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Medline Plus Health Information
*Heart Transplantation
*Organ Donation
The Journal of Immunology, 2006, 176: 2190-2196.
Copyright © 2006 by The American Association of Immunologists

CD8 T Cells Specific for a Donor-Derived, Self-Restricted Transplant Antigen Are Nonpathogenic Bystanders after Vascularized Heart Transplantation in Mice1

Anna Valujskikh*, Qiwei Zhang* and Peter S. Heeger2,*,{dagger}

* Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; and {dagger} Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8 T cell cross-priming, an established mechanism of protective antiviral immunity, was originally discovered during studies involving minor transplantation Ags. It is unclear whether or how cross-primed CD8 T cells, reactive to donor-derived, but recipient class I MHC-restricted epitopes, could injure a fully MHC-disparate, vascularized transplant. To address this question we studied host class I MHC-restricted, male transplantation Ag-reactive T cell responses in female recipients of fully MHC-disparate, male heart transplants. Cross-priming to the immune-dominant determinant HYUtyp occurred at low frequency after heart transplantation. CD8 T cell preactivation through immunization with HYUtyp mixed in CFA did not alter the kinetics of acute rejection. Furthermore, neither HYUtyp immunization nor adoptive transfer of HYUtyp-specific TCR-transgenic T cells affected outcome in 1) a model of chronic rejection in the absence of immunosuppression or 2) a model of allograft acceptance induced by costimulatory blockade. The results support the contention that CD8 T cells reactive to host-restricted, but donor-derived, Ags are highly specific and are nonpathogenic bystanders during rejection of MHC-disparate cardiac allografts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytokine production and cytotoxicity are functions of CD8 T lymphocytes that need to be carefully controlled to prevent bystander destruction of healthy host tissues. Specificity is required during two critical stimulatory events: the activation step that initiates the response of a naive or memory CD8 T cell to differentiate into an effector cell, and a re-encounter/triggering step that engages effector machinery of a primed CD8 cell, leading to cytotoxicity and/or cytokine release (1, 2, 3). For naive CD8 T cells, the activation step occurs only in secondary lymphoid organs (4) and requires costimulatory signals offered by appropriately licensed, APCs (5, 6). In contrast, the secondary recognition event can occur in the periphery on essentially any Ag-expressing cell and has significantly less stringent (and perhaps different) costimulatory requirements (7, 8).

Antiviral T cell immunity is often initiated by directly infected, professional APCs that have processed intracytoplasmic viral proteins and presented at least one dominant peptide determinant in the context of a class I MHC (MHC-I),3 to the specific CD8 T cell (9). Studies have also shown that in response to some viruses that do not directly infect APCs, professional APCs can take up exogenous Ags (derived from other infected cells) and process and present these Ags on MHC-I to CD8 T cells (10). This latter process is known as cross-priming, or priming through the indirect pathway, and can be essential for induction of protective antiviral immunity. Once activated, cross-primed CD8 T cells migrate to the site of infection where they make cognate interactions with peptide/MHC complexes expressed directly on infected cells, thereby engaging their effector machinery and leading to specific destruction of the infected cells (10).

Although cross-primed T cells are now recognized as important effectors within the antiviral immune response, the process was originally discovered as an unanticipated consequence of an experiment with minor transplantation Ags (11, 12). Immunization of F1(MHCaxb) females with MHCa male cells led to the activation of two sets of CTL: one that could kill MHCa, but not MHCb, male targets, and one with the converse specificity. Other studies, including those from our group, have demonstrated similar results using skin graft models (13, 14).

In contrast to a well-defined effector function of cross-primed CD8 T cells in antiviral immunity (directly killing infected parenchymal cells and clearing infection), a role for cross-primed CD8 T cells as effectors in MHC-disparate transplant injury remains controversial. Because the MHC restriction elements expressed on fully allogeneic grafts differ from those in the recipient, it has been unclear whether or how a cross-primed CD8 T cell could impact a transplanted organ. Studies performed in the 1960s (15) and repeated in the 1980s (16) showed that B6 mice selectively rejected the A/J component of a B6 plus A/J allophenic graft, and A/J mice rejected only the B6 component. Because the B6 and A/J cells in these grafts were intimately mixed, these results showed that long-range nonspecific effector molecules (such as cytokines) were unlikely to be involved in graft rejection. In contrast, we showed that cross-primed CD8 T cells could reject allogeneic skin grafts despite lacking TCRs capable of cognate interactions with any Ag expressed on donor cells (14, 17). This effect required the responding T cell to interact with host (recipient) vascular endothelium that has processed and presented donor Ag to the primed CD8 T cell. The pathogenic target of this cross-primed immune response was the vasculature feeding the graft, rather than donor graft cells (18).

Fully vascularized, MHC-disparate organs commonly transplanted into humans differ from skin grafts in part because the vascular endothelium is overwhelmingly derived from donor rather than recipient. Because in this situation the vasculature expresses the incorrect MHC (donor), the donor endothelium cannot be a target of self-restricted, cross-primed effector CD8 T cells. Nonetheless, cross-primed CD8 T cells could still theoretically influence the strength of the induced antidonor alloresponse at the priming stage and/or mediate nonspecific injury at the effector stage through bystander mechanisms upon encounter with cross-presented Ag expressed on a graft-infiltrating APCs.

To assess the potential effector roles of cross-primed CD8 T cells as mediators of MHC-disparate vascularized organ graft injury, we performed a series of experiments using a murine cardiac transplant model. Our findings support the contention that CD8 T cells reactive to host-restricted, but donor-derived, Ags are nonpathogenic bystanders during vascularized allograft rejection and during costimulatory blockade-induced prolongation of graft survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Female and male C57BL/6 (H-2b), C3H (H-2k), BALB/c (H-2d), and (B6 x C3H)F1 mice, ages 6–8 wk, were purchased from The Jackson Laboratory. Male and female Marilyn (CD4 TCR-transgenic specific for HYDby peptide plus I-Ab, H-2b, RAG2–/–) and MataHari (CD8 TCR-transgenic specific for Db + HYUty, RAG1–/–, H-2b) were originally provided as gifts from P. Matzinger (National Institutes of Health, Bethesda, MD). 2C mice were a gift from G. Hadley (University of Maryland, Baltimore, MD). All animals were maintained and bred in the pathogen-free facility at the Cleveland Clinic Foundation. All animal studies were reviewed and approved by the institutional animal care and use committee at the Cleveland Clinic Foundation.

Peptides

Peptides HYUtyp (WMHHNMDLI), HYDbyp (NAGFNSNRANSSRSS), HYKk (TENSGKDI), chicken OVA peptide 323–339 (KISQAVHAAHAEINEAG), and beta-galactosidase (betagal) peptide 96–103 (DAPIYTNV) were synthesized by Research Genetics at a purity >95% by HPLC. Immunizations were performed by mixing peptides with CFA (500 µM) and injecting 100–200 µl (50–100 µmol) s.c. as previously described (19, 20).

Placement and evaluation of skin and cardiac transplants

Full-thickness skin grafts were placed as customarily performed by our laboratory (18). Bandages were removed on day 7, and grafts were inspected daily. Rejection was defined as >90% necrosis. Vascularized heterotopic cardiac allografts were placed in the abdomen as previously described (18, 21, 22) and palpated daily for evidence of a heartbeat. Rejection was defined as loss of a palpable heartbeat. Grafts were harvested at the time of rejection or at predetermined time points after transplant. CTLA4Ig (0.5 mg) and MR1 (0.5 mg; purchased from BioExpress) were administered on days 0, 2, and 4 after transplantation.

T cell isolation

Splenic and lymph node T cells were isolated by negative selection using commercially available murine T cell isolation columns (R&D Systems), following the instructions supplied by the manufacturer. Resultant cells were washed in HBSS medium, counted by trypan blue exclusion, and resuspended at appropriate concentrations for use in the various assays or adoptive transfers (via tail vein injections). The purified T cells were stained with PE-conjugated anti-CD3 (2C11; BD Pharmingen), or PE-conjugated anti-TNP as an isotype-matched control (BD Pharmingen). Flow cytometry using a BD Biosciences FACScan revealed that the purification resulted in >92% CD3+ cells (data not shown).

Histology

Formalin-fixed paraffin sections of graft tissues were stained with H&E and for elastin as previously described (18, 21, 22). The total number of blood vessels and the number of blood vessels with vasculopathy were counted in four or five elastin-stained sections per graft as previously described (22, 23). The results were represented as an average percentage of partially (10–90% luminal occlusion) or fully (>90% luminal occlusion) occluded blood vessels.

ELISPOT assays

Assays were performed as previously outlined in detail (18, 21, 22). Briefly, ELISPOT plates (Millipore) were coated overnight with the capture Abs (obtained from BD Pharmingen) in sterile PBS, blocked with sterile 1% BSA in PBS, and washed three times with sterile PBS. Spleen cells (0.2–1 x 106/well) were plated in HL-1 medium (Cambrex) with or without mitomycin C-treated stimulator cells (400,000/well) and/or soluble Ags (HYDbyp and OVA at 0.1–1 µM), then incubated at 37°C in 5% CO2 for 24 h. After washing with PBS followed by PBST (PBS/0.025% Tween 20), detection Abs (obtained from BD Pharmingen) were added overnight. After washing with PBST, alkaline phosphatase-conjugated anti-biotin Ab (Vector Laboratories) diluted 1/2000 in PBST was added for 2 h at room temperature. The plates were developed as previously described. The resulting spots were counted on an ImmunoSpot series 1 analyzer (Cellular Technologies).

Isolation of organ-infiltrating lymphocytes

Animals were anesthetized and injected i.v. with 10 ml of sterile PBS until all organs were visibly blanched. Heart grafts were individually harvested, cut into pieces with a sterile razor, and incubated with 25 mg of collagenase A (Roche) in 25 ml of sterile HBSS at 37°C for 10 min with intermittent gentle vortexing. Resultant cells were filtered through a 40-µm pore size cell strainer to remove larger pieces of residual tissue. RBCs were lysed from the filtrate, and the organ-infiltrating cells were used in ELISPOT assays as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cross-priming occurs after minor Ag-disparate, but not fully allogeneic, heart transplantation

We first confirmed our published studies showing that cross-priming occurs to minor Ag HY-disparate grafts using a murine heart transplant model. Although B6 females do not acutely reject syngeneic male heart transplants, graft placement primes a low frequency HY-reactive T cell immune response that results in chronic graft injury (23). The immunodominant MHC-I-restricted antigenic peptide determinant in B6 mice has been identified as the Db-restricted peptide derived from the H-Y Ag, Uty, WMHHNMDLI (hereafter called HYUtyp) (24, 25, 26, 27). To test whether cross-priming of CD8 T cells occurred in vivo after heart transplantation, we used an approach analogous to previous work (18) and transplanted male C3H hearts into female (B6 x C3H)F1 recipients. In this combination, if graft placement results in CD8 T cell cross-priming, then placement of male C3H heart grafts should lead to processing and presentation of the donor H-Y Ag by recipient, Db-expressing APCs, thus priming a detectable recall immune response to HYUtyp. We used a cytokine ELISPOT assay to evaluate T cell priming on day 14 after graft placement. C3H male heart grafts primed T cell responses to the Db-(host)-restricted HYUtyp (92 ± 34/million spleen cells; day 14 after transplant; data not shown), consistent with cross-priming. The detected response was comparable to the immune-dominant determinant found on donor H-2k cells (TENSGKDI + Kk; 61 ± 17/million spleen cells; not shown) (18).

We next tested whether we could detect cross-primed CD8 T cells reactive to the immune-dominant determinant HYUtyp after transplantation of fully allogeneic heart grafts. C3H male hearts were transplanted into B6 female recipients, and a kinetic analysis of recall responses was performed by ELISPOT (Fig. 1) using spleen cells and graft-infiltrating lymphocytes (GILs). Strong IFN-{gamma} and IL-2 recall responses were noted to donor C3H alloantigens (direct recognition of donor Ags on donor cells) and to indirectly primed CD4 T cells reactive to HYDbyp (21) at all time points both in spleen and within GILs. In contrast, we did not detect a response to the indirectly presented HYUtyp at any time point either in the secondary lymphoid organ or within the grafts.


Figure 1
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  		FIGURE 1. Acute cardiac allograft rejection results in indirect (cross) priming of CD4, but not CD8, T cells reactive to H-2b-restricted HY-derived peptides. Spleen cells (A) or GILs (B) were isolated from B6 female recipients of C3H male hearts at the time points stated (day 4, 6, or 8 after transplant) and tested in IFN-{gamma} or IL-2 ELISPOT assays for reactivity to HYUtyp, HYDbyp, and donor C3H stimulator cells. No responses were detected to control peptides OVA323–339 or betagal96–103, and no responses to any peptides were detected in naive, untransplanted mice (not shown). Each point represents a response from a single animal (n = 3–5/group). Note the log scale in A.

 
Pre-existing CD8 effector T cells reactive to donor-derived, but recipient-restricted, Ag do not influence acute or chronic injury to a transplanted heart

To assess the role of CD8 T cells reactive to donor-derived, but self-restricted, Ag as effector cells during fully MHC-disparate cardiac allograft rejection, we next primed mice with HYUtyp and OVA323–339 mixed in CFA, followed 3 wk later by placement of a C3H male heart graft. OVA323–339 was included in the immunization as a control Ag that induces a potent CD4 T cell immune response (28). Graft survival was not detectably altered by the preimmunization, because all grafts were rejected on days 7–8 (n = 3/group), and histologic examination showed typical cellular rejection in all grafts (not shown). Recall ELISPOT assays confirmed that the immunization specifically primed HYUtyp- and OVA323–339-reactive T cells (Fig. 2). In addition, there was no significant difference in the frequency of antidonor C3H T cells in mice preimmunized with HYUtyp vs control mice, suggesting that the presence of HYUtyp-specific CD8 T cells did not have a bystander effect that influenced the strength of the transplant-induced, antidonor T cell immune response.


Figure 2
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  		FIGURE 2. Immunization with the donor-derived, recipient MHC-I-restricted peptide, HYUtyp, induces peptide-specific immunity, but does not alter the kinetics of graft rejection. Groups of mice were immunized with HYUtyp plus OVA323–339 in CFA or control betagal96–103 plus OVA323–339 in CFA. At the time of rejection (days 7–8 after transplant), spleen cells were isolated and tested for reactivity to peptides and donor cells in IFN-{gamma} ELISPOT assays. Results represent the means of three animals per group. There was no statistical difference in graft survival between groups. *, The frequencies of betagal-reactive and HYUtyp-reactive IFN-{gamma}-producers were significantly different between the two groups of mice (p < 0.05).

 
Because the relatively low frequency of HYUtyp-specific cells may not be sufficient to significantly influence the high frequency direct response during acute allograft rejection, we next assessed the effects of CD8 T cells responding via the indirect pathway using a chronic rejection model. We immunized (B6 x C3H)F1 females with HYUtyp mixed in CFA; an unimmunized group of animals served as the control. Two weeks later, the mice were transplanted with C3H male hearts. In this MHC-matched model, the HY minor Ag disparity is sufficient to drive T cell-mediated chronic injury/transplant vasculopathy by 60–90 days (23). In this experiment, host (female) HY-reactive T cells (H-2bxk) responsive to the immune-dominant, H-2k-restricted HY peptide can directly interact with this Ag expressed on donor H-2k male graft cells. In contrast, host T cells reactive to HYUtyp plus Db (activated in this experiment by immunization) can only interact with this Ag cross presented on recipient (H-2b) cells (Fig. 3A).


Figure 3
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  		FIGURE 3. Immunization with donor-derived, recipient MHC-I-restricted peptide HYUtyp induces peptide-specific immunity, but does not induce accelerated cardiac vasculopathy in (b x k)F1 female recipients of H-2k male heart grafts. Recipient (b x k)F1 females were immunized with HYUtyp/OVA/CFA or were unimmunized. Three weeks later, the animals were transplanted with H-2k male heart grafts. All grafts were beating for >60 days (n = 4–5/group). A, Schematic diagram depicting class I-restricted HY Ag processing and presentation on donor and recipient APCs. B, Representative elastin-stained sections of heart grafts (magnification, x40) obtained on days 60–70 after transplant in unimmunized (left) and immunized (middle and right) recipients. C, Quantification of the extent of vasculopathy in immunized and unimmunized mice (no statistical difference between groups). D, Recall IFN-{gamma} ELISPOTs performed at the time of death (days 60–70; n = 4–5/group), demonstrating HYUtyp-specific immunity in all immunized mice, but not in controls. Each dot represents the mean result of duplicate IFN-{gamma} ELISPOT wells for a single animal.

 
Immunization with HYUtyp/CFA did not precipitate acute rejection, because all grafts were beating for >60 days (n = 4–5/group). Histologic examinations of the heart grafts (Fig. 3, B and C) revealed transplant vasculopathy in both groups of animals, and there was no significant difference between the groups in terms of extent or severity of the vasculopathy. HYUtyp-specific immunity was detectable in the immunized group, confirming the efficacy of the immunization and thus the presence of primed HYUtyp-specific CD8 cells (Fig. 3D).

Pre-existing CD8 effector T cells reactive to donor-derived, but recipient-restricted, Ag do not influence costimulatory blockade-induced prolongation of graft survival

As a complementary approach to assessing a role for effector CD8 T cells reactive to this donor-derived, but self-restricted, transplant Ag, we tested how preimmunization with HYUtyp affected graft survival after costimulation blockade. Groups of B6 female mice were immunized with 1) the MHC-I-restricted determinant HYUtyp (plus OVA323–339) mixed in CFA, 2) the MHC-II-restricted determinant HYDbyp mixed in CFA, or 3) control MHC-I-Kb-restricted betagal96–103 or were not immunized. Three weeks later, animals were transplanted with allogeneic male C3H heart grafts and were given CTLA4Ig plus anti-CD154 mAb MR1. Preimmunization with MHC-I-restricted peptide HYUtyp had no effect on graft survival, because grafts in all control and HYUtyp-immunized mice had detectable heartbeats for >60 days (Fig. 4). In contrast, preimmunization with the MHC-II-restricted peptide, HYDbyp, led to rapid rejection despite CTLA4Ig plus MR1 administration. To confirm the efficacy of the immunization and to evaluate potential effects of the immunizations on the antidonor immune response, recall ELISPOT assays were performed on day 60 after transplant or at the time of rejection in HYDbyp-immunized mice. As shown in Fig. 5, spleen cells from naive mice treated with CTLA4Ig plus MR1 (and exhibiting prolonged graft survival) exhibited essentially no detectable response to either HYUtyp (MHC-I restricted) or HYDbyp (MHC-II restricted). Spleen cells from these animals responded only weakly to donor C3H Ags, and the magnitude of this response was not different from that reactive to third-party BALB/c cells. This low frequency antidonor immunity was significantly weaker than the response detected in untreated mice that underwent rejection (Fig. 5), where anti-C3H immunity and anti-HYDbyp (CD4) indirect responses were readily detected. A strong recall response to HYUtyp was detected in animals preimmunized with MHC-I-restricted HYUtyp despite the costimulatory blockade-induced prolonged graft survival, thereby confirming the efficacy of the initial immunization. Notably, the detected frequency of direct antidonor immunity was low and similar in magnitude to the response detected to third-party BALB/c Ags in these mice (Fig. 5). The antidonor immunity in this group was not statistically different from that in naive mice given a transplant and treated with costimulatory blockade. Together with the data in Figs. 2 and 3, these findings suggest that CD8 effector T cells reactive to a donor-derived, self-MHC-I-restricted peptide are bystanders that do not influence the strength of the direct antidonor immune response and do not contribute to heart graft injury.


Figure 4
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  		FIGURE 4. Immunization with donor-derived, recipient MHC-I-restricted peptide HYUtyp induces peptide-specific immunity, but does not prevent the effects of costimulatory blockade on allograft survival. Groups of five or six B6 females were immunized with the indicated Ags (or not immunized); 3 wk later, the mice were transplanted with C3H male heart grafts and treated with CTLA4Ig plus MR1. HYDbyp-immunized mice treated with CTLA4Ig/MR1 and untreated controls rejected grafts with similar kinetics (not statistically different by Kaplan-Meier analysis). All grafts in unimmunized mice given CTLA4Ig/MR1 and in mice immunized with HYUtyp or a control peptide plus CTLA4Ig/MR1 were beating for >60 (p < 0.05 vs no treatment or HYDbyp plus CTLA4Ig/MR1).

 

Figure 5
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  		FIGURE 5. Primed CD8 T cells responding through the indirect pathway do not significantly influence the strength of the antidonor alloresponse. Recall IFN-{gamma} ELISPOT assays were performed on spleen cells from untreated mice that rejected allografts (A), unimmunized mice given CTLA4Ig/MR1 (B), mice immunized with HYDbyp/CFA plus CTLA4Ig/MR1 (C), and mice immunized with HYUtyp/CFA plus CTLA4Ig/MR1 (D) at the time points indicated. The mean values of three to five animals per group are depicted.

 
In contrast to the results with the MHC-I-restricted peptide, preimmunization with the MHC-II-restricted HYDbyp (that precipitated rejection despite costimulatory blockade) was associated with strong and specific antidonor T cell immunity (low third-party response), which was significantly higher in frequency than that in HYUtyp-immunized cells treated with CTLA4Ig/MR1 (Fig. 5). These data are consistent with previous work by our group showing that effector/memory CD4 T cells reactive to indirectly presented donor Ags overcome the effects of costimulatory blockade on prolonging graft survival (18, 29) and probably function through enhancing the strength of the direct antidonor T cell response.

Histologic examination of the heart tissue on day 60 revealed minimal graft vasculopathy in control-immunized or HYUtyp-immunized mice treated with costimulatory blockade (Fig. 6, A and B), and there was no significant difference in the extent of vasculopathy between these two groups. Histologic examination of the grafts from HYDbyp-immunized mice revealed typical features of acute cellular rejection (Fig. 6C). Antidonor alloantibodies, as determined by flow cytometry using donor thymocytes as targets (18, 29), were not detected in the serum of any animal with prolonged graft survival (not shown).


Figure 6
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  		FIGURE 6. Primed CD8 T cells responding through the indirect pathway do not induce allograft vasculopathy. Representative photomicrographs of elastin-stained sections (A, B, and D) or H&E-stained sections (C) of cardiac allografts in control mice preimmunized with betagal/OVA and treated with CTLA4Ig/MR1 (A), mice preimmunized with HYUtyp and treated with CTLA4Ig/MR1 (B), mice preimmunized with HYDbyp and treated with CTLA4Ig/MR1 (rejected; C), and mice given 6 x 106 preactivated Matahari T cells and treated with CTLA4Ig/MR1 (D). Magnification, x40.

 
Previous studies by others and by our laboratory revealed that the frequency of effector T cells influences the outcome of a transplant (23), raising the possibility that the number of antidonor CD8 T cells primed by immunization in the previous experiments was insufficient to have an effect on the graft rejection process. To circumvent this issue, we exploited Matahari TCR-transgenic T cells (reactive to HYUtyp/Db). In previous work we showed that primed Matahari T cells efficiently induce rejection of male (but not female) H-2b heart grafts through the direct pathway and that Matahari T cells do not induce acute or chronic injury of an H-2k male heart graft via an indirect effector mechanism (14, 23).

We repeated the costimulatory blockade experiments after adoptive transfer of 6 million TCR-transgenic Matahari T cells (reactive to HYUtyp + Db) that were preactivated in vivo by i.p. injection of Matahari females with 10 x 106 B6 male spleen cells. CTLA4Ig plus MR1 treatment resulted in >35-d graft survival, recall assays confirmed reactivity to HYUtyp (>30 IFN-{gamma} ELISPOTs/800,000 spleen cells detected; not shown), and the grafts were devoid of vasculopathy (Fig. 6D). Although it remains possible that a high frequency of Matahari T cells could indirectly mediate chronic injury in costimulatory blockade-treated animals over a longer period of time (>60 days), we believe that this is unlikely, because we previously showed that primed Matahari T cells did not induce chronic injury in unmanipulated mice followed for 70 days (14).

As a control to demonstrate that the adoptively transferred Matahari T cells could function after adoptive transfer in vivo, we repeated the experiments, but placed skin grafts, rather than heart transplants. In this model of skin transplantation we have previously shown that primed Matahari T cells can reject C3H male skin grafts through cognate interaction with cross-presented male Ag expressed on recipient endothelium (14). It is important to note that administration of CTLA4Ig plus MR1 does not induce indefinite skin graft survival in this strain combination, but routinely prolongs survival from 10 to >20 days (30). The adoptively transferred Matahari T cells precipitated skin graft rejection by day 14 despite CTLA4Ig/MR1 treatment (Fig. 7), significantly faster than grafts placed in control mice (not given adoptive transfers) treated with CTLA4Ig plus MR1. As an additional control we transferred 2C TCR transgenic T cells (CD8, reactive to Ld, but not cross-reactive to C3H Ags) into a group of three animals, followed by CTLA4Ig plus MR1 and a C3H skin graft. The grafts on these animals were visually intact (no evidence of rejection) on day 15 when they were killed to perform recall assays for comparison with Matahari T cell-transferred animals. Recall assays confirmed specific reactivity to HYUtyp in mice given Matahari T cells (1270 HYUtyp-reactive IFN-{gamma} ELISPOTs/million spleen cells; not shown), but not in control mice (<10/million spleen cells; not shown). Together the results show that a high number of preactivated, adoptively transferred CD8 T cells responding to a donor-derived, but self-restricted, transplant Ag can reject skin, but not heart, grafts despite costimulatory blockade.


Figure 7
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  		FIGURE 7. Primed Matahari CD8 T cells responding through the indirect pathway specifically abrogate male skin graft survival induced by costimulatory blockade. C3H male skin grafts were placed on B6 females (n = 5/group) 1 day after adoptive transfer of 6 x 106 Matahari or 2C TCR-transgenic T cells or onto recipients without T cell transfers and were treated with CTLA4Ig/MR1 (a control group was untreated). *, Mice given Matahari T cells and treated with CTLA4Ig/MR1 rejected grafts significantly faster than mice given 2C T cells or no T cells plus CTLA4Ig/MR1 (p < 0.05, by Kaplan-Meier analysis). The mice given Matahari T cells plus CTLA4Ig/MR1 rejected grafts with kinetics similar to untreated controls (p > 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CD4 T cells reactive to donor-derived, self-restricted transplant Ags play a vital role in the immune response that develops after organ transplantation. Multiple studies performed by a number of investigators over the last 15–20 years have shown that CD4 T cells responding through the indirect pathway mediate acute and chronic graft injury and are important for the induction and maintenance of allograft tolerance (21, 29, 31, 32, 33, 34, 35, 36, 37, 38).

The results of the present studies provide new information about CD8 T cells responding through the indirect pathway to transplant Ags, specifically focusing on their ability to mediate injury at the effector stage. Our results show that in contrast to CD4 T cells responding to an indirectly present Ag (22, 38), CD8 T cells reactive to a donor-derived, self-restricted transplant Ag are nonpathogenic bystanders within the alloimmune repertoire induced to a fully MHC-disparate heart graft. Using a defined host-restricted, minor transplantation Ag, we showed that preactivated Ag-specific CD8 T cells did not influence the kinetics of acute cardiac allograft rejection, did not influence the development of chronic graft injury, and did not adversely affect costimulatory blockade-induced prolongation of graft survival. The latter was true in the context of in vivo primed endogenous immune cells and after adoptive transfer of large numbers of Ag-specific CD8 T cells. Furthermore, HY-specific CD8 T cells responding via the indirect pathway had no influence on the induced strength of the direct, antidonor immune response after CTLA4Ig plus MR1 treatment.

These results argue against the possibility that preactivated CD8 T cells responding via the indirect pathway nonspecifically enhance antidonor responses via bystander reactions (i.e., via cytokine secretion) or efficiently function at the effector stage in the absence of cognate interactions with donor cells. This latter conclusion is in contradistinction to the effect of indirectly primed CD8 T cells on allogeneic skin graft rejection, in which the target Ag is expressed on host (recipient) endothelium (14, 17). The vascular endothelium feeding a heart graft is overwhelmingly comprised of donor, rather than recipient, cells (39, 40). Whether the small numbers of host-derived endothelial cells detected in animal and human cardiac allografts can function as targets of a cross-primed CD8 response remains unclear from this work. Although it is also theoretically possible that recipient-derived, graft-infiltrating monocyte/macrophages that cross-presented donor Ag on recipient MHC-I could function as immune targets for the cross-primed CD8 T cells, the absence of graft injury in our studies suggests that such a mechanism of injury occurs inefficiently and/or that there is minimal bystander damage to the graft despite such cognate interactions. It should be noted that our studies used full MHC disparity, and that indirectly primed CD8 T cells are likely to contribute to graft injury at the effector stage if the transplant shares MHC-I alleles with the recipient (in such a situation the target Ag will be expressed on the donor cells).

We find it intriguing that cross-priming of CD8 T cells occurred reproducibly after minor Ag disparate heart transplantation, but that essentially no cross-priming to this Ag was detectable after fully allogeneic heart transplantation (C3H male to B6 female; Fig. 1). It is well established that an immune repertoire induced by any antigenic stimulus is directed at specific immune-dominant epitopes, rather than being polyreactive to all potential MHC-binding determinants derived from the target Ag (41, 42, 43). If dominant epitopes are removed from the system, then the immune repertoire shifts toward subdominant or so-called cryptic determinants. Mechanisms underlying this phenomenon are not well understood, but may involve time-dependent and inflammation-induced alterations in Ag processing, presentation, and costimulatory molecule expression that permit T cells of lower affinity to be recruited later into a given response (41, 42, 43). Our data suggest that the cross-presented determinant, HYUtyp, functions as a cryptic epitope during the potent alloimmune response directed predominantly at donor MHC. Only when the immune dominant alloantigens (i.e., donor MHC) are removed from the donor-recipient disparity does this cross-presented epitope become apparent.

The results of our work have implications relevant to clinical transplantation. It is now clear that effector/memory T cells reactive to donor Ags can accelerate graft injury and are possible barriers to tolerance induction (44, 45, 46). This suggests that immune monitoring to assess frequencies and specificities of the peripheral, preactivated, antidonor immune repertoire might be useful for predicting incipient acute and/or chronic graft injury so as to ultimately direct therapeutic decision-making. The present work demonstrates that cross-primed CD8 T cells are not pathogenic in the context of transplantation if the donor and recipient do not share MHC-I alleles. Thus, evaluating the strength of the cross-primed CD8 response under these circumstances may not be required, because it would be of minimal diagnostic or therapeutic value.


   Acknowledgments
 
We thank Shanzhong Zhang for his technical assistance with the cardiac transplantation, and Earla Biekert for her editorial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 supported by National Institutes of Health Grant R01 AI43578 (to P.S.H.). A.V. is the recipient of a bridge award from the American Society of Transplantation. Back

2 Address correspondence and reprint requests to Dr. Peter S. Heeger, Department of Immunology, Cleveland Clinic Foundation, NB30, 9500 Euclid Avenue, Cleveland OH 44195. E-mail address: heegerp{at}ccf.org Back

3 Abbreviations used in this paper: MHC-I, class I MHC; betagal, beta-galactosidase; GIL, graft-infiltrating lymphocyte. Back

Received for publication August 31, 2005. Accepted for publication December 8, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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