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The Journal of Immunology, 1999, 162: 1206-1214.
Copyright © 1999 by The American Association of Immunologists

Rejection of Cardiac Xenografts by CD4+ or CD8+ T Cells

Yuan Lin1, Miguel P. Soares, Koichiro Sato, Ko Takigami, Eva Csizmadia, Josef Anrather and Fritz H. Bach2

Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently showed that brief complement inhibition induces accommodation of hamster cardiac transplants in nude rats. We have reconstituted nude rats carrying an accommodated xenograft with syngeneic CD4+ or CD8+ T cells to investigate the cellular mechanism of xenograft rejection. We show that CD4+ T cells can initiate xenograft rejection (10 ± 1.7 days) by promoting production of IgG xenoreactive Abs (XAb). These XAb are able to activate complement as well as to mediate Ab-dependent cell-mediated cytotoxicity. Adoptive transfer of these XAb into naive nude rats provoked hyperacute xenograft rejection (38 ± 13 min). The rejection was significantly (p < 0.001) delayed by cobra venom factor (CVF; 11 ± 8 h in four of five cases) but was still more rapid than in control nude rats (3.3 ± 0.5 days). CVF plus NK cell depletion further prolonged survival (>7 days in four of five cases; p < 0.01 vs CVF only). CD8+ T cell-reconstituted nude rats rejected their grafts later (19.4 ± 5.8 days) and required a larger number of cells for transfer as compared with CD4+ T cell-reconstituted nude rats. However, second xenografts were rejected more rapidly than first xenografts in CD8+ T cell-reconstituted nude rats (9 ± 2 days), indicating that the CD8+ T cells had been activated. This study demonstrates that CD4+ and CD8+ T cells can both reject xenografts. The CD4+ cells do so at least in part by generation of helper-dependent XAb that act by both complement-dependent and Ab-dependent cell-mediated cytotoxicity mechanisms; the CD8+ cells do so as helper-independent cytotoxic T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the absence of hyperacute rejection, xenografts are rejected primarily by cellular mechanisms. T cells are a major mediator of the rejection of neovascularized xenografts (1, 2). For immediately vascularized xenografts, however, other immune cells such as NK cells and macrophages may initiate delayed xenograft rejection (DXR)3 before T cell activation occurs (3, 4). The concept that DXR can proceed independently of T cells is supported by its occurrence in nude rats (5) and by the ineffectiveness of T cell-directed immunosuppressive agents in suppression of DXR (6).

Recent progress in prevention of DXR has allowed vascularized xenografts to survive until T cell-dominated rejection occurs. In several concordant xenografting models, long-term survival of xenografts depends on continuous administration of an anti-T cell immunosuppressant, cyclosporin A (CyA) or FK506 (7, 8). Previous studies also showed that T cells participate in both cellular and humoral responses by generation of cytotoxic T cells (9) and T cell-dependent xenoreactive Abs (XAb) (7). However, it has been difficult to evaluate precisely which features of T cell-dependent XAb responses participate in xenograft rejection, since T cell-independent XAb responses can also reject xenografts (7). Moreover, T cell immunosuppressants, such as CyA, suppress both CD4+ and CD8+ T cell helper function (10), making it difficult to dissect the relative contribution of each T cell subset in xenograft rejection.

Long-term survival of hamster cardiac xenografts in rats has been previously induced by transient depletion of complement activity with cobra venom factor (CVF) plus daily and continuous administration of CyA (11, 12). These grafts survive indefinitely in the presence of antigraft IgM and normal complement levels, a phenomenon referred to as "accommodation" (13). Using the same CVF protocol, we recently induced long-term survival and accommodation of hamster hearts in nude rats without further immunosuppression.4

To analyze directly the mechanism underlying T cell-mediated xenograft rejection, we reconstituted nude rats with syngeneic rat T cells 30 days after xenografts had accommodated in those rats. This model allows investigation of T cell-mediated rejection in the absence of certain other factors that would complicate the interpretation of results, such as immunosuppressants. Moreover, it permits analysis of the relative contributions of unique T cell subsets. We demonstrate that CD4+ and CD8+ T cells can independently mediate rejection of xenografts. The CD4+ cells do so by generation of helper-dependent XAb that function to contribute to rejection both by complement-dependent mechanisms and by Ab-dependent cell-mediated cytotoxicity (ADCC). The CD8+ cells that contribute to rejection are helper-independent CTL that produce their own growth factors. Both of these CD4+ and CD8+ mechanisms involve cytokine synthesis that is sensitive to CyA, suggesting that the T cell immunosuppression will be important for achieving long-term xenograft survival.


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

Golden Syrian hamsters weighing 60–80 g were used as organ donors. Inbred male nude rats (PVG-rnu/rnu, RT1c) weighing 100–200 g were used as recipients. Inbred PVG rats (RT1c) (Harlan Sprague-Dawley, Indianapolis, IN) were used as donors in T cell transfer experiments. All animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care. The research protocols were approved by the International Animal Care and Use Committees of the Beth Israel Deaconess Medical Center.

Heterotopic heart transplantation

Cervical heterotopic hamster-to-rat cardiac xenografts were performed using a technique described previously (7). The function of the grafts was monitored by daily inspection and palpation. Rejection was diagnosed by cessation of visible and palpable ventricular contraction and confirmed by histology.

Immunosuppressive agents

CVF (Quidel, San Diego, CA) was diluted in normal saline and administered by i.p. injection at day -1 (60 U/kg) and day 0 (20 U/kg) of transplantation. Anti-asialo GM-1 (AAGM-1) antiserum (Wako, Richmond, VA) (100 µl) was diluted in normal saline and was injected i.v. into some animals 12 h before CVF injection.

Preparation and fractionation of T cells

T cells for adoptive transfer were prepared from pooled cervical and mesenteric lymph nodes from normal PVG rats. Briefly, freshly removed lymph nodes were placed in sterile petri dishes containing ice-cold, serum-free RPMI 1640 and were subsequently minced. The single-cell suspension was passed through a sterile 53-µm stainless steel mesh screen. Monocytes and macrophages were depleted by two-round adhesion to cell culture dishes at 37°C in 5% CO2 as described previously (14). Purified CD4+ or CD8+ T cells were prepared by depletion of B cells, NK cells, and CD8+ or CD4+ T cells using indirect panning techniques. Briefly, cells were labeled with the mAbs MARM4 (anti-rat IgM; a gift of Prof. H. Bazin, University of Louvain, Louvain, Belgium) and 3.2.3 (anti-rat NK cells; Serotec, Oxford, U.K.) together with OX8 (anti-rat CD8; PharMingen, San Diego, CA) or OX38 (anti-rat CD4; a kind gift of Prof. C. G. Fathman, Stanford University, Stanford, CA); after labeling, the cells (1 x 107/ml in complete RPMI 1640) were incubated in anti-mouse Ig-coated petri dishes at room temperature for 30 min. The unbound cells were collected by gently washing with complete RPMI 1640. Following depletion, the purity of the T cell fractions was examined by FACS analysis. Purity of CD4+ or CD8+ T cells was consistently >95% with, respectively, <0.5% contamination by CD8+ or CD4+ T cells. When contamination was >0.5%, the cells were further rosetted with anti-mouse Ig-coated magnetic Dyna-Beads (Dynal, New Ferry, U.K.) followed by magnetic depletion. After purification, various T cell fractions were injected i.v. into nude rats carrying a long term (30 days)-surviving xenograft.

Phenotype analysis

PBMC (0.5 x 106) were double-stained with phycoerythrin-conjugated mouse mAb specific for rat TCR-{alpha}ß (R73) and FITC-conjugated mouse mAb specific for rat CD4 (OX35) or CD8 (OX8) (PharMingen). The stained cells were analyzed by flow cytometry. Fluorescence-conjugated, isotype-matched irrelevant Abs were used as negative controls.

Assay of elicited xenoantibodies

The IgM and IgG isotype anti-hamster XAb were measured by flow cytometry using hamster PBMC as target cells, as described previously (7). Briefly, aliquots of 0.5 x 106 cells were incubated for 30 min at 4°C with 100 µl of 1:10 diluted serum taken from recipient rats at various time intervals after transplantation. To determine IgG XAb, the cells were further incubated with a mouse mAb directed against rat IgG1, IgG2a, IgG2b, or IgG2c (mouse IgG1 was a gift of Prof. H. Bazin). After addition of FITC-goat anti-rat IgM antiserum (Cappel, Aurora, OH) or FITC-rat anti-mouse IgG1 antiserum (Zymed, South San Francisco, CA), the cells were examined by flow cytometry. Results were expressed as the mean channel fluorescence of stained cells divided by the mean channel fluorescence of cells incubated with control serum and FITC-Abs.

Transfer of rejecting serum

Whole blood (1–2 ml) was taken by heart puncture from nude rats having rejected a hamster graft 1 day earlier. After 40 min at room temperature, the serum was separated by centrifugation and stored at -70°C until use. Before transfer, the serum was heat inactivated at 56°C for 30 min and injected i.v. (0.5 ml).

Histopathology and immunohistochemistry

Graft samples for histology were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for light microscopy. Graft samples for immunohistochemistry were snap frozen in prechilled isopentane and stored at -70°C. The frozen specimens were cut into 4-µm sections in a cryostat at -25°C and air dried. Rat anti-hamster Igs in the xenografts were detected by mAb directed against rat IgM (MARM-4), IgG1 (MARG-1), IgG2a (MARG2a-1), IgG2b (MRAG2b-8), and IgG2c (MARG2c-5) (a kind gift from Prof. H. Bazin). Intragraft complement deposition was detected by staining with an anti-rat C3 mAb (ED11) (Serotec). Expression of P-selectin was detected using a rabbit anti-rat P-selectin polyclonal Ab (CD62P) (PharMingen). Rat leukocyte populations were analyzed using mAb specific for leukocyte common Ag (OX-1), T cells (TCR-{alpha}ß-chains, R73), NK cells (NKR-P1A) (PharMingen), and macrophages (CD68, ED-1) (Serotec). Intragraft cytokines were stained with mAb directed against rat IL-2 (A38-3), IL-4 (OX81), IL-10 (A5-4) (PharMingen), and IFN-{gamma} (DB-1, Biosource, Camarillo, CA). Goat polyclonal Abs were used to detect rat IL-13 (Santa Cruz Biotechnology, Santa Cruz, CA) and TNF-{alpha} (R&D Systems, Minneapolis, MN). For study of leukocytes, Abs, and complement, cryostat sections were fixed in paraformaldehyde-lysine-periodate or were fixed in acetone for localization of cytokines and stained as previously described (11). Isotype-matched mAbs or purified Ig were included in each experiment.

Statistics

The results were statistically analyzed by the Student’s t test or by Fisher’s Exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Graft survival after T cell reconstitution

Survival of accommodated hamster heart grafts in nude rats after T cell reconstitution is shown in Table IGo. We recently induced accommodation of ~50% of hamster hearts in nude rats by transient depletion of complement activity with CVF (data not shown). Thirty days after accommodation occurred, reconstitution of nude rats with whole T cells (2 x 107) or CD4+ T cells (1 x 107) provoked acute rejection of xenografts within a few days (9.3 ± 2.3 days (n = 6) and 10 ± 1.7 days (n = 6), respectively). Reconstitution with 1 x 107 or 2 x 107 CD8+ T cells resulted in rejection of three of six xenografts (22.7 ± 2.3 days) and six of six xenografts (19.4 ± 5.8 days), respectively.


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  		Table I. Survival of accommodated xenografts in nude rats after T cell reconstitution

 
Detection of adoptively transferred T cells in nude rats

Nude rats normally have no detectable circulating CD4+ or CD8+ T cells (Fig. 1Go, A and B). One week after reconstitution with total T cells, PBMC of these nude rats contained 10–15% CD4+ and 1–4% CD8+ T cells (Fig. 1Go, C and D). In CD4+ T cell-reconstituted nude rats, CD4+ and CD8+ T cells comprise 10–15% and <1% of PBMC, respectively (Fig. 1Go, E and F). Nude rats reconstituted with CD8+ T cells (10 x 106) showed 4–6% CD8+ and <1% CD4+ T cells in their PBMC (Fig. 1Go, H and G).



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  		FIGURE 1. Circulating T cells in nude rats after T cell reconstitution. Nude rats normally have no detectable circulating CD4+ or CD8+ T cells (A and B). One week after T cell reconstitution, whole T cell-reconstituted nude rats showed both CD4+ and CD8+ T cells in their peripheral blood (C and D), whereas CD4+ T cells and CD8+ T cells were only present in nude rats reconstituted with CD4+ (E and F) and CD8+ (G and H) T cells, respectively. Data are shown as one representative of three individual rats from each group.

 
Xenoantibody formation after T cell reconstitution

Production of IgM and IgG anti-graft XAb after T cell reconstitution is shown in Fig. 2Go. Without T cell reconstitution, nude rats carrying an accommodated xenograft transplanted 30 days earlier had low, but increased, levels of anti-graft IgM as compared with pretransplantation. These nude rats had no detectable IgG (Fig. 2Go, a and b). Nude rats reconstituted with either total T cells (Fig. 1GoD) or CD4+ T cells (Fig. 1GoF) developed high levels of IgG2a and IgG2b and relatively lower levels of IgG1 and IgG2c. IgM levels were also slightly increased in these two groups as compared with nonreconstituted nude rats (Fig. 1Go, C and E). In contrast, nude rats reconstituted with CD8+ T cells, even at the higher number (2 x 107), did not produce significant levels of additional anti-graft IgM or IgG XAb (Fig. 1Go, G and H) as compared with nonreconstituted nude rats.



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  		FIGURE 2. Xenoantibody formation after T cell reconstitution. Before T cell reconstitution, nude rats carrying an accommodated xenograft transplanted 30 days earlier had low levels of anti-donor IgM elevated from first xenotransplantation and no detectable IgG (a and b). Nude rats reconstituted with either whole T cells or CD4+ T cells developed high levels of IgG (d and f) and low levels of IgM (c and e), whereas neither IgG nor IgM XAb increased in CD8+ T cell-reconstituted nude rats (g and h). Data are shown as one representative of three individual rats from each group.

 
Survival of second xenografts

A second hamster heart was transplanted to T cell-reconstituted nude rats 1 day after they rejected a first hamster xenograft (Table IIGo). Nude rats reconstituted with CD4+ T cells rejected second hamster heart grafts in 38 ± 22 min. In contrast, nude rats reconstituted with CD8+ T cells (2 x 107) rejected second hamster heart grafts in 9 ± 2 days.


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  		Table II. Survival of second xenografts in T cell-reconstituted nude rats rejecting a first hamster heart

 
Graft rejection caused by adoptive transfer of serum

Serum (0.5 ml) taken from CD4+ T cell-reconstituted nude rats 1 day after the rats rejected their accommodated xenografts was injected into naive nude rats that had received hamster heart grafts 1 h earlier (Table IIIGo). This serum transfer provoked hyperacute rejection of hamster heart grafts (38 ± 13 min; n = 6). When the naive nude rats were depleted of complement by administration of CVF (given at day -1 and day 0 with respect to the time of serum transfer), graft survival was significantly prolonged to 11 ± 8 h (n = 5), with one graft surviving for >5 days. Treatment with both CVF and AAGM-1 to deplete NK cells led to further prolongation of graft survival in four of five nude rats (>7 days), with one xenograft rejected at day 2. AAGM-1 alone did not prolong graft survival after serum transfer (data not shown).


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  		Table III. Survival of hamster hearts in naive nude rats after adoptive transfer of rejecting serum

 
Histopathology and immunohistochemistry

Accommodated xenografts exhibited healthy-appearing cardiac fibers and less mononuclear cell infiltration than found in rejected xenografts (Fig. 3GoA). Grafts rejected in CD4+ T cell-reconstituted nude rats showed interstitial edema, hemorrhage, myocardial necrosis, vascular thrombosis, and dense mononuclear cell infiltration (Fig. 3GoB). Grafts rejected in CD8+ T cell-reconstituted nude rats showed mainly mononuclear cell infiltration and associated myocardial destruction (Fig. 3GoC).



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  		FIGURE 3. Histology and immunoperoxidase staining for Ig and complement in accommodated vs rejected grafts. Grafts were harvested after rejection or 40 days after transplantation. Hematoxylin and eosin-stained paraffin sections show that accommodated grafts exhibited healthy-appearing myocardial fibers and less mononuclear cell infiltration than rejected grafts (A); rejection in CD4+ T cell-reconstituted nude rats was associated with interstitial edema and hemorrhage, myocardial necrosis, and vascular thrombosis, with dense mononuclear cell infiltration (B); and rejection in CD8+ T cell-reconstituted nude rats was associated with mononuclear cell infiltration and myocardial destruction (C). Immunoperoxidase staining of cryostat sections shows that accommodated grafts and grafts rejected by CD8+ T cell-reconstituted nude rats exhibited comparable deposition of IgM (D and F) and C3 (T and U) but no IgG (G, J, M, and P; I, L, O, and R), whereas grafts rejected by CD4+ T cell-reconstituted nude rats showed deposition of IgM (E), C3 (T), and IgG (H, K, N, and Q). The arrow shows positive staining when appropriate.

 
Immunohistochemistry demonstrated vascular deposition of IgM and C3 but no IgG in accommodated xenografts (Fig. 3Go, D, G, J, M, P, and S). Grafts rejected by CD4+ T cell-reconstituted nude rats showed deposition of IgM, C3, and all IgG subclasses (Fig. 3Go, E, H, K, N, Q, and T). In contrast, grafts rejected by CD8+ T cell-reconstituted nude rats showed only IgM and C3 deposition but no IgG (Fig. 3Go, F, I, L, O, R, and U).

Immunohistochemical analysis demonstrated little infiltration into accommodated xenografts by leukocytes, consisting mainly of macrophages and a small number of NK cells, without TCR+, CD8+, or CD45RC+ T cells (Fig. 4Go, A, D, G, J, M, and P). These infiltrating cells produced a small amount of IFN-{gamma} and TNF-{alpha} without detectable IL-2 (Fig. 5Go, A, D, and G). Importantly, these cells showed strong staining for IL-10 (Fig. 5GoJ). Expression of P-selectin within grafts was essentially undetectable (Fig. 5GoM).



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  		FIGURE 4. Immunoperoxidase staining for infiltrating cells in accommodated vs rejected grafts. Grafts were harvested after rejection or 40 days after transplantation. Accommodated grafts showed less leukocyte infiltration (A) consisting of NK cells (D) and macrophages (G) without T cells (J, M, and P). Grafts rejected by CD4+ or CD8+ T cell-reconstituted nude rats showed dense leukocyte infiltration (B and C) consisting mainly of macrophages (H and I), a small number of NK cells (E and F), and TCR+ (K and L) or CD45RC+ (Q and R) T cells. In CD8+ but not CD4+ T cell-reconstituted nude rats, rejected grafts showed labeling of some infiltrating cells for anti-CD8 mAb (N and O). The arrow shows positive staining when appropriate.

 


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  		FIGURE 5. Immunoperoxidase staining for intragraft cytokines in accommodated vs rejected grafts. Grafts were harvested after rejection or 40 days after transplantation. Accommodated grafts showed labeling of fewer infiltrating cells for IFN-{gamma} or TNF-{alpha} and no IL-2 (A, D, and G), whereas the infiltrating cells in these grafts strongly expressed IL-10 (J). Grafts rejected by CD4+ or CD8+ T cell-reconstituted nude rats exhibited a larger number of infiltrating cells expressing IFN-{gamma} (B and C), TNF-{alpha} (E and F), and IL-2 (H and I) but essentially no IL-10 (K and L). Both sets of rejected grafts, but not accommodated grafts, expressed P-selectin (M, N, and O). The arrow shows positive staining when appropriate.

 
Grafts rejected by CD4+ or CD8+ T cell-reconstituted nude rats showed dense infiltration of leukocytes (Fig. 4Go, B and C) consisting of macrophages (Fig. 4Go, H and I), a small number of NK cells (Fig. 4Go, E and F), and TCR+ (Fig. 4Go, K and L) or CD45RC+ (Fig. 4Go, Q and R) T cells. In contrast to grafts rejected by CD4+ T cell-reconstituted nude rats, grafts rejected by CD8+ T cell-reconstituted nude rats showed anti-CD8 mAb labeling of some infiltrating cells (Fig. 4Go, N and O). In both sets of grafts, the infiltrating cells expressed high levels of IFN-{gamma} (Fig. 5Go, B and C), TNF-{alpha} (Fig. 5Go, E and F), and IL-2 (Fig. 5Go, H and I) but essentially no IL-10 (Fig. 5Go, K and L). P-selectin was strongly expressed on endothelial cells in both sets of grafts (Fig. 5Go, N and O).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T cells participate in xenograft rejection and may become the dominant rejection factor at later times of rejection (7, 8, 15). Consistent with this notion, we have previously shown that survival of accommodated hamster hearts in rats can be maintained by the T cell-directed immunosuppressant CyA (11). Moreover, this maintenance therapy is not required when accommodation is induced in nude rats.4 The present study reveals that reconstitution of nude rats with syngeneic rat T cells results in rejection of the accommodated xenografts.

In an effort to analyze the relative contribution of unique T cell subsets in xenograft rejection, we reconstituted nude rats with CD4+ or CD8+ T cell subpopulations. Whereas reconstitution with either CD4+ or CD8+ T cells caused nude rats to reject their accommodated xenografts, rejection in CD4+ T cell-reconstituted nude rats was accompanied by generation of anti-donor, predominantly IgG XAb. These XAb played a major role in graft rejection. First, second hamster hearts transplanted into CD4+ T cell-reconstituted nude rats that had rejected first accommodated xenografts 1 day earlier underwent hyperacute rejection. Second, hyperacute rejection of freshly transplanted xenografts could be achieved in naive nude rats by adoptive transfer of sensitized serum from CD4+ T cell-reconstituted nude rats that had rejected a hamster heart. This finding is consistent with our previous study in euthymic rats showing that adoptive transfer of anti-hamster IgG or IgG subclasses from sensitized rats provoked hyperacute rejection of hamster cardiac xenografts in naive rats (16, 17). Previous work from other groups has also shown that xenoreactive IgG are potent complement-fixing Abs capable of mediating xenograft rejection in rodents (18) and in nonhuman primates (19).

In the absence of T cells, nude rats reject hamster cardiac grafts by production of T cell-independent IgM XAb that activate lytic complement (14). After T cell reconstitution, nude rats started to produce IgG XAb. We demonstrated that nude rats reconstituted with CD4+ T cells, but not those reconstituted with CD8+ T cells, produced anti-graft IgG XAb, consistent with the known functions of these cells (20).

Another mechanism by which IgG XAb may initiate xenograft rejection may involve their ability to mediate ADCC. Several lines of evidence show that IgG XAb enhance in vitro non-T cell-mediated cytotoxicity against xenogeneic target cells, such as cultured endothelial cells, through an ADCC mechanism (21, 22). An ADCC mechanism is also consistent with xenograft rejection in vivo involving adoptive transfer of IgG XAb or sensitized B cells; blockade of action of either the ADCC-associated Abs or the ADCC-associated effector cells is associated with prevention of transfer-induced acceleration of xenograft rejection (14, 23).

To test the possibility that ADCC is involved in xenograft rejection in the present study, IgG XAb serum was transferred into naive nude rats that had received a fresh heart 1 h earlier and whose complement activity had been inhibited by CVF. While hyperacute rejection occurred after serum transfer without CVF treatment (38 ± 13 min), graft survival was significantly (p < 0.001) prolonged when CVF was administered (11 ± 8 h in four of five cases). However, rejection still occurred significantly earlier than in control nude rats without serum transfer (3.3 ± 0.5 days, p < 0.001). Graft survival was further prolonged to >7 days in four of five cases (p < 0.01 vs CVF treatment only) when the recipients were also depleted of NK cells, which are major ADCC-associated effector cells. These results led to the conclusion that IgG XAb are involved in xenograft rejection both by activating complement (the graft had accommodated to the IgM that was present) and by mediating ADCC. The fact that monocytes, which are also capable of mediating ADCC (24), were not depleted may account for the rejection of one graft on day 2 when the rats were given CVF plus AAGM-1. More importantly, the ADCC mechanism may explain why accommodated grafts appear more resistant to IgM XAb than to IgG XAb. First, the established accommodation in the nude rats was broken when IgG XAb were present; second, we previously showed that adoptive transfer of IgG, but not IgM, XAb serum provoked rejection of accommodated hamster hearts in euthymic rats (25).

CD4+ T cells are believed to be crucial to initiating cellular rejection of xenografts (26, 27). In addition to providing assistance for generation of CTL (28), a major effector in T cell-mediated immune responses, CD4+ T cells are able to mediate graft rejection in the absence of CTL (28, 29). We showed previously that T cell-dependent XAb can be important in mediating rejection of xenografts (7). Our present study provides direct evidence that T cell-dependent XAb are a major mediator of xenograft rejection in the absence of CD8+ CTL. Our data do not rule out the possibility that CD4+ T cells may have direct cytotoxic effects through mechanisms such as the Fas-mediated pathway (28) or T cell-derived cytokines (29).

In CD8+ T cell-reconstituted nude rats, rejection occurred without XAb formation. The rejection was associated with dense mononuclear cell infiltration including, in particular, a small number of CD8+ T cells. The latter cells produced T cell-derived cytokines such as IL-2 (Fig. 5Go). Moreover, second hamster hearts were rejected more rapidly than the initial ones, indicating that CD8+ T cells were at an activated or expanded state.

CD8+ T cells are considered to be CTL, but their activation normally requires help from CD4+ T cells. One explanation of our finding is that the CD8+ T cells are helper-independent CTL that produce their own help (30). An additional finding arguing for the presence of this type of helper-independent CTL is that the helper function of the CD8+ cells is sensitive to CyA suppression, which interferes with helper/inducer T cell function (10). This phenomenon was also observed in the present study: CyA suppressed completely both CD4+ T cell and CD8+ T cell helper responses during xenograft accommodation in euthymic rats. Similarly, previous experiments showed that CD8+ T cells directly recognize xeno-MHC Ags in vitro (31, 32). Furthermore, skin and cardiac xenografts were rejected in mice that have only CD8+ T cells (33, 34). The mechanism underlying the activation of helper-independent CD8+ CTL is unclear. Previous studies indicate that this effect may be mediated by cytokines such as IL-1 (30) and IL-6 (35); interaction between APCs and CD8+ T cells through CD40/CD40-L signaling (36); or so-called "cross-priming," in which exogenous Ags are processed and presented by class I Ags on APC (37).

In addition to mediating the MHC-restricted effector mechanism, both activated CD4+ and CD8+ T cells seemed to contribute indirectly to xenograft rejection by producing Th1 cytokines. The infiltrating T cells within rejected xenografts expressed the CD45RC phenotype, a characteristic of Th1 cells (38), and they produced Th1 cytokines such as IL-2, IFN-{gamma}, and TNF-{alpha}. In contrast, accommodated xenografts, despite the absence of Th2 cells, showed a Th2-type cytokine response characterized by expression of monocyte-associated IL-10. This finding is consistent with our previous observation in euthymic rats (11). Proinflammatory cytokines might, in turn, activate other immune effector cells such as NK cells and macrophages, as evidenced by profound infiltration and cytokine production by these cells (Fig. 4Go). These cytokines might also activate graft endothelial cells, characterized by expression of P-selectin (Fig. 3Go).

In summary, the present study shows that T cell immune responses may become dominant in xenotransplantation after DXR is avoided. CD4+ T cells initiate rejection of xenografts even in the absence of CD8+ CTL by promoting generation of helper-dependent IgG XAb. These XAb can activate complement as well as mediate ADCC; we present evidence that both of these mechanisms contribute to rejection. There are also CD4+ T cell-independent CTL that produce their own help. Since both helper responses are sensitive to CyA suppression, conventional immunosuppressants can be evaluated for their potential application in eventual clinical xenotransplantation.


   Footnotes
 
1 Address correspondence and reprint requests Dr. Yuan Lin, Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215. E-mail address: Back

2 F.H.B. is the Lewis Thomas Professor at Harvard Medical School, and a paid consultant for Novartis Pharma. Back

3 Abbreviations used in this paper: DXR, delayed xenograft rejection; CyA, cyclosporin A; XAb, xenoreactive antibodies; CVF, cobra venom factor; ADCC, antibody-dependent cell-mediated cytotoxicity; AAGM-1, anti-asialo GM-1. Back

4 Y. Lin, M. Soares, K. Sato, K. Takigami, E. Csizmadia, J. Anrather, and F. H. Bach. Submitted for publication. Back

Received for publication August 19, 1998. Accepted for publication September 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Faustman, D., C. Coe. 1991. Prevention of xenograft rejection by masking donor HLA class I antigens. Science 252:1700.[Abstract/Free Full Text]
  2. Zhao, Y., K. Swenson, J. J. Sergio, J. S. Arn, D. H. Sachs, M. Sykes. 1996. Skin graft tolerance across a discordant xenogeneic barrier. Nat. Med. 2:1211.[Medline]
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