survive for >4 mo in rats based on histological examination of the brains at autopsy (46). The graft survival rate in rats after masking of the porcine neuronal cell MHC class I was significantly improved as compared with no treatment control and was not statistically different from that obtained by systemic immunosuppression with cyclosporin. In a human clinical trial of porcine fetal neural cells for Parkinson’s disease, we have observed clinical improvement in patients who received masked cells (47). At 6 mo after transplantation the clinical improvement was at least as great in the patients receiving masked cells as for patients receiving cyclosporin based on the United Parkinson’s Disease Rating Scale.

Inhibition of graft rejection with Ab against the donor class I Ags has been reported in the immunoprivileged environment of the central nervous system (46, 48). The data from our in vitro study suggest an explanation for this enhanced graft survival. As compared with peripheral sites, the central nervous system environment is skewed toward a type 2 cytokine profile (49, 50), and this accounts in part for the immunoprivilege of the brain. It is attractive to conclude that the preexisting environment is susceptible to a further shift toward a type 2 profile when the graft is treated with Ab to MHC class I Ag. The slow rejection of xenografts in the brain without immunosuppression is thereby converted to a tolerant state in response to the MHC class I-blocked graft.

The increased level of IL-10 that we find in these mixed cultures shows that the Ab to MHC class I induces an active response in the responding T cells and does not simply block activation by preventing access to MHC class I Ag. The increase in IL-10 could be the key to explaining the effect of MHC class I Ab masking in vivo. IL-10 is an immunosuppressive cytokine that has been shown to impair T cell effector function and to alter Ag presentation such that APCs induce tolerance (12, 13, 14, 15, 51). Moreover, the indirect pathway of porcine Ag presentation by human cells is abrogated by pretreatment with IL-10 (18). We have not yet identified the cell(s) secreting IL-10, but the decreased reactivity of the PBMCs to anti-CD3/CD28 suggests that the cytokine environment influences the outcome of the immune response regardless of the source of the cytokines. This could be due in part to a bystander effect (52, 53).

In allogeneic transplantation combinations, numerous studies have reported that, as in the case of autoimmunity, a shift from a type 1 cytokine environment to a type 2 pattern of secretion promotes tolerance and helps to prevent graft rejection (9, 10). Type 2 cytokines appear to correlate with graft acceptance in the case of xenografts as well (16, 17, 54). This correlation between cytokine type and graft survival is less clear in the case of xenografts, in which IL-4 has been suggested to be involved in graft rejection (19). The answer to this question will require in vivo studies of correlation between graft survival and cytokine profile.

We hypothesize that the altered binding of CD8+ T cells to masked porcine targets changes molecular signaling in the human CD8+ T cells, thereby leading to the decreased type 1 cytokines. Since PT-85 mAb cross-reacts with human HLA class I Ags, human HLA class I-specific mAb W6/32 was used as control. The results from these experiments showed that W6/32 had no effect on either proliferation or cytokine production. Thus, the inhibition observed in the production of IFN-{gamma} and IL-2 was most likely due to interference of T cell interaction with porcine MHC class I in the presence of the porcine reactive anti-MHC class I Abs. Further elucidation of the epitopes recognized by the blocking Abs and definition of the alterations in the T cell phenotype after interacting with masked xenogeneic cells will provide us with a detailed explanation for the induction of tolerance at a molecular level. The ability of 9.3 mAb to inhibit human PBMC and CD8+ T cell proliferation in response to porcine cells (Fig. 3Go) suggests that Abs against the {alpha}3 domain of MHC class I are equally effective as PT-85, directed against the {alpha}1/{alpha}2 domains. This could be due to a similar mechanism or to a distinct mechanism mediated by the {alpha}3-specific Ab.

It is reasonable to propose that a spectrum of CD8+ T cell responder populations recognize the MHC class I/Ag peptides with varying affinities and that these cells produce cytokines of both the type 1 and type 2 profiles. During masking, however, the spectrum of cells becomes skewed functionally to the type 2 cytokine profile because of alterations in the recognition of MHC class I molecules. This could occur either by decreasing the functional affinity of the TCR for MHC class I or by altering Ag concentration on target cells as reviewed by Constant and Bottomly (8). In the former possibility, the TCR would still bind to MHC class I masked with Ab, but the binding affinity would be decreased or altered, as has been shown for a number of altered ligand models. Alternatively, MHC class I Ab binding would block access to that crucial epitope and decrease the avidity of the total interaction between the T cells and the target cells. Either mechanism could affect intracellular signaling and lead to altered cytokine gene expression. It is of interest to elucidate the signaling mechanism responsible for the change in the responding T cells. Transcriptional regulation of cytokine expression as a result of altered TCR interaction with its target is the most likely explanation. We plan to examine calcium uptake and phosphorylation patterns of specific TCR-associated proteins in vitro and cytokine gene expression in vivo after activation with and without masking. This will allow us to determine how masking alters signal transduction in human T cells that encounter porcine cells.


   Acknowledgments
 
We thank Dr. David H. Sachs for kindly providing porcine aorta for SMC isolations, Dr. Douglas Jacoby and Judson Ratliff for generously providing porcine EBCs, and Katie Crosby for generating the murine F(ab')2 fragments. We also thank Drs. Jay Berzofsky, Hugh Auchincloss, Laurie Glimcher, and David Sachs for critical review of the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Harout DerSimonian, Department of Molecular and Cellular Biology, Diacrin, Inc., 13th Street, Building 96, Charlestown, MA 02129. E-mail address: Back

2 Abbreviations used in this paper: SMC, smooth muscle cells; EBC, embryonic brain cells. Back

Received for publication January 20, 1999. Accepted for publication March 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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